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Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa (Medicago sativa L.)
Abstract
Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impact of specifically reducing nodule NADH-GOGAT activity on symbiotic performance of alfalfa (Medicago sativa L.). Four independent transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for GOGATantisense), containing an antisense NADH-GOGAT cDNA fragment under the control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA plants were fertile and showed normal growth in non-symbiotic conditions. The NADH-GOGAT antisense transgene was heritable and the T1 plants showed phenotypic alterations - similar to primary transformants. Clonally propagated plants were inoculated with Sinorhizobium meliloti after rooting and the symbiotic phenotype was analyzed 21 days post-inoculation. Nodules of each GA line had reduced NADH-GOGAT activity, ranging from 33 to 87% of control plants, that was accompanied by comparable decreases in RNA and protein. Plants from the GA89 line, with the lowest NADH-GOGAT activity (c. 30%), presented a strikingly altered symbiotic phenotype: concomitantly activities of key enzyme for carbon and nitrogen assimilation decreased; nodule amino acids and amides were reduced while sucrose accumulated. Antisense GOGAT plants were chlorotic, reduced in fresh weight, and had a lower N content than control plants. Photosynthesis was also impaired in antisense plants. Specifically, reducing NADH-GOGAT in nodules resulted in plants having impaired nitrogen assimilation and altered carbon/nitrogen metabolic flux.
... In this study, the authors show that the entire process is dependent on the O 2 concentration, the low O 2 content of which protects nitrogenase from inhibition, reduces the assimilation of NH 4 + into glutamate in the bacteroids, and promotes the export of NH 4 + and alanine to the plant cell (Schulte et al., 2021). NH 4 + exported by the bacteroid is transported into the cytosol of the infected host cells for its assimilation in amino acids, via the combined action of GS/ GOGAT ( Figure 4; Cordoba et al., 2003;Seabra et al., 2012). This assimilation process requires an additional flux of C skeleton (aketoglutarate) provided by photosynthesis to match the NH 4 + flux. ...
... SUNN, HAR1, and NARK loci encode these AON LRR-RLK in M. truncatula, L. japonicus, and soybean, respectively (Krusell et al., 2002;Nishimura et al., 2002;Searle et al., 2003;Schnabel et al., 2005). The receptors exist as homodimers or heterodimers formed with truncated co-receptors (LjCLV2, LjKLV in L. japonicus or MtCLV2 and MtCRN in M. truncatula; Miyazawa et al., 2010;Krusell et al., 2011;Crook et al., 2016). CLE peptides are encoded by large gene families in legumes and non-legume plants (Yamaguchi et al., 2016). ...
Symbiotic nodules formed on legume roots with rhizobia fix atmospheric N2. Bacteria reduce N2 to NH4 + that is assimilated into amino acids by the plant. In return, the plant provides photosynthates to fuel the symbiotic nitrogen fixation. Symbiosis is tightly adjusted to the whole plant nutritional demand and to the plant photosynthetic capacities, but regulatory circuits behind this control remain poorly understood. The use of split-root systems combined with biochemical, physiological, metabolomic, transcriptomic, and genetic approaches revealed that multiple pathways are acting in parallel. Systemic signaling mechanisms of the plant N demand are required for the control of nodule organogenesis, mature nodule functioning, and nodule senescence. N-satiety/N-deficit systemic signaling correlates with rapid variations of the nodules’ sugar levels, tuning symbiosis by C resources allocation. These mechanisms are responsible for the adjustment of plant symbiotic capacities to the mineral N resources. On the one hand, if mineral N can satisfy the plant N demand, nodule formation is inhibited, and nodule senescence is activated. On the other hand, local conditions (abiotic stresses) may impair symbiotic activity resulting in plant N limitation. In these conditions, systemic signaling may compensate the N deficit by stimulating symbiotic root N foraging. In the past decade, several molecular components of the systemic signaling pathways controlling nodule formation have been identified, but a major challenge remains, that is, to understand their specificity as compared to the mechanisms of non-symbiotic plants that control root development and how they contribute to the whole plant phenotypes. Less is known about the control of mature nodule development and functioning by N and C nutritional status of the plant, but a hypothetical model involving the sucrose allocation to the nodule as a systemic signaling process, the oxidative pentose phosphate pathway, and the redox status as potential effectors of this signaling is emerging. This work highlights the importance of organism integration in plant biology.
... To study the function of NADH-GOGAT, its activity was reduced by up to 87% in transgenic alfalfa plants, using antisense constructs controlled either by an AAT-2 promoter with enhanced expression in nodules, or by a nodule-specific leghemoglobin promoter (Cordoba et al., 2003;Schoenbeck et al., 2000). The transgenic plants were chlorotic and exhibited altered symbiotic phenotypes compared to controls. ...
... In contrast to the well-documented catabolic functions of plant GDH, it is possible that the enzyme may also operate in parallel to GOGAT in the aminating direction of glutamate biosynthesis. Analyses of plants with reduced GOGAT activity, either due to genetic mutation or due to expression of GOGAT antisense constructs (Cordoba et al., 2003;Coschigano et al., 1998;Ferrario-Mery et al., 2000Lancien et al., 2002), suggested that GOGAT is the major enzyme responsible for glutamate biosynthesis in plants. Hence, a possible anabolic (aminating) activity of GDH, if it exists, contributes relatively little to overall glutamate biosynthesis. ...
Amino acids are not only building blocks of proteins but also participate in many metabolic networks that control growth and adaptation to the environment. In young plants, amino acid biosynthesis is regulated by a compound metabolic network that links nitrogen assimilation with carbon metabolism. This network is strongly regulated by the metabolism of four central amino acids, namely glutamine, glutamate, aspartate, and asparagine (Gln, Glu, Asp, and Asn), which are then converted into all other amino acids by various biochemical processes. Amino acids also serve as major transport molecules of nitrogen between source and sink tissues, including transport of nitrogen from vegetative to reproductive tissues. Amino acid metabolism is subject to a concerted regulation by physiological, developmental, and hormonal signals. This regulation also appears to be different between source and sink tissues. The importance of amino acids in plants does not only stem from being central regulators of plant growth and responses to environmental signals, but amino acids are also effectors of the nutritional quality of human foods and animal feeds. Since mammals cannot synthesize about half of the 20‐amino acid building blocks of proteins, they rely on obtaining them from foods and feeds. Yet, the major crop plants contain limited amounts of some of these so‐called “essential amino acids,” which decreases nutritional value. Recent genetic engineering and more recently genomic approaches have significantly boosted our understanding of the regulation of amino acid metabolism in plants and their participation in growth, stress response, and reproduction. In addition, genetic engineering approaches have improved the content of essential amino acids in plants, particularly the contents of lysine and methionine, which are often most limiting.
... truncatula; [43,44]) or endogenous Lotus retrotransposon 1 [45,46] for both forward and reverse genetics. Antisense RNA/RNAi methods began contributing to a better understanding of BNF's genetic background at the beginning of the 21st century [47,48], and over the years these have enabled the identification of many genes associated with BNF (see review by Arthikala et al. [49]). In recent years, CRISPR/Cas9 mediated genome editing has been established in legumes such as G. max [50], L. japonicus [51], M. truncatula [52], and Life 2022, 12, 1975 3 of 26 Cicer arietinum [53] and enabled targeted mutagenesis of BNF-associated genes. ...
Commonly studied in the context of legume–rhizobia symbiosis, biological nitrogen fixation (BNF) is a key component of the nitrogen cycle in nature. Despite its potential in plant breeding and many years of research, information is still lacking as to the regulation of hundreds of genes connected with plant–bacteria interaction, nodulation, and nitrogen fixation. Here, we compared root nodule transcriptomes of red clover (Trifolium pratense L.) genotypes with contrasting nitrogen fixation efficiency, and we found 491 differentially expressed genes (DEGs) between plants with high and low BNF efficiency. The annotation of genes expressed in nodules revealed more than 800 genes not yet experimentally confirmed. Among genes mediating nodule development, four nod-ule-specific cysteine-rich (NCR) peptides were confirmed in the nodule transcriptome. Gene duplication analyses revealed that genes originating from tandem and dispersed duplication are significantly over-represented among DEGs. Weighted correlation network analysis (WGCNA) organized expression profiles of the transcripts into 16 modules linked to the analyzed traits, such as nitrogen fixation efficiency or sample-specific modules. Overall, the results obtained broaden our knowledge about transcriptomic landscapes of red clover’s root nodules and shift the phenotypic description of BNF efficiency on the level of gene expression in situ.
... 22: 1549-1559.1271Cordoba, E., Shishkova, S., Vance, C.P., andHernández, G. (2003). Antisense 1272 inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in 1273 nodules of alfalfa (Medicago sativa L.). ...
Since 1999, various forward- and reverse-genetic approaches have uncovered nearly 200 genes required for symbiotic nitrogen fixation (SNF) in legumes. These discoveries advanced our understanding of the evolution of SNF in plants and its relationship to other beneficial endosymbioses, signaling between plants and microbes, control of microbial infection of plant cells, control of plant cell division leading to nodule development, autoregulation of nodulation, intracellular accommodation of bacteria, nodule oxygen homeostasis, control of bacteroid differentiation, metabolism and transport supporting symbiosis, and control of nodule senescence. This review catalogs and contextualizes all of the plant genes currently known to be required for SNF in two model legume species, Medicago truncatula and Lotus japonicus, and two crop species, Glycine max (soybean) and Phaseolus vulgaris (common bean). We also consider, briefly, the future of SNF genetics in the era of pan genomics and genome editing.
... Custom-made antisera against Nlj16 were generated in guinea pig (BIONEEDS Preclinical Services, Bangalore, India) using affinity purified His-tagged protein. The antileghemoglobin antibody was obtained from Dr. C. P. Vance, University of Minnesota, St. Paul, USA (Cordoba et al. 2003). Additionally, we have also raised antisera against purified His-tagged LegH in rabbit. ...
Nodulin proteins are expressed in legume root cells after infection with rhizobia. Nodulins have been classified as early and late, reflecting the developmental time points of their expression. Leghemoglobin (LegH), which is a classical example of a late nodulin, sequesters oxygen inside the nodule to protect the nitrogenase from oxygen toxicity to sustain symbiotic nitrogen fixation (SNF). Post-translational modification and/or protein–protein interaction are known to regulate activity of proteins. To elucidate the role of post-translational modification of LegH on its oxygen sequestration activity, earlier we have shown that phosphorylation at its S45 imparts most structural disruption of the porphyrin binding pocket responsible for its oxygen binding. In the present report, in an attempt to characterize the protein(s) that may interact with LegH to regulate its activity, it is demonstrated that LegH interact in vitro with Nodulin 16 of Lotus japonicus (Nlj16), another late nodulin. These two interacting proteins resulted in a bigger sized particle which shows higher diffusion coefficient as measured by dynamic light scattering. Interestingly, it was also shown that in vitro oxygen sequestration by LegH is stimulated by this interaction. Furthermore, this interaction is validated by the fact that LegH and Nlj16 could be co-immunoprecipitated from the nodule lysate. Most importantly, fluorescent immunohistochemistry of post-infected nodule sections show perceivable co-localization of these two proteins in the nodule symbiosomes. Thus, this work is a foundation for further investigation on these two interacting late nodulins as one of the plausible regulations for the oxygen sequestration by LegH during SNF.
... pathway (Gene et al. 1981;Cordoba et al. 2003). Nodules export the assimilated ammonia in the form of glutamine, which is metabolised in leaves (Mus et al. 2016). ...
Legume plants are able to interact with soil bacteria from the Rhizobiaceae family. This interaction leads to the development of a specialized organ called root nodule. Inside the symbiotic nodule cells, rhizobia are capable to fix atmospheric nitrogen and convert it to ammonia, which is a usable nitrogen source for the plant. In the legume Medicago truncatula the symbiotic cells produce high amounts of Nodule-Specific Cysteine-Rich (NCR) peptides which induce differentiation of the rhizobia into enlarged, polyploid and non-cultivable bacterial cells. NCRs are similar to innate immunity antimicrobial peptides. The NCR gene family is extremely large in Medicago with about 600 genes. The expression analysis of 334 NCR genes in 267 different experimental conditions using the Medicago truncatula Gene Expression Atlas (MtGEA) revealed that all the NCR genes except five are exclusively expressed in nodules. No NCR expression is induced in any other plant organ or in response to biotic, abiotic stress tested or to Nod factors. The NCR genes are activated in consecutive waves during nodule organogenesis, which correlated with a specific spatial localization of their transcripts from the apical to the proximal nodule zones. Moreover, we showed that NCRs are not induced during nodule senescence. According to their Shannon entropy, a metric for tissue specificity, NCR genes are among the most specifically and highest expressed genes in M. truncatula. Thus, NCR gene expression is subject to an extreme tight regulation since they are only activated during nodule organogenesis in the polyploid symbiotic cells. This analysis suggested the involvement of epigenetic regulation of the NCR genes. The formation of the symbiotic cells is driven by endoreduplication and is associated with transcriptional reprogramming. Using sorted nodule nuclei according to their DNA content, we demonstrated that the transcriptional waves correlate with growing ploidy levels and investigated how the epigenome changes during endoreduplication cycles. We studied genome-wide DNA methylation and chromatin accessibility as well as the presence of repressive H3K27me3 and activating H3K9ac histone tail modifications on selected genes. Differential DNA methylation was found only in a small subset of symbiotic nodule-specific genes, including over half of the NCR genes, while in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes’ active or repressed state. On the other hand, expression of these genes correlated with ploidy-dependent opening of the chromatin and in a subset of tested genes with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in differentiation of symbiotic cells.
... It was observed that a high number of the intermediates of central carbon metabolism, such as glycolysis and TCA cycle, were present in lateral roots in both species. This event is associated with decreases in energy consumption and transformation rates from C metabolism to N metabolism [33] and we proposed that the increased N metabolism likely associated with ginsenosides synthesis in lateral roots. Glycerate-3-P and Acetyl-CoA are particular precursors to generate numerous antioxidants [34], such as flavonoids, phenols and ginsenosides. ...
The traditional medicine Ginseng mainly including Panax ginseng and Panax quinquefolius is the most widely consumed herbal product in the world. Despite the extensive investigation of biosynthetic pathway of the active compounds ginsenosides, our current understanding of the metabolic interlink between ginsenosides synthesis and primary metabolism at the whole-plant level. In this study, the tissue-specific profiling of primary and the secondary metabolites in two different species of ginseng were investigated by gas chromatography- and liquid chromatography coupled to mass spectrometry. A complex continuous coordination of primary- and secondary-metabolic network was modulated by tissues and species factors during growth. The results showed that altogether 149 primary compounds and 10 ginsenosides were identified from main roots, lateral roots, stems, petioles and leaves in P. ginseng and P. quinquefolius. The partial least squares-discriminate analysis (PLS-DA) revealed obvious compounds distinction among tissue-specific districts relative to species. To survey the dedication of carbon and nitrogen metabolism in different tissues to the accumulation of ginsenosides, we inspected the tissue-specific metabolic changes. Our study testified that the ginsenosides content was dependent on main roots and lateral roots energy metabolism, whereas independent of leaves and petiole photosynthesis during ginsenosides accumulation. When tow species were compared, the results indicated that high rates of C assimilation to C accumulation are closely associated with ginsenosides accumulation in P. ginseng main roots and P. quinquefolius lateral roots, respectively. Taken together, our results suggest that tissue-specific metabolites profiling dynamically changed in process of ginsenosides biosynthesis, which may offer a new train of thoughts to the mechanisms of the ginsenosides biosynthesis at the metabolite level.
... Ammonium is thought to be further transported across the SM by either ion or aquaporin-like channels, although the molecular identity of an NH 4 + channel in the SM has yet to be identified (Udvardi and Poole 2013). Ammonium is primarily assimilated by the plant using asparagine and glutamine synthetases to convert ammonia into amino acids (Cordoba et al. 2003;Barsch et al. 2006). Alternately, in a number of nodules that exhibit determinate morphology, the ammonia is assimilated into purine derivatives in the plant fraction of the nodule and subsequently exported to the plant (Sprent 1980). ...
Nitrogen is a building block of life. Molecular nitrogen is the relatively inert atmospheric form of this element, and it must be fixed into more biologically accessible forms in order to be used for organic processes. In total, approximately 380 teragrams of nitrogen per year are fixed by atmospheric, biological, and industrial nitrogen fixation processes. Whereas the Haber–Bosch process currently accounts for the majority of the reduced nitrogen that is used agriculturally with the world’s increasing dependence on agriculture to feed its population, the use of reduced nitrogen derived from energy provided by fossil fuels in not likely to be sustainable. Biological nitrogen fixation is mediated by diazotrophic microorganisms that are capable of fixing atmospheric nitrogen using the enzyme nitrogenase. Much of this is carried out as a symbiotic association between plants and some diazotrophic bacteria. The study of symbiotic nitrogen fixation is an area of research that spans both microbiology and plant biology. Since this is an area that has had a great deal of renewed interest, this chapter reviews what is currently understood about the process of symbiotic nitrogen fixation at the molecular and physiological level from both the plant and bacterial perspective.
... NADH-GOGAT specific activity was determined in nodules, since it is involved in the main ammonium assimilation pathway in the nodule tissue (Chen and Cullimore 1988;Cordoba et al. 2003;Trepp et al. 1999). In our case, NADH-GOGAT specific activity was not significantly affected by cyanotoxins in nodules of faba bean plants inoculated with the different rhizobial strains (Fig. 6). ...
Irrigation of crops with microcystins (MCs)-containing waters-due to cyanobacterial blooms-affects plant productivity and could be a way for these potent toxins entering the food chain. This study was performed to establish whether MC-tolerant rhizobia could benefit growth, nodulation, and nitrogen metabolism of faba bean plants irrigated with MC-containing waters. For that, three different rhizobial strains-with different sensitivity toward MCs-were used: RhOF96 (most MC-sensitive strain), RhOF125 (most MC-tolerant strain), or Vicz1.1 (reference strain). As a control, plants grown without rhizobia and fertilized by NH4NO3 were included in the study. MC exposure decreased roots (30-37 %) and shoots (up to 15 %) dry weights in un-inoculated plants, whereas inoculation with rhizobia protects plants toward the toxic effects of MCs. Nodulation and nitrogen content were significantly impaired by MCs, with the exception of plants inoculated with the most tolerant strain RhOF125. In order to deep into the effect of inoculation on nitrogen metabolism, the nitrogen assimilatory enzymes (glutamine synthetase (GS) and glutamate synthase (GOGAT)) were investigated: Fertilized plants showed decreased levels (15-30 %) of these enzymes, both in shoots and roots. By contrast, inoculated plants retained the levels of these enzymes in shoots and roots, as well as the levels of NADH-GOGAT activity in nodules. We conclude that the microcystin-tolerant Rhizobium protects faba bean plants and improves nitrogen assimilation when grown in the presence of MCs.
... While use of promoters from other plant species has been successful in alfalfa (examples include Brears et al., 1991;Cordoba et al., 2003), the specificity of transacting factors to promoter cis elements might complicate the expression dynamics of promoters used across species. The development of Medicago truncatula as a model legume, the extensive monetary support given to the study of this species, and its genetic closeness to alfalfa, can be exploited in the development of alfalfa promoters. ...
Alfalfa is a very challenging crop to breed because of its complicated genetics and self-breeding restrictions. Therefore, development of new cultivars is usually done as a population. Strategies have included breeding for resistance to pests and diseases, resistance to abiotic stresses like drought, salinity, and heat, and breeding for increased biomass, protein content, and forage quality. Successes have been variable, but quality-breeding programs are successfully introducing important genetic traits into local populations that are beginning to address some of these problems.
... GS catalyzes the ATP-dependent incorporation of NH 3 to glutamate to produce glutamine (Cullimore and Bennett 1988 ), and at least two cytosolic and one plastid isoforms have been described in M. truncatula (Carvalho et al. 2000 ). NADH-GOGAT is responsible for the transfer of the amide group from glutamine to α-ketoglutarate leading to the production of glutamate (Ta et al. 1986 ). NADH-GOGAT is primarily found in non-photosynthetic plant tissues, such as roots and legume root nodules, where it is located in plastids (Trepp et al. 1999a , b ), being essential for N assimilation (Cordoba et al. 2003 ). Furthermore, in certain legume species such as pea and common bean, an amino-acid cycling system has been shown to be necessary for an effective N 2 -fi xing symbiosis (Lodwig et al. 2003 ;Prell et al. 2010 ). ...
Legumes include important agricultural crops, as their high protein content is of primary importance for human food and animal feed. In addition, the ability of most of them to establish symbiotic relationships with soil bacteria allows them to obtain their N requirements from nitrogen fixation in nodules and, therefore, avoids the use of nitrogen fertilizers. Thus, legumes are also essential to improve the soil fertility and quality of agricultural lands and to reclaim eroded or barren areas, making them crucial for agricultural and environmental sustainability. However, legume nitrogen fixation in crop species is very sensitive to environmental constraints and drought, in particular. The present contribution reviews our current knowledge on the processes involved in this inhibition, with particular emphasis on oxygen, nitrogen and carbon physiology. Emerging aspects such as oxidative damage, C/N interactions and sulphur metabolism together with future prospects are also discussed.
... GS catalyzes the ATP-dependent incorporation of NH 3 to gluta- mate to produce glutamine ( Cullimore and Bennett 1988 ), and at least two cytosolic and one plastid isoforms have been described in M. truncatula (Carvalho et al. 2000 ). NADH-GOGAT is responsible for the transfer of the amide group from glutamine to α-ketoglutarate leading to the production of glutamate ( Ta et al. 1986 ). NADH- GOGAT is primarily found in non-photosynthetic plant tissues, such as roots and legume root nodules, where it is located in plastids ( Trepp et al. 1999a , b ), being essential for N assimilation ( Cordoba et al. 2003 ). Furthermore, in certain legume species such as pea and common bean, an amino-acid cycling system has been shown to be necessary for an effective N 2 -fi xing symbiosis ( Lodwig et al. 2003 ;Prell et al. 2010 ). ...
According to global climate model predictions, environmental conditions such as temperature are going to be altered. Plants will be facing hightemperature conditions that affect their development. Within this context, it is crucial to identify the target processes that infl uence N2 fixation and crop production under elevated temperature conditions. As it is described in this book chapter, while N2 fixation has been well characterized under other adverse environmental conditions (drought, salinity, elevated CO2 concentration, etc.), very little is known about the effect of heat stress on nodule functioning. While there are a few reports about high-temperature effect on nodule carbohydrate and amino acid contents, there is not any study analyzing oxidative stress in those nodules. Regulation of these three factors is essential for optimized N2 fixation; thus, this is a topic that should be studied in more detail. Available information confi rms that high temperature strongly affects N2 fixation and plant growth, especially when plants are exposed to temperature higher than 25°C. High temperature decreased the growth of plants due to its negative effects not only on plants’ photosynthetic performance, but also on nodule growth and development which result in decreased nodule biomass and depletion of nodule total soluble protein content. It is also remarkable that N2 fixation has been showed to be more sensitive to high temperature than photosynthesis. In this chapter, we highlight the variability in performance of various bacterial strains and plant species under high-temperature environments, and discuss about the importance of the identification of target plants and rhizobium cultivars to form optimal symbiotic combinations that will be better adapted to predicted climate change conditions. This in turn will enable higher N2 fixation efficiency and consequently plant growth under adverse environmental conditions, including high temperature.
... GS catalyzes the ATP-dependent incorporation of NH 3 to glutamate to produce glutamine (Cullimore and Bennett 1988 ), and at least two cytosolic and one plastid isoforms have been described in M. truncatula (Carvalho et al. 2000 ). NADH-GOGAT is responsible for the transfer of the amide group from glutamine to α-ketoglutarate leading to the production of glutamate (Ta et al. 1986 ). NADH-GOGAT is primarily found in non-photosynthetic plant tissues, such as roots and legume root nodules, where it is located in plastids (Trepp et al. 1999a , b ), being essential for N assimilation (Cordoba et al. 2003 ). Furthermore, in certain legume species such as pea and common bean, an amino-acid cycling system has been shown to be necessary for an effective N 2 -fi xing symbiosis (Lodwig et al. 2003 ;Prell et al. 2010 ). ...
A significant decline in the content of water in soils provokes a water deficit at the plant level. In plant physiology, water deficit can be defined as the water content of a tissue or cell below the highest water content under the optimum hydrated state. The basis of the fundamental mechanism involved in stress tolerance, although intensively explored, is still matter of debate. Cell growth is the physiological process first affected as cell water content decreases when plants encounter mild water-deficit levels, followed by an inhibition of cell wall and protein biosynthesis. Although stomatal conductance and photosynthesis are affected in more intense water-deficit stages, most research efforts have focused on the study of these processes. In legume plants grown under symbiotic conditions, one of the primary effects of water deficitis a decline in the rates of symbiotic nitrogen fixation (SNF). The causes of this inhibition, which occurs even before a measurable decline in the rates of photosynthesis, have been explored in detail in the last decades, although the molecular mechanism involved are yet not fully understood.In the present chapter, we summarize our current understanding of the factors involved in the regulation of SNF in different legume species, including crops such as soybean (Glycine max), alfalfa (Medicago sativa), bean (Phaseolus vulgaris), and pea (Pisum sativum) but also model legumeslike Medicago truncatula. Finally, an overview of the available resources and applications of molecular system-based approaches for understanding the complex responses of legumes to drought stress is provided.
... Until recently, the main approaches to determining the functions of specific genes in planta involved RNAi or antisense RNA, which trigger the degradation of target gene transcripts and reduce or eliminate the production of the cognate protein. These approaches have shown that the following enzymes (among others) are necessary for effective SNF: PEPC (127) and GOGAT (25) in Medicago sativa (alfalfa), and sucrose synthase (5) and γ-glutamylcysteine synthetase (involved in glutathione biosynthesis) (38) in M. truncatula. Interestingly, overexpression of γ-glutamylcysteine synthetase led to enhanced SNF in Medicago, at least under laboratory conditions (38), indicating that there may be scope to improve SNF in this and other legume species. ...
Symbiotic nitrogen fixation by rhizobia in legume root nodules injects approximately 40 million tonnes of nitrogen into agricultural systems each year. In exchange for reduced nitrogen from the bacteria, the plant provides rhizobia with reduced carbon and all the essential nutrients required for bacterial metabolism. Symbiotic nitrogen fixation requires exquisite integration of plant and bacterial metabolism. Central to this integration are transporters of both the plant and the rhizobia, which transfer elements and compounds across various plant membranes and the two bacterial membranes. Here we review current knowledge of legume and rhizobial transport and metabolism as they relate to symbiotic nitrogen fixation. Although all legume-rhizobia symbioses have many metabolic features in common, there are also interesting differences between them, which show that evolution has solved metabolic problems in different ways to achieve effective symbiosis in different systems. Expected final online publication date for the Annual Review of Plant Biology Volume 64 is April 29, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
... Knockout mutations of NADH-GOGAT1 in rice decreased yield, overall biomass and panicle production, while maintaining individual spikelet weight (Tamura et al., 2010). In alfalfa, antisense inhibition of NADH-GOGAT in nodules has been shown to alter concentrations of key metabolites in C and N pathways, resulting in plants with lower fresh weight and N content, than control plants (Cordoba et al., 2003). Using three independent alfalfa NADH-GOGAT over-expressing tobacco lines, Chichkova et al. (2001) showed increased total C and N concentrations in overexpressing plants just prior to flowering, in both shoots and roots. ...
... The PvTRE1-RNAi transgenic nodules displayed 71.01 ± 7.19% more nitrogenase activity than control nodules (Fig. 5c). Just as acetylene reduction is indicative of nitrogenase activity in bacteroids, the increase in transcript accumulation of genes involved in nitrogen assimilation (GOGAT (NADH-Glutamate synthase II )) provides information about the amount of fixed nitrogen mobilized into plant cells (Silvente et al., 2002;Cordoba et al., 2003;Blanco et al., 2008). GOGAT transcript abundance was increased in PvTRE1-RNAi transgenic nodules by 82 ± 0.02% compared with that of control nodules (Fig. 5d). ...
Legume–rhizobium interactions have been widely studied and characterized, and the disaccharide trehalose has been commonly detected during this symbiotic interaction. It has been proposed that trehalose content in nodules during this symbiotic interaction might be regulated by trehalase. In the present study, we assessed the role of trehalose accumulation by down‐regulating trehalase in the nodules of common bean plants.
We performed gene expression analysis for trehalase ( Pv TRE 1 ) during nodule development. Pv TRE 1 was knocked down by RNA interference ( RNA i) in transgenic nodules of the common bean.
Pv TRE 1 expression in nodulated roots is mainly restricted to nodules. Down‐regulation of Pv TRE 1 led to increased trehalose content (78%) and bacteroid number (almost one order of magnitude). In addition, nodule biomass, nitrogenase activity, and GOGAT transcript accumulation were significantly enhanced too.
The trehalose accumulation, triggered by Pv TRE 1 down‐regulation, led to a positive impact on the legume–rhizobium symbiotic interaction. This could contribute to the agronomical enhancement of symbiotic nitrogen fixation.
... These results revealed the existence of alterations in carbon metabolism of mutant nodules, under both photorespiratory-active and -suppressed conditions. Recent reports established that antisense inhibition of NADH-GOGAT activity impairs carbon and nitrogen metabolism in alfalfa nodules [93]. The requirement of carbon skeletons for ammonium condensation and the supply of reducing equivalents as products of photosynthesis, respiration, and photorespiration pathways are well known [3,94]. ...
Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N(2) fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1) or plastidic (GS2) isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photorespiration and water stress, in this latter case by means of proline production. Functional genomic analysis using GS2-minus mutant reveals the key role of GS2 in the metabolic control of the plants and, more particularly, in carbon metabolism.
... Knockout mutations of NADH-GOGAT1 in rice decreased yield, overall biomass and panicle production, while maintaining individual spikelet weight (Tamura et al., 2010). In alfalfa, antisense inhibition of NADH-GOGAT in nodules has been shown to alter concentrations of key metabolites in C and N pathways, resulting in plants with lower fresh weight and N content, than control plants (Cordoba et al., 2003). Using three independent alfalfa NADH-GOGAT over-expressing tobacco lines, Chichkova et al. (2001) showed increased total C and N concentrations in overexpressing plants just prior to flowering, in both shoots and roots. ...
In the last 40 years the amount of synthetic nitrogen (N) applied to crops has risen drastically, resulting in significant increases in yield but with considerable impacts on the environment. A requirement for crops that require decreased N fertilizer levels has been recognized in the call for a 'Second Green Revolution' and research in the field of nitrogen use efficiency (NUE) has continued to grow. This has prompted a search to identify genes that improve the NUE of crop plants, with candidate NUE genes existing in pathways relating to N uptake, assimilation, amino acid biosynthesis, C/N storage and metabolism, signalling and regulation of N metabolism and translocation, remobilization and senescence. Herein is a review of the approaches taken to determine possible NUE candidate genes, an overview of experimental study of these genes as effectors of NUE in both cereal and non-cereal plants and the processes of commercialization of enhanced NUE crop plants. Patents issued regarding increased NUE in plants as well as gene pyramiding studies are also discussed as well as future directions of NUE research.
... These results are in agreement with those found by Cordovilla et al. (1999) which demonstrated that GS appeared to be more tolerant to salinity than GOGAT. Besides, it has been reported that GOGAT reduction in nodules resulted in plant having impaired nitrogen assimilation and altered carbon/nitrogen metabolic flux (Cordoba et al., 2003). GDH did not show any change at 50 and 100 mM NaCl, but decreased by about 50% when plants were treated with 200 mM NaCl (Table 3). ...
The behaviour of enzymes involved in nitrogen metabolism, as well as oxidative stress generation and heme oxygenase gene and protein expression and activity, were analysed in soybean (Glycine max L.) nodules exposed to 50, 100 and 200 mM NaCl concentrations. A significant increase in lipid peroxidation was found with 100 and 200 mM salt treatments. Moreover, superoxide dismutase, catalase and peroxidase activities were decreased under 100 and 200 mM salt. Nitrogenase activity and leghemeoglobin content were diminished and ammonium content increased only under 200 mM NaCl. At 100 mM NaCl, glutamine synthetase (GS) and NADH-glutamate dehydrogenase (GDH) activities were similar to controls, whereas a significant increase (64%) in NADH-glutamate synthase (GOGAT) activity was observed. GS activity did not change at 200 mM salt treatment, but GOGAT and GDH significantly decreased (40 and 50%, respectively). When gene and protein expression of GS and GOGAT were analysed, it was found that they were positively correlated with enzyme activities. In addition, heme oxygenase (HO) activity, protein synthesis and gene expression were significantly increased under 100 mM salt treatment. Our data demonstrated that the up-regulation of HO, as part of antioxidant defence system, could be protecting the soybean nodule nitrogen fixation and assimilation under saline stress conditions.
... Considering that Ljgln2-1 and Ljgln2-2 mutants are defective in photorespiratory ammonium assimilation, our results emphasize how a "nitrogen" assimilation defect affects "carbon" metabolism and nodule function in L. japonicus. Other recent reports established that antisense inhibition of NADH-GOGAT impairs carbon and nitrogen metabolism in nodules of alfalfa (Cordoba et al. 2003). Values are the means of two independent determinations for each of three biological replicates. ...
Two photorespiratory mutants of Lotus japonicus deficient in plastid glutamine synthetase (GS(2)) were examined for their capacity to establish symbiotic association with Mesorhizobium loti bacteria. Biosynthetic glutamine synthetase (GS) activity was reduced by around 40% in crude nodule extracts from mutant plants as compared with the wild type (WT). Western blot analysis further confirmed the lack of GS(2) polypeptide in mutant nodules. The decrease in GS activity affected the nodular carbon metabolism under high CO(2) (suppressed photorespiration) conditions, although mutant plants were able to form nodules and fix atmospheric nitrogen. However, when WT and mutant plants were transferred to an ordinary air atmosphere (photorespiratory active conditions) the nodulation process and nitrogen fixation were substantially affected, particularly in mutant plants. The number and fresh weight of mutant nodules as well as acetylene reduction activity showed a strong inhibition compared with WT plants. Optical microscopy studies from mutant plant nodules revealed the anticipated senescence phenotype linked to an important reduction in starch and sucrose levels. These results show that, in Lotus japonicus, photorespiration and, particularly, GS(2) deficiency result in profound limitations in carbon metabolism that affect the nodulation process and nitrogen fixation.
... It appears that plants are able to compensate for inefficient nodules, caused either by plant and/or bacteria genetic lesions, at least to some extent, by increasing nodule number, although this does little good when mutations completely abolish nitrogen fixation (Hirsch and Smith 1987;Gordon et al. 1999;Suganuma et al. 2003). The pattern of nodulation of the LjAMT1;1 antisense lines, with a low density of nodules distributed in a wider zone of the root system (Fig. 5a) as well as their nodulation kinetics (Fig. 3d–f) are reminiscent of the phenotype exhibited by many of the nitrogen fixation-impaired plant mutants, which exhibit normal nodulation kinetics up to the time when nitrogen fixation usually begins but enhanced nodulation compared to the wild-type afterwards (Cordoba et al. 2003;Gordon et al. 1999;Suganuma et al. 2003;Krusell et al. 2005). Interestingly, it appears that the reduced ARA activity detected in the antisense nodules (Fig. 5b), that was not associated to a reduced biomass (Fig. 5c), was compensated for by increased nodulation, since shoots of LjAMT1;1 antisense were at least as large as those of control plants (Fig. 5d). ...
Plant ammonium transporters of the AMT1 family are involved in N-uptake from the soil and ammonium transport, and recycling within the plant. Although AMT1 genes are known to be expressed in nitrogen-fixing nodules of legumes, their precise roles in this specialized organ remain unknown. We have taken a reverse-genetic approach to decipher the physiological role of LjAMT1;1 in Lotus japonicus nodules. LjAMT1;1 is normally expressed in both the infected zone and the vascular tissue of Lotus nodules. Inhibition of LjAMT1;1 gene expression, using an antisense gene construct driven by a leghemoglobin promoter resulted in a substantial reduction of LjAMT1;1 transcript in the infected tissue but not the vascular bundles of transgenic plants. As a result, the nitrogen-fixing activity of nodules was partially impaired and nodule number increased compared to control plants. Expression of LjAMT1;1-GFP fusion protein in plant cells indicated a plasma-membrane location for the LjAMT1;1 protein. Taken together, the results are consistent with a role of LjAMT1;1 in retaining ammonium derived from symbiotic nitrogen fixation in plant cells prior to its assimilation.
... In summary, as a result of our multifaceted analysis of GmPpcK1-4, GmPpc7 and PEPC-phosphorylation in the soybean root nodule (this study; Xu et al., 2003;Zhang and Chollet, 1997;Zhang et al., 1995), it can reasonably be proposed that the nearly identical GmPpcK2/3 homologs and GmPpc7 together constitute the key molecular 'downstream players' in this regulatory protein-phosphorylation system within the mature nodule's central zone. Functional genomics experiments have been initiated recently in our laboratories to critically assess this working hypothesis and the apparent duplication in nodule-enhanced PEPC-kinase with respect to a single, nodule-enhanced target PEPC by the generation and subsequent analysis of down-regulated transgenic lines of GmPpcK3 and GmPpc7, and of the control GmPpcK1 gene, by an RNA interference-based approach that exploits the strong, nodule-specific promoter of soybean Lbc3 (Cordoba et al., 2003;Stougaard et al., 1990). Conversely, GmPpcK3 is also being overexpressed specifically within the central infected zone under the control of this same heterologous promoter. ...
Phosphoenolpyruvate carboxylase (PEPC) is a widely distributed metabolic enzyme among plant and prokaryotic species. In vascular plants, the typical PEPC is regulated post-translationally by a complex interplay between opposing metabolite effectors and reversible protein phosphorylation. This phosphorylation event is controlled primarily by the up-/down-regulation of PEPC-kinase (PpcK), an approximately 31-kDa Ser/Thr-kinase. As a sequel to earlier investigations related to PEPC phosphorylation in N(2)-fixing nodules of Glycine max, we now present a detailed molecular analysis of the PpcK multigene family in nodulated soybeans. Although the GmPpcK1-4 transcripts are all expressed throughout nodule development, only the nearly identical GmPpcK2/3 homologs are nodule-enhanced and up-/down-regulated in vivo by photosynthate supply from the shoots. In contrast, GmPpcK1 is a 'housekeeping' gene, and GmPpcK4 is a highly divergent member, distantly removed from the legume PpcK subfamily. Real-time qRT-PCR analysis indicates that GmPpcK2/3 are overwhelmingly the dominant PpcKs expressed and up-/down-regulated throughout nodule development, mirroring the expression properties of nodule-enhanced PEPC (GmPpc7). In situ RT-PCR investigation of the spatial localization of the GmPpcK1-4 and GmPpc7 transcripts in mature nodules is entirely consistent with this view. Complementary histochemical and related RNA gel-blot findings with nodulated, GmPpcK1/3 promoter::GUS-expressing T(2) plants provide direct experimental evidence that (i) PpcK gene expression is controlled primarily at the transcriptional level; and (ii) the contrasting expression properties of GmPpcK1/3 are conferred largely by regulatory element(s) within the approximately 1.4-kb 5'-upstream region. As a result of our multifaceted analyses of GmPpcK1-4, GmPpc7 and PEPC-phosphorylation in the soybean nodule, it is proposed that the GmPpcK2/3 homologs and GmPpc7 together comprise the key molecular 'downstream players' in this regulatory phosphorylation system within the mature nodule's central zone.
... Transcriptome, proteomic , and metabolite profiling by gas chromatographymass spectrometry of L. japonicus supported the biosynthesis of Gln and Asn and identified induction of two genes encoding GS enzymes and two genes encoding Asn synthetases (Wienkoop and Saalbach, 2003; Colebatch et al., 2004; Desbrosses et al., 2005). Consistent with the assimilation of ammonium by the plant, antisense inhibition of NADH GOGAT in alfalfa caused severe inhibition of nitrogen fixation, with the formation of chlorotic plants (Cordoba et al., 2003). Finally, in most temperate legumes the assimilated Gln is exported out of the nodule, principally as Asn, which requires the concerted activity of a Gln-dependent Asn synthetase and Asp aminotransferase. ...
In this review, we consider the exchange of nutrients between the host plant and the bacterial microsym- biont in nitrogen-fixing legume root nodules. During nodule formation, the host tissues and the bacterial microsymbiont develop in response to each other to formaspecializedtissuethatmaintains anenvironment where nitrogen fixation can occur (Brewin, 2004; Mergaert et al., 2006; Prell and Poole, 2006). This com- plex development will not be considered here but, at the end of the process, specialized, nitrogen-fixing forms of the bacteria, known as bacteroids, reside in the plant cytosol, enclosed within plant-derived mem- branes. These organelle-like structures are known as symbiosomes; the plant-derived membrane that sur- rounds the bacteroid is the symbiosome (or peribacte- roid) membrane and the space between the two is the symbiosome (or peribacteroid) space. An infected plant cell may be packed with thousands of symbiosomes. The exchange of nutrients that is fundamental to N2 fixation therefore involves metabolism in the plant to provide carbon and nitrogen compounds to the bacte- roids and to assimilate the metabolites that bacteroids release. Nutrients transferred between the symbionts must traverse both the symbiosome and the bacteroid membranes. It is clear that there is more than one pattern whereby successful nutrient exchange can take place: There are two basic types of legume nodules, determinate and indeterminate, and there are funda- mental differences between the two in how they de- velop and in their carbon and nitrogen metabolism. This review focuses on the metabolism of carbon and nitrogen compounds in the symbionts and on the ex- change of nutrients across the bacteroid and symbio- some membranes. Particular attention is paid to the movementofprimarycarbonandnitrogensourcesand how they are utilized by both bacteroids and the plant.
The objective of this study was to evaluate the effect of a mix of pectic oligosaccharides in the common bean-rhizobia symbiosis. Cuba Cueto-25-9-N bean seeds were inoculated with Rhizobium tropici CIAT 899 and treated with a mix of oligogalacturonides, either by application to the seed (10 mg L-1, 1 ml per seed) at sowing and inoculation or by foliar spray (5 or 100 mg L-1, 1.5 ml per plant) to plants with two trefoils. Plant growth, nodulation, nitrogenase activity, and the gene expression of glutamine synthetase and glutamate synthase in nodules were evaluated in inoculated plants and oligogalacturonide-treated inoculated plants at 18 d postinoculation. The oligogalacturonide-treated plants showed increases in shoot and root growth and number of nodules. Regarding nodule function, the oligogalacturonides increased the nitrogenase activity and the expression level of the gene coding for NADH-dependent glutamate synthase. The positive effects of oligogalacturonides resulted in higher effectiveness of the symbiotic nitrogen fixation. The application of oligogalacturonides can offer an alternative to increasing the symbiotic nitrogen fixation in common bean plants.
Excess sludge is rich in organic matter, nitrogen, and phosphorus. However, in the disposal of industrial wastewater with high concentrations of organic matter, the toxic organics accumulate in the sludge will cause sludge toxicity. Microalgae have strong adaptability to toxic environments. Therefore, in the present study, Chlorella vulgaris was cultured in sludge extract and BG11 medium (blank group) with the objectives of reducing both sludge yield and organic toxicity. The results show that the toxic sludge extract could maintain the stable growth of Chlorella vulgaris, and on the 10th day of culture, Chlorella vulgaris obtained higher biomass and photosynthesis efficiency. The BOD/COD (B/C) ratio of the sludge extract increased from 0.12 to 0.37 while the carbon/nitrogen ratio increased by a factor of 4.9, which significantly improved its biodegradability. Excitation emission matrix analysis showed that C. vulgaris removed soluble microbial products and reduced some humic acids. The isobaric tags for relative and absolute quantitation revealed the presence of differential proteins affecting the processes of small molecule metabolism, energy metabolism, and photosynthesis in different media.
The sections in this article are
Introduction
Carbon Assimilation
Photorespiration
Nitrogen Assimilation and Amino Acid Biosynthesis
Synthesis of Fatty Acids
Starch Metabolism
Glycolysis
The Oxidative Pentose–Phosphate Pathway
Plastid Metabolite Transport Systems
Conclusion
Plant nitrate transporters were first identified and functionally characterized more than 20 years ago. They are encoded at least by four gene families, NRT1 (NPF), NRT2, CLC, and SLAC1/SLAH. In this review, we overview the functions of the nitrate transporters in relation to their potential use as targets for improving crop nitrogen use efficiency. These functions include their roles in root architecture and nutrient acquisition; vacuole nitrate and protein storage; nutrient allocation from source to sink; sensing both abiotic and biotic stresses; the ionic balance of nitrate with potassium, chloride and cellular pH; and the circadian clock-regulated carbon and nitrogen balance. We provide and discuss some examples of the use of nitrate transporter genes and their regulators in improving plant growth and development, nitrogen use efficiency, and resistance to some abiotic stresses. We propose several strategies for effectively using nitrate transporters to achieve higher crop yields and nitrogen use efficiency by using gene transformation or genome editing or molecular marker-assisted breeding. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
Growth in production and productivity of wheat (Triticum aestivum) in the past relied heavily on the application of high doses of nitrogen and phosphorous fertilizers. However, increasing use of chemical fertilizers over time has resulted in diminishing returns in terms of either no increase or a mere marginal increase in wheat productivity. High doses of chemical fertilizers have also contributed to climate change and eutrophication of terrestrial and aquatic ecosystems, and added to the cost of crop production. Furthermore, the currently available rock phosphate, which is the only source of P fertilizers, is expected to last for only 50–100 years. Hence, as a viable and environment-friendly alternative, efforts are needed for the development of wheat cultivars with improved N and P use efficiency (NUE and PUE, respectively). In this review, we first give a brief account of the present status of our knowledge about the physiology of the uptake and assimilation of N and P and then consider the need of precise and high throughput phenotyping for traits related to N/PUE. We also provide a summary of genes and quantitative trait loci (QTL) involved in N/P signaling, uptake and metabolism, and for other traits related to NUE and PUE in wheat. The role of microRNA (miRNA) in NUE and PUE in wheat is also summarized. Finally, we discuss the possible future wheat breeding approaches for improvement in NUE and PUE involving conventional and molecular breeding approaches.
In order to investigate the mechanisms of nitrogen (N) re-distribution in response to proteolytic enzyme (PE), glutamine synthetase (GS) and glutamate synthetase (GOGAT) activities and the effects on N use efficiency (NUE). Two oilseed rape genotypes were grown in sand culture in a greenhouse under normal (15.0 mmol L-1 NO3-) and limited-N (7.5 mmol L-1 NO3-) levels. Isotope (15N) labeling and enzyme inhibitors against the PE, GS and GOGAT enzymes were used. We found that, when the two genotypes were subjected to specific inhibitors of PE, GS, and GOGAT, the activities of these enzymes were significantly decreased, resulting in reduced N re-distributed from leaf to grain, as well as reduced NUE. Lglutamine and free amino acid contents in the phloem sap were primarily influenced by PE and GS activities, whereas grain yield was primary regulated by GOGAT activity during the later growth stages. These findings suggest that PE, GS, and GOGAT are key enzymes for the regulation of N re-distribution in plant tissues during later growth stages, with grain yield and NUE of oilseed rape being positively regulated by PE, GS and GOGAT activities.
As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope-assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate-limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS-GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate-free and less ammonium-affinity GDH pathway, or alternatively the energy-consuming GS-GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.
Improved nitrogen use efficiency (NUE: capacity to produce a supplement of yield for each added unit of N fertilizer) has the potential to enhance yield under low nitrogen (N) and thereby improve crop nutritional quality while reducing ground water contamination by nitrates. In order to realize potential benefits with respect to sustainable agriculture, we need to identify the physiological, biochemical and molecular mechanisms controlling component processes, including inorganic N uptake, N reduction, N partitioning between roots and shoots and N-incorporation into organic molecules as a function of carbon availability. Moreover, sustained decreases in fertilizer input and improved or stabilized yield require an improved understanding of the transition between N assimilation and N remobilization during plant development. Recent advances in plant molecular biotechnology, combined with modern physiological and biochemical studies, have expanded our understanding of the regulatory mechanisms controlling the primary steps of inorganic N assimilation and the subsequent biochemical pathways involved in N supply for secondary metabolism. In parallel, a number of physiological and agronomic studies have been undertaken to identify which are the limiting steps in the control of N uptake, assimilation and recycling during plant growth and development. Although they have been very informative in defining key elements of the economy of N, our knowledge of the regulation of N acquisition and management is still fragmentary. This is partly because most biochemical and molecular studies were designed to identify the key physiological and morphological traits involved, but were rarely placed in a whole plant or an agronomic context. To help fill the gap that still exists between molecular and agronomic studies, we describe in this chapter how the understanding of the control of N assimilation has increased through the use of whole plant physiology, transgenic technology, quantitative genetics and agronomic approaches. In addition, one of the goals of this review is to obtain a more integrated view of the regulation of N management from the gene to the field within the plant or within a particular organ. Prospects for future development and applications in crop improvement are explored.
The literature on the relations between plant nitrogen (N) assimilation enzymes and plant/crop N assimilation, growth and yield is reviewed to assess if genetic manipulation of the activities of N assimilation enzymes can result in increased yield and/or increased N use efficiency. The available data indicate that (I) levels of N assimilation enzymes do not limit primary N assimilation and hence yield; (II) root or shoot nitrate assimilation can have advantages under specific environmental conditions; (III) for cereals, cytosolic glutamine synthetase (GS1) is a key enzyme in the mobilisation of N from senescing leaves and its activity in senescing leaves is positively related to yield; and (IV) for rice (Oryza sativa), NADH-glutamate synthase (NADH-GOGAT) is important in the utilisation of N in grain filling and its activity in developing grains is positively related to yield. In our opinion, selection of plants, from either a genetically manipulated population or genetic resources, with expression of nitrate reductase/nitrite reductase primarily in the root or shoot should increase plant/crop growth and hence yield under specific environmental conditions. In addition for cereals the selection of plants with high GS1 in senescing leaves and in some cases high NADH-GOGAT in developing grains could help maximise the retrieval of plant N in seeds.
Crop productivity relies heavily on nitrogen (N) fertilization. Production and application of N fertilizers consume huge amounts of energy, and excess is detrimental to the environment; therefore, increasing plant N use efficiency (NUE) is essential for the development of sustainable agriculture. Plant NUE is inherently complex, as each step-including N uptake, translocation, assimilation, and remobilization-is governed by multiple interacting genetic and environmental factors. The limiting factors in plant metabolism for maximizing NUE are different at high and low N supplies, indicating great potential for improving the NUE of current cultivars, which were bred in well-fertilized soil. Decreasing environmental losses and increasing the productivity of crop-acquired N requires the coordination of carbohydrate and N metabolism to give high yields. Increasing both the grain and N harvest index to drive N acquisition and utilization are important approaches for breeding future high-NUE cultivars.
There is considerable confusion about N use efficiency (NUE) in the plant literature. We would like to propose the simple and ubiquitous definitions described by Good et al. (2004) as a starting point for studies of NUE. Based on this terminology, there is evidence from breeding programs for variation in both uptake efficiency (UpE) and utilization efficiency (UtE). Molecular physiology studies typically address mechanisms for improving NUE, but often do not calculate NUE or even acquire appropriate data for calculating NUE. Herein, we report in detail on recent studies involving molecular approaches for improving NUE, and calculate changes in NUE where possible. The evidence suggests that there is potential for improving usage index and UpE in dicots and UpE and UtE in monocots by overexpressing enzymes for N assimilation, specifically glutamine synthetase1, glutamate synthase, and alanine aminotransferase. If decreased fertilizer-N input and improved NUE are truly goals of the plant biology community, researchers are encouraged to (i) consider the use of both wild type and azygous controls, (ii) compare general NUE (on the basis of grain or biomass yield per unit of applied N) of overexpression mutants and controls at both limiting and non-limiting N levels, (iii) select an appropriate type of specific NUE for assessing the physiological mechanisms involved (uptake versus internal utilization), and (iv) confirm promising results under field conditions.
The Fabaceae comprise the third largest family of higher plants, and numerous legume species are spread worldwide in a variety
of climates from the artic circle to the tropics. Many legumes obtain their nitrogen sources via a symbiotic interaction with
nitrogen-fixing rhizobia and are therefore important natural fertilizers and pioneer plants on eroded soils. Moreover, some
legumes have protein-rich seeds, making them essential for food and feed. Medicago truncatula and Lotus japonicus are the model species for legume research as well as for nodulation, the process that results in the symbiotic organs, the
nodules, in which the rhizobia reside for nitrogen fixation. Via biotechnological tools such as genome analysis, bioinformatics,
mutagenesis, transcriptomics, proteomics and metabolomics, researchers are investigating the molecular and biological principles
of the nodulation process. These studies will ultimately lead to the improvement of existing symbioses and eventually to the
extension of the process to non-nodulating crops. In this chapter, an overview is given of the current knowledge of the nodulation
process in the model legumes.
The research findings in the field of Rhizobium-legume symbiosis reported worldwide during the years 2002 and 2003 (up to September) have been summarized. The information is presented under the various topics, viz., isolation and characterization of rhizobial strains, physiological aspects of nitrogen fixation, rhizosphere interactions and root surface signals, genomics and proteomics, plant genes involved in nodule formation, bioremediation and biocontrol, and review articles and conference reports. The postal and e-mail addresses of the concerned scientists have also been included.
Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.
Ammonium ion assimilation constitutes a central metabolic pathway in many organisms, and glutamate synthase, in concert with glutamine synthetase (GS, EC 6.3.1.2), plays the primary role of ammonium ion incorporation into glutamine and glutamate. Glutamate synthase occurs in three forms that can be distinguished based on whether they use NADPH (NADPH-GOGAT, EC 1.4.1.13), NADH (NADH-GOGAT, EC 1.4.1.14) or reduced ferredoxin (Fd-GOGAT, EC 1.4.7.1) as the electron donor for the (two-electron) conversion of L-glutamine plus 2-oxoglutarate to L-glutamate. The distribution of these three forms of glutamate synthase in different tissues is quite specific to the organism in question. Gene structures have been determined for Fd-, NADH- and NADPH-dependent glutamate synthases from different organisms, as shown by searches in nucleic acid sequence data banks. Fd-glutamate synthase contains two electron-carrying prosthetic groups, the redox properties of which are discussed. A description of the ferredoxin binding by Fd-glutamate synthase is also presented. In plants, including nitrogen-fixing legumes, Fd-glutamate synthase and NADH-glutamate synthase supply glutamate during the nitrogen assimilation and translocation. The biological functions of Fd-glutamate synthase and NADH-glutamate synthase, which show a highly tissue-specific distribution pattern, are tightly related to the regulation by the light and metabolite sensing systems. Analysis of mutants and transgenic studies have provided insights into the primary individual functions of Fd-glutamate synthase and NADH-glutamate synthase. These studies also provided evidence that glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) does not represent a significant alternate route for glutamate formation in plants. Taken together, biochemical analysis and genetic and molecular data imply that Fd-glutamate synthase incorporates photorespiratory and non-photorespiratory ammonium and provides nitrogen for transport to maintain nitrogen status in plants. Fd-glutamate synthase also plays a role that is redundant, in several important aspects, to that played by NADH-glutamate synthase in ammonium assimilation and nitrogen transport.
Nitrogen-fixing rhizobial bacteria and leguminous plants have evolved complex signal exchange mechanisms that allow a specific bacterial species to induce its host plant to form invasion structures through which the bacteria can enter the plant root. Once the bacteria have been endocytosed within a host-membrane-bound compartment by root cells, the bacteria differentiate into a new form that can convert atmospheric nitrogen into ammonia. Bacterial differentiation and nitrogen fixation are dependent on the microaerobic environment and other support factors provided by the plant. In return, the plant receives nitrogen from the bacteria, which allows it to grow in the absence of an external nitrogen source. Here, we review recent discoveries about the mutual recognition process that allows the model rhizobial symbiont Sinorhizobium meliloti to invade and differentiate inside its host plant alfalfa (Medicago sativa) and the model host plant barrel medic (Medicago truncatula).
The effects of ultraviolet-B (UV-B) radiation on water relations, leaf development, and gas-exchange characteristics in pea (Pisum sativum L. cv Meteor) plants subjected to drought were investigated. Plants grown throughout their development under a high irradiance of UV-B radiation (0.63 W m 2) were compared with those grown without UV-B radiation, and after 12 d one-half of the plants were subjected to 24 d of drought that resulted in mild water stress. UV-B radiation resulted in a decrease of adaxial stomatal conductance by approximately 65%, increasing stomatal limitation of CO 2 uptake by 10 to 15%. However, there was no loss of mesophyll light-saturated photosynthetic activity. Growth in UV-B radiation resulted in large reductions of leaf area and plant biomass, which were associated with a decline in leaf cell numbers and cell division. UV-B radiation also inhibited epidermal cell expansion of the exposed surface of leaves. There was an interaction between UV-B radiation and drought treatments: UV-B radiation both delayed and reduced the severity of drought stress through reductions in plant water-loss rates, stomatal conductance, and leaf area.
In this article we discuss the ways in which our understanding of the nature of the molecular controls of nitrogen assimilation
have been increased by the use of leguminous and non-leguminous plants with modified capacities for ammonium assimilation.
These modifications have been achieved through genetic engineering and breeding. An improved understanding of nitrogen assimilation
will be vital if improvements in crop nitrogen use efficiency are to be made to reduce the need for excessive input of fertilisers.
In this review we present an overall view of past work and more recent studies on this topic. In our work, using tobacco and
Lotus as model plants, glutamine synthetase and glutamate synthase activites have been altered by stimulating or inhibiting in
an organ- or tissue-specific manner the expression of the corresponding genes. The physiological impact of these genetic manipulations
has been studied on plants grown under different nitrogen regimes.
Glutamate synthase (GOGAT), a key enzyme in ammonia (NH4+) assimilation, occurs as two forms in plants: a ferredoxin-dependent form (Fd-GOGAT) and an NADH-dependent form (NADH-GOGAT). These enzymes are encoded by distinct genes as evidenced by their cDNA and deduced amino acid sequences. This paper reports the isolation and characterization of a NADH-GOGAT gene from alfalfa (Medicago sativa L.), the first GOGAT gene to be isolated from a eukaryote. RNase protection and primer extension experiments map the transcription start site of NADH-GOGAT to nearly identical positions. The transcribed region of this gene, 12 214 bp, is comprised of 22 exons separated by 21 introns. The 2.7 kbp region 5′ from the translation initiation site confers nodule-specific reporter gene activity when used in a chimeric β-glucuronidase (GUS) construct and transformed into Lotus corniculatus and Medicago sativa. Both infected and uninfected cells display GUS activity. The abundance of NADH-GOGAT transcripts increases substantially in developing nodules of plants infected with effective rhizobia. However, this increase is not observed when nodules are induced by a variety of ineffective rhizobial strains. Thus, unlike many other plant genes involved in root nodule NH4+ assimilation, high levels of NADH-GOGAT expression are strictly associated with effective nodules indicating that NADH-GOGAT plays a central role in the functioning of effective root nodules. An alfalfa Fd-GOGAT PCR product showing greater than 85% identity to maize Fd-GOGAT was isolated and used to investigate the contribution of this enzyme to NH4+ assimilation in nodules. Fd-GOGAT mRNA was abundant in leaves and cotyledons but was not detected in alfalfa root nodules. Fd-GOGAT in alfalfa does not appear to play a significant role in symbiotic N2 fixation.
Transgenic alfalfa plants expressinBacillus licheniformis alpha-amylase and mangaese-dependent lignin peroxidase (Mn-P) from Phanerochaete chrysosporium were produced using the Agrobacterium tumefaciens transformation system. In each case, there was a range of expression of the introduced gene among independent transgenic
plants. Plants producing alpha-amylase showed no alteration of phenotype. Production of Mn-P in alfalfa, howeven, in most
cases adversely affected plant growth and development. Affected plants were stunted with yellowing foliage, but survived and
produced seed. Results from field trials showed that Mn-P production in transgenic alfalfa reduced dry matter yield and plant
height. The extent of these symptoms and yield reduction was, for the most part, related to the level of foreign protein production
as estimated by Western analysis. Field data from transgenic plants expressing alpha-amylase showed that there was no effect
of foreign protein production on plant performance. Expression of Mn-P was shown to segregate in sexual progeny derived from
transgenic plants.
Transformed plants of Nicotiana plumbaginifolia Viv. constitutively expressing nitrate reductase (35S-NR) or β-glucuronidase (35S-GUS) and untransformed controls were grown
for two weeks in a CO2-enriched atmosphere. Whereas CO2 enrichment (1000 μl · l−1) resulted in an increase in the carbon (C) to nitrogen (N) ratio of both the tobacco lines grown in pots with vermiculite,
the C/N ratio was only slightly modified when plants were grown in hydroponic culture in high CO2 compared to those grown in air. Constitutive nitrate reductase (NR) expression per se did not change the C/N ratio of the
shoots or roots. Biomass accumulation was similar in both types of plant when hydroponic or pot-grown material, grown in air
or high CO2, were compared. Shoot dry matter accumulation was primarily related to the presence of stored carbohydrate (starch and sucrose)
in the leaves. In the pot-grown tobacco, growth at elevated CO2 levels caused a concomitant decrease in the N content of the leaves involving losses in NO−
3 and amino acid levels. In contrast, the N content and composition were similar in all plants grown in hydroponic culture.
The 35S-NR plants grown in air had higher foliar maximum extractable NR activities and increased glutamine levels (on a chlorophyll
or protein basis) than the untransformed controls. These increases were maintained following CO2 enrichment when the plants were grown in hydroponic culture, suggesting that an increased flux through nitrogen assimilation
was possible in the 35S-NR plants. Under CO2 enrichment the NR activation state in the leaves was similar in all plants. When the 35S-NR plants were grown in pots, however,
foliar NR activity and glutamine content fell in the 35S-NR transformants to levels similar to those of the untransformed
controls. The differences in NR activity between untransformed and 35S-NR leaves were much less pronounced in the hydroponic
than in the pot-grown material but the difference in total extractable NR activity was more marked following CO2 enrichment. Foliar NR message levels were decreased by CO2 enrichment in all growth conditions but this was much more pronounced in pot-grown material than in that grown hydroponically.
Since β-glucuronidase (GUS) activity and message levels in 35S-GUS plants grown under the same conditions of CO2 enrichment (to test the effects of CO2 enrichment on the activity of the 35S promoter) were found to be constant, we conclude that NR message turnover was specifically
accelerated in the 35S-NR plants as well as in the untransformed controls as a result of CO2 enrichment. The molecular and metabolic signals involved in increased NR message and protein turnover are not known but possible
effectors include NO3
−, glutamine and asparagine. We conclude that plants grown in hydroponic culture have greater access to N than those grown
in pots. Regardless of the culture method, CO2 enrichment has a direct effect on NR mRNA stability.
Nodulated soybean plants (Glycine max (L.) Merr. cv. Clarke) were supplied with 10 mol m-3 nitrate at the vegetative stage. This treatment caused a rapid decline in nitrogen fixation (acetylene reduction) activity
and a consequent decline in ureides in the xylem sap. However, there was virtually no effect on the nitrogenase complex, according
to Western blots against components 1 and 2. The effect on nitrogen fixation was matched by a decrease in nitrogenase-linked
respiration and increases in nodule oxygen diffusion resistance and the carbon cost of nitrogen fixation. The addition of
nitrate had little effect on protein content from either nodule plant or bacteroid fractions. Activities of nitrate reductase
(NR) and nitrite reductase (NiR) from either the plant fraction or the bacteroids were affected in different ways during 8
d of supply. Nodule plant NR and bacteroid NiR were not affected. However, nodule plant NiR increased 5-fold within 2 d of supplying
Bacteroid NR only increased after 6 d. These results could be interpreted in terms of a restricted nitrate access into the
infected region of nodules. However, denitrification was detected within 2 d of nitrate supply in soybean nodules. The results
are discussed in relation to possible causes of the nitrate-induced decline in nitrogenase activity.
Transgenic alfalfa plants harboring a gene fusion between the soybean leghemoglobin (lbc3) promoter region and the chloramphenicol acetyl transferase (cat) gene were used to determine the influence of rhizobial mutants on lb gene expression in nodules. The promoter region of the Sesbania rostrata glb3 (Srglb3) leghemoglobin gene was examined for the presence of conserved motifs homologous to binding site 1 and 2 of the soybean lbc3 promoter region, found to interact with a trans-acting factor present in soybean nodule nuclear extracts (Jensen EO, Marcker KA, Schell J, de Bruijn FJ, EMBO J 7:1265-1271, 1988). Subfragments of the S. rostrata glb3 (Srglb3) promoter region were examined for binding to trans-acting factors from nodule nuclear extracts. In addition to the binding sites previously identified (Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ, Mol Gen Genet 214: 181-191), several other sites were found to interact with trans-acting factors. In most cases the same trans-acting factor(s) were shown to be involved. One fragment (202) was found to bind specifically to a different factor (protein) which was extremely heat-resistant (100 degrees C). The appearance of this factor was shown to be developmentally regulated since the expected protein-DNA complexes were first observed around 12 days after infection, concomitant with the production of leghemoglobin proteins. Fragments of the Srglb3 5' upstream region were fused to the beta-glucuronidase reporter gene with its own CAAT and TATA box region or those of the cauliflower mosaic virus 35S and nopaline synthase (nos) promoters.(ABSTRACT TRUNCATED AT 250 WORDS)
Versatile DNA polylinkers of more than 300 bp were constructed. They contain the recognition sequences of all restriction enzymes--whether known or still to be discovered--that recognize palindromic hexamers. In addition to these 64 uninterrupted hexameric recognition sites, a number of sites containing interrupted palindromes and nonpalindromic sequences and two recognition sequences with 8 bp are present. Polylinkers (in several variants) were inserted into frequently utilized Escherichia coli cloning vectors such as pBluescript (yielding pSLJ10, pSL250, pSL260, pSL270, and pSL300), pUC18/pUC19 (yielding pSL180 and pSL190, respectively), or pUC118/pUC119 (yielding pSL1180 and pSL1190, respectively). A subtle color discrimination between presence and absence of insert in pSL300 (mid-blue to light-blue or white) was seen in a number of test ligations. The mid-blue color that is generated by pSL300 is presumably due to translational restarts. A different intergenic region for translational restarts was used in plasmids pSL251, pSL261, pSL271, and pSL301. The polylinker was also inserted into expression vector pUC120, yielding pSE1200, and into expression vector pKK233-2, yielding pSE220 and a shortened version thereof, pSE280. Finally, the polylinker was inserted into pTrc99A, resulting in pSE380, which carries a lac repressor gene. This expands the use of the expression system beyond lacIq strains to other bacterial hosts. These versatile vectors have broad applications in genetic engineering.
Recent studies of chloroplast architecture have emphasized the segregation of photosystem I and photosystem II in different regions of the lamellar membrane. The apparent localization of photosystem II reaction centers in regions of membrane appression and of photosystem I reaction centers in regions exposed to the chloroplast stroma has focused attention on the intervening electron carriers, carriers which must be present to catalyze electron transfer between such spatially separated reaction sites. Information regarding the stoichiometries of these intermediate carriers is essential to an understanding of the processes that work together to establish the mechanism and to determine the rate of the overall process. We have reinvestigated the numbers of photosystem I and photosystem II reaction centers, the numbers of intervening cytochrome b6/f complexes, and the numbers of molecules of the relatively mobile electron carriers plastoquinone and plastocyanin that are actively involved in electron transfer. Our investigations were based on a new experimental technique made possible by the use of a modified indophenol dye, methyl purple, the reduction of which provides a particularly sensitive and accurate measure of electron transfer. Using this dye, which accepts electrons exclusively from photosystem I, it was possible to drain electrons from each of the carriers. Thus, by manipulation of the redox condition of the various carriers and through the use of specific inhibitors we could measure the electron storage capacity of each carrier in turn. We conclude that the ratio of photosystem I reaction centers to cytochrome b6/f complexes to photosystem II reaction centers is very nearly 1:1:1. The pool of rapid donors of electrons to P700 includes not only the 2 reducing equivalents stored in the cytochrome b6/f complex but also those stored in slightly more than 2 molecules of plastocyanin per P700. More slowly available are the electrons from about 6 plastoquinol molecules per P700.
The symbiotic pattern of expression of Rhizobium meliloti N2-fixation genes is tightly coupled with the histological organization of the alfalfa root nodule and thus is under developmental control. N2-fixation gene expression is induced very sharply at a particular zone of the nodule called interzone II-III that precedes the zone where N2 fixation takes place. We show here that this coupling can be disrupted, hereby resulting in ectopic expression of N2-fixation genes in the prefixing zone II of the nodule. Uncoupling was obtained either by using a R. meliloti strain in which a mutation rendered N2-fixation gene expression constitutive with respect to oxygen in free-living bacterial cultures or by placing nodules induced by a wild-type R. meliloti strain in a microoxic environment. These results implicate oxygen as a key determinant of the symbiotic pattern of N2-fixation gene expression.
Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules. Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants. A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT. Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence. The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT. DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene or a small gene family. The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules. A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R. meliloti strains. Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis.
Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD+/NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.
Symbiotic nitrogen fixation, the process whereby nitrogen-fixing bacteria enter into associations with plants, provides the major source of nitrogen for the biosphere. Nitrogenase, a bacterial enzyme, catalyzes the reduction of atmospheric dinitrogen to ammonium. In rhizobia-leguminous plant symbioses, the current model of nitrogen transfer from the symbiotic form of the bacteria, called a bacteroid, to the plant is that nitrogenase-generated ammonia diffuses across the bacteroid membrane and is assimilated into amino acids outside of the bacteroid. We purified soybean nodule bacteroids by a procedure that removed contaminating plant proteins and found that alanine was the major nitrogen-containing compound excreted. Bacteroids incubated in the presence of 15N2 excreted alanine highly enriched in 15N. The ammonium in these assays neither accumulated significantly nor was enriched in 15N. The results demonstrate that a transport mechanism rather than diffusion functions at this critical step of nitrogen transfer from the bacteroids to the plant host. Alanine may serve only as a transport species, but this would permit physiological separation of the transport of fixed nitrogen from other nitrogen metabolic functions commonly mediated through glutamate.
Al toxicity is a severe impediment to production of many crops in acid soil. Toxicity can be reduced through lime application to raise soil pH, however this amendment does not remedy subsoil acidity, and liming may not always be practical or cost-effective. Addition of organic acids to plant nutrient solutions alleviates phytotoxic Al effects, presumably by chelating Al and rendering it less toxic. In an effort to increase organic acid secretion and thereby enhance Al tolerance in alfalfa (Medicago sativa), we produced transgenic plants using nodule-enhanced forms of malate dehydrogenase and phosphoenolpyruvate carboxylase cDNAs under the control of the constitutive cauliflower mosaic virus 35S promoter. We report that a 1.6-fold increase in malate dehydrogenase enzyme specific activity in root tips of selected transgenic alfalfa led to a 4.2-fold increase in root concentration as well as a 7.1-fold increase in root exudation of citrate, oxalate, malate, succinate, and acetate compared with untransformed control alfalfa plants. Overexpression of phosphoenolpyruvate carboxylase enzyme specific activity in transgenic alfalfa did not result in increased root exudation of organic acids. The degree of Al tolerance by transformed plants in hydroponic solutions and in naturally acid soil corresponded with their patterns of organic acid exudation and supports the concept that enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and Al toxicity.
Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules. Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants. A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT. Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence. The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT. DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene or a small gene family. The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules. A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R. meliloti strains. Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis.
This paper reviews recent efforts to understand carbon metabolism in legume nodules. Because of space limitations the review is limited, with a few exceptions, to papers published within the past 2 years (1983–85). Also, because of the emphasis on nodules, papers dealing with carbon metabolism in cultured Rhizobium have, for the most part, been omitted. Within the past few years, papers on the identification and metabolism of carbon compounds in actinorhizal nodules have begun to appear, but this work is also not reviewed here. Several topics relevant to this subject have recently been reviewed in another symposium volume (Ludden, Burris, 1985).
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Microarray and RNA gel blot analyses were performed to identify Arabidopsis genes that responded to nitrate at both low (250 μM) and high (5 to 10 mM) nitrate concentrations. Genes involved directly or indirectly with nitrite reduction were the most highly induced by nitrate. Most of the known nitrate-regulated genes (including those encoding nitrate reductase, the nitrate transporter NRT1, and glutamate synthase) appeared in the 40 most strongly nitrate-induced genes/clones on at least one of the microarrays of the 5524 genes/clones investigated. Novel nitrate-induced genes were also found, including those encoding (1) possible regulatory proteins, including an MYB transcription factor, a calcium antiporter, and putative protein kinases; (2) metabolic enzymes, including transaldolase and transketolase of the nonoxidative pentose pathway, malate dehydrogenase, asparagine synthetase, and histidine decarboxylase; and (3) proteins with unknown functions, including nonsymbiotic hemoglobin, a senescence-associated protein, and two methyltransferases. The primary pattern of induction observed for many of these genes was a transient increase in mRNA at low nitrate concentrations and a sustained increase when treated with high nitrate concentrations. Other patterns of induction observed included transient inductions after both low and high nitrate treatments and sustained or increasing amounts of mRNA after either treatment. Two genes, AMT1;1 encoding an ammonium transporter and ANR1 encoding a MADS-box factor, were repressed by nitrate. These findings indicate that nitrate induces not just one but many diverse responses at the mRNA level in Arabidopsis.
The role of sucrose synthase (SS) in the fixation of N was examined in the rug4 mutant of pea (Pisum sativum L.) plants in which SS activity was severely reduced. When dependent on nodules for their N supply, the mutant plants were not viable and appeared to be incapable of effective N fixation, although nodule formation was essentially normal. In fact, N and C resources invested in nodules were much greater in mutant plants than in the wild-type (WT) plants. Low SS activity in nodules (present at only 10% of WT levels) resulted in lower amounts of total soluble protein and leghemoglobin and lower activities of several enzymes compared with WT nodules. Alkaline invertase activity was not increased to compensate for reduced SS activity. Leghemoglobin was present at less than 20% of WT values, so O2 flux may have been compromised. The two components of nitrogenase were present at normal levels in mutant nodules. However, only a trace of nitrogenase activity was detected in intact plants and none was found in isolated bacteroids. The results are discussed in relation to the role of SS in the provision of C substrates for N fixation and in the development of functional nodules.
In root nodules of alfalfa (Medicago sativa L.), N2 is reduced to NH4⁺ in the bacteroid by the nitrogenase enzyme and then released into the plant cytosol. The NH4⁺ is then assimilated by the combined action of glutamine synthetase (EC 6.3.1.2) and NADH-dependent Glu synthase (NADH-GOGAT; EC 1.4.1.14) into glutamine and Glu. The alfalfa nodule NADH-GOGAT protein has a 101-amino acid presequence, but the subcellular location of the protein is unknown. Using immunocytochemical localization, we determined first that the NADH-GOGAT protein is found throughout the infected cell region of both 19- and 33-d-old nodules. Second, in alfalfa root nodules NADH-GOGAT is localized predominantly to the amyloplast of infected cells. This finding, together with earlier localization and fractionation studies, indicates that in alfalfa the infected cells are the main location for the initial assimilation of fixed N2.
During root nodule development several key genes involved in nitrogen fixation and assimilation exhibit enhanced levels of expression. However, little is known about the temporal and spatial distribution patterns of these transcripts. In a systematic study the transcripts for 13 of the essential enzymes involved in alfalfa (Medicago sativa) root nodule nitrogen and carbon metabolism were localized by in situ hybridization. A serial section approach allowed the construction of a map that reflects the relative distribution of these transcripts. In 33-day-old root nodules, the expression of nifH, NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) and a cytosolic isoform of glutamine synthetase (GS13; GS; EC 6.3.1.2) were localized predominantly in a 5- to 15-cell-wide region in the distal part of the nitrogen-fixing zone. This zone was also the region of high expression for leghemoglobin, a second cytosolic glutamine synthetase isoform (GS100), aspartate aminotransferase-2 (AAT-2; EC 2.6.1.1), asparagine synthetase (AS; 6.3.5.4), phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), and sucrose synthase (SuSy; EC 2.4.1.13). This suggests that, in 33-day-old alfalfa root nodules, nitrogen fixation is restricted to this 5-to 15-cell-wide area. The continued significant expression of the GS100 subclass of GS and AS in the proximal part of the nitrogen-fixing zone implicates these gene products in nitrogen remobilization. A low constitutive expression of NADH-dependent glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) was observed throughout the nodule. The transcript distribution map will be used as a navigational tool to assist in developing strategies for the genetic engineering of alfalfa root nodules for enhanced nitrogen assimilation.
Tobacco (Nicotiana tabacum) plants expressing a
partial ferredoxin-dependent glutamine-2-oxoglutarate aminotransferase (Fd-GOGAT) cDNA in the antisense orientation under
the control of the 35S promoter, were used to study the metabolism of amino acids, 2-oxoglutarate and ammonium following the
transition from CO2 enrichment (where photorespiration is inhibited) to air (where photorespiration is a major process of ammonium production
in leaves). The leaves of the lowest Fd-GOGAT expressors accumulated more foliar glutamine (Gln) and α-ketoglutarate (α-KG)
than the untransformed controls in both growth conditions. Photorespiration-dependent increases in foliar ammonium, glutamine,
α-KG and total amino acids were proportional to the decreases in foliar Fd-GOGAT activity. No change in endoprotease activity
was observed following transfer to air in the Fd-GOGAT transformants or the untransformed controls which has similar activities
over a broad range of pH values. We conclude that several pathways of amino acid biosynthesis are modified when NH3
+ and Gln accumulate in leaves.
Characterizing plants following Agrobacterium ‐mediated transformation has become an important consideration in the creation of transgenic plants. As an alternative to Southern blot analysis, polymerase chain reaction (PCR) was used to screen for the simultaneous integration of two foreign genes for neomycin phosphotransferase II (NFT II) and β‐glucuronidase (GUS) in alfalfa ( Medicago saliva L.) transformed with Agrobacterium tumefaciens . We were able to distinguish between plants that contained only the NFT II gene and those that had both the NFT II and GUS genes. Of our putative transformants selected on kanamycin, 28% contained only the NFT II gene and apparently had lost the GUS gene during the transformation process. Requiring only nanogram amounts of DNA, PCR provided a rapid method to determine which genes had been integrated to our putative transformants much earlier in the plant regenerative process than was feasible by Southern analysis.
A vector molecule for the efficient transformation of higher plants has been constructed with several features that make it efficient to use. It utilizes the trans acting functions of the vir region of a co-resident Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by left and right T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene that confers high levels of resistance to kanamycin, and a lac alpha-complementing region from M13mp19 that contains several unique restriction sites for the positive selection of inserted DNA.
Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) plays a paramount role in nodule metabolism and several reports have shown that PEPC provides substantial carbon for N2-fixation and N assimilation. To study the short- and long-term implications of reduced nodule CO2 fixation for N2 fixation in alfalfa, PEPC enzyme expression was reduced through an antisense strategy. The full-length root nodule-enhanced PEPC cDNA in antisense orientation driven by the nodule-enhanced AAT-2 promoter was transformed into alfalfa. Out of 105 transformed plants, 14 showed reduced in vitro nodule PEPC activity. Three plants were selected for further evaluation. RNA and protein blots showed reduced PEPC transcript and protein. Nodules of these plants also displayed reducedin vivo CO2 fixation. Total nitrogenase activity as measured by H2 evolution was reduced, although there was no change in apparent nitrogenase. The nodule electron allocation coefficient of antisense plants was reduced. All antisense plants accumulated less dry matter and nitrogen in a 6-week growing period under controlled conditions. The data confirm a strong interdependence of nodule PEPC and nitrogenase activity.
When a chimaeric soybean leghaemoglobin gene was introduced into the genome of another legume species, Lotus corniculatus, nodule-specific expression of the chimaeric gene was found in root nodules formed on fully regenerated plants inoculated with the Lotus microsymbiont, Rhizobium loti. Expression under control of the 5′ upstream region of the soybean gene was regulated at the level of RNA and followed the correct developmental timing. This indicates a conserved induction mechanism for leghaemoglobin genes in legumes.
Mutants of higher plants with alterations in amino acid metabolism have now been available for 20 years. Following the realization that at least four distinct classes of herbicides (phosphinothricins, glyphosates, imidazolinones and sulphonylureas) act by the inhibition of amino acid biosynthesis, mutants resistant to the herbicides have also been obtained. More recently, transgenic plants containing altered levels of enzymes of amino acid biosynthesis have been constructed. In this article, we have attempted to review several areas of amino acid biosynthesis including ammonia assimilation, the aspartate pathway, branched chain amino acids, aromatic amino acids and proline.
Full length cDNAs encoding alcohol dehydrogenase (ADH), fructose-1,6-biphosphate aldolase (ALD), nodule-enhanced malate dehydrogenase (neMDH), phosphoenolpyruvate carboxylase (PEPC), and nodule-enhanced sucrose synthase (neSS) were isolated from a pea (Pisum sativum L.) root nodule cDNA library and characterized. Transcript abundance and cellular expression patterns for each gene were examined at different stages of nodule development. All the genes were expressed prior to the induction of nitrogenase suggesting a developmental signal as the initial trigger for expression. RNA tissue blots demonstrated that all the genes except ALD exhibit enhanced expression in effective nodules. In situ hybridization studies showed contrasting patterns of gene expression within various nodule zones. The highest expression of ADH was observed in interzone. ALD was expressed predominantly in nodule meristem, invasion zone and interzone. The neSS transcripts were found rather uniformly throughout the nodule. Expression of neMDH and PEPC was also detected throughout the nodule, but the highest levels were associated with interzone and N2-fixing zone.
The NH2-terminal sequences of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) purified from barley (Hordeum vulgare L.) and Chlamydomonas reinhardtii (Dangeard), and of a barley peptide, were determined and the barley sequences were used to design oligonucleotide primers for the polymerase chain reaction. A specific 1.3-kilobase (kb) cDNA fragment specifying the NH2-terminal one-third of the mature barley polypeptide, was amplified, cloned and sequenced. The NH2-terminus of plant Fd-GOGAT is highly conserved and homologous to the NH2-terminus of the heavy subunit of Escherichia coli NADPH-GOGAT. Based on sequence homologies, we tentatively identified the NH2-terminal region of Fd-GOGAT as the glutamine-amidotransferase domain, which is related to the corresponding domain of the purF-type amidotransferases. The Fd-GOGAT cDNA clone, and polyclonal antibodies raised against the barley enzyme, were used to analyse four Fd-GOGAT-deficient photorespiratory mutants. Three mutants (RPr 82/1, RPr 82/9 and RPr 84/82) had no detectable Fd-GOGAT protein in leaves, while the fourth (RPr 84/42) had a small amount of cross-reacting material. Hybridization to Northern blots of total leaf RNA revealed that both RPr 82/9 and RPr 84/82 were indistinguishable from the parental line (Maris Mink), having normal amounts of a 5.7-kb mRNA species. On the other hand, RPr 82/2 and RPr 84/42 each contained two distinct hybridizing RNA species, one of which was larger than 5.7 kb, the other smaller. Using a set of wheat-barley telosomic addition lines we have assigned the Fd-GOGAT structural locus to the short arm of chromosome 2.
A vector system, based on copper controllable gene expression, has been developed to give control over place as well as time of expression of an introduced gene. This system consists of two elements: (1) the yeastace1 gene encoding a metallo-regulatory transcription factor, ACE1, under control of either an organ-specific or a constitutive promoter; and (2) a gene of interest under control of a chimaeric promoter consisting of the 46 bp TATA fragment of the CaMV 35S RNA promoter linked to four repeats of the ACE1 binding site. The functioning of the system in an organ-specific manner was tested in nodulatedLotus corniculatus plants which consisted of non-transformed shoots plus transformed hairy root tissue wild-type tops/transgenic roots. After addition of copper ions to the plant nutrient solution, -glucuronidase (GUS) expression was visualized either specifically in nodules or in both roots and nodules when theace1 gene was placed under control of thenod45 promoter or the CaMV 35S RNA promoter, respectively. The nodule-specific system was used to express antisense constructs of aspartate aminotransferase-P2 in transgenicLotus corniculatus plants. When expression was induced by the addition of copper ions to the plant nutrient solution aspartate aminotranferase-P2 activity declined dramatically, and a decrease of up to 90% was observed in nodule asparagine concentration.
The primary assimilation of symbiotically fixed nitrogen (N) in alfalfa root nodules involves complex intermingling with carbon (C) metabolism. Integrated functioning of both cytosolic and organelle-associated enzymes is required to link N assimilation with C metabolism. Understanding how N and C metabolism are controlled in root nodules requires fundamental knowledge of how the plant genes involved are regulated. While significant progress has been made in understanding the regulation of glutamine synthetase (GS), much less is known about the genes controlling other enzymatic steps in this process. To that end we have isolated, purified and characterized the root nodules enzymes aspartate aminotransferase (AAT), phosphoenolpyruvate carboxylase (PEPC) and glutamate synthase (NADH-GOGAT). Moreover the cDNAs encoding these crucial enzymes were isolated and characterized. While the most prominent forms of GS associated with N assimilation in nodules are located in the cytosol, AAT and NADH-GOGAT appears to be organelle-associated. The deduced amino acid sequence suggested and immunogold labeling showed that nodule-enhanced AAT-2 is located in amyloplasts. Comparison of the deduced amino acid sequence of nodule-enhanced NADH-GOGAT to the N-terminal sequence of the processed protein indicated that NADH-GOGAT has a 101 amino acid presequence. However, it is unclear as to which organelle ADH-GOGAT is targeted. Cytosolic phosphoenolpyruvate carboxylase (PEPC), which can be expressed in legume root nodules at levels comparable to those detected in leaves of C4 plants, provides a substantial amount of carbon for malate, aspartate and asparagine biosyntheses. RNA blots showed that GS, AAT, PEPC, and NADH-GOGAT mRNAs were enhanced about 15-fold during the development of effective alfalfa nodules. By comparison, the expression of GS, AAT and PEPC mRNAs was reduced by 65% in ineffective nodules. NADH-GOGAT was different from GS, AAT, and PEPC in that expression had an absolute requirement for a factor(s) related to effective nodules. The data suggest that NADH-GOGAT plays a key role in regulating N assimilation. Moreover, plastids in nodules play a major role not only in C metabolism but also in N metabolism.
The process of assimilation of inorganic nitrogen into organic form is essential both for plant growth and development as nitrogen deprivation in plants can cause a number of metabolic deficiencies in plants. Thus, the study of the enzymes involved in ammonium assimilation have an impact on both basic and applied plant research. Ammonium is first assimilated into the amino acids glutamine and glutamate by the concerted actions of glutamine synthetase (GS), glutamine-oxoglutarate aminotransferase (GOGAT), and glutamate dehydrogenase (GDH). The glutamate and glutamine are then channeled into aspartate and asparagine by aspartate amino transferase (AspAT) and asparagine synthetase (AS). However, the actual biology of the ammonium assimilation pathway has been obscured by the fact that most reactions are catalyzed by multiple isoenzymes, located in distinct tissues and/or subcellular compartments. Therefore, standard biochemical methods used to define rate-limiting enzymes in a given pathway may lead to misleading interpretations when employed to study metabolic pathways in plants. Here we discuss how the availability of genetic and molecular tools, especially in the model plant Arabidopsis thaliana, have made it possible to start delineating the mechanisms of genetic regulation of the ammonium assimilatory pathway, and to destine the in vivo role of each isoenzyme. The basic knowledge obtained on the genes involved in the process of ammonium assimilation may be applied in attempts to increase the efficiency with which nitrogen is incorporated into organic form which may have marked effects on plant productivity, biomass, and crop yield.
The assimilation of ammonia by a wide variety of organisms is the primary route for the introduction of nitrogen into the biosphere. The assimilatory enzymes glutamine synthetase and glutamate synthase catalyze reactions that convert α-ketoglutarate and ammonia to glutamate, which is then used in a wide variety of biosynthetic reactions. These enzymes also play a major role in the reassimilation of ammonia derived from photorespiration in C3 plants. Recent biochemical, molecular and genetic studies are leading to a better understanding of the factors that determine the activity and function of glutamate synthase.
The CO2 released in photorespiration is believed to result
from a complex mitochondrial reaction in which glycine is converted to
equimolar amounts of CO2, NH3 and the C-1 group of
N5,N10-methylene-tetrahydrofolate 1-3.
Because photorespiratory CO2 production may amount to 80
μmol per h per g fresh weight4,5 the stoichiometry of the
decarboxylation reaction indicates that NH3 release from
glycine exceeds primary NO3-
reduction6,7. Leaf cells must therefore be capable of rapid
NH3 reassimilation in photorespiratory conditions. It has
been suggested8 that NH3 could be refixed directly
into glutamate by mitochondrial glutamate dehydrogenase (GDH), utilizing
the NADH generated during glycine decarboxylation1,9,10. This
seems unlikely11,12 because of the high Km for
NH3 exhibited by GDH in vitro. An alternative
suggestion11,12, that photorespiratory NH3 could
be re-assimilated into glutamate by the sequential action of cytoplasmic
glutamine synthetase (GS) and the chloroplast enzyme glutamate synthase
(GOGAT)13,14, is supported by circumstantial evidence from in
vitro studies12,15. Here we provide direct evidence for such
a pathway, based on the results of experiments with mutants of
Arabidopsis thaliana (L.) Heynh which are deficient in leaf GOGAT
activity.
Changes in key nodule proteins following defoliation of white clover plants were assessed by measurements of enzyme activities and by use of antibodies to specific nodule proteins.
Defoliation caused major declines in protein and leghaemoglobin levels and in the activities of invertase, sucrose synthase, UDP glucose pyrophosphorylase, aspartate amino transferase, glutamine synthetase, phosphoenolpyruvate carboxylase, and malate dehydrogenase.
In continuously defoliated plants the activities of these nodule enzymes continued to decline throughout the course of the 17 d experiment. In plants defoliated once and then allowed to regrow new leaves, nodule enzyme activities declined for 7 to 10 d before increasing again to control levels by the end of the experiment. In this single defoliation/recovery treatment only the activities of PEP carboxylase and glutamine synthetase declined to a greater extent than the general decline in protein content. Alcohol dehydrogenase, increased in specific activity following a single defoliation and only declined in nodules of continuously defoliated plants.
Amino peptidase activity declined in concert with other enzyme activities described above. Endopeptidase activity, in contrast, increased significantly after 4 d following either a single or continuous defoliation. In the plants allowed to regrow new leaves endopeptidase activity declined to control levels again, whereas in plants continuously defoliated the activity rose 5-fold. However, endopeptidase increased only after significant declines in protein and other enzyme activities had already occurred.
Western immunoblotting confirmed that the declines in glutamine synthetase activity and leghaemoglobin levels were due to the disappearance of antigen. Declines in nitrogenase components I and II indicated that bacteroid proteins were affected by defoliation over the same time-scale as host plant encoded proteins. As new leaves grew and nodule N2 fixation was reestablished, these specific nodule proteins were again detectable by immunoblotting.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Bacteroid differentiation was examined in developing and mature alfalfa nodules elicited by wild-type or Fix- mutant strains of Rhizobium meliloti. Ultrastructural studies of wild-type nodules distinguished five steps in bacteroid differentiation (types 1 to 5), each being restricted to a well-defined histological region of the nodule. Correlative studies between nodule development, bacteroid differentiation, and acetylene reduction showed that nitrogenase activity was always associated with the differentiation of the distal zone III of the nodule. In this region, the invaded cells were filled with heterogeneous type 4 bacteroids, the cytoplasm of which displayed an alternation of areas enriched with ribosomes or with DNA fibrils. Cytological studies of complementary halves of transversally sectioned mature nodules confirmed that type 4 bacteroids were always observed in the half of the nodule expressing nitrogenase activity, while the presence of type 5 bacteroids could never be correlated with acetylene reduction. Bacteria with a transposon Tn5 insertion in pSym fix genes elicited the development of Fix- nodules in which bacteroids could not develop into the last two ultrastructural types. The use of mutant strains deleted of DNA fragments bearing functional reiterated pSym fix genes and complemented with recombinant plasmids, each carrying one of these fragments, strengthened the correlation between the occurrence of type 4 bacteroids and acetylene reduction. A new nomenclature is proposed to distinguish the histological areas in alfalfa nodules which account for and are correlated with the multiple stages of bacteroid development.
We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
1. The finding that the plant is the genetic determinant of leghaemoglobin production in legume nodules was further tested by inoculating snake beans with two strains of Rhizobium selected to give large genetic differences. Carbohydrate requirement patterns, immunological techniques and DNA base ratio determinations were used to demonstrate genetic differences between the two rhizobial strains. 2. Partially purified preparations of the haemoglobins from the nodules produced by the two strains showed no differences when examined by electrophoresis, isoelectric focusing or ion-exchange chromatography. 3. Two different leghaemoglobins from each type of nodule were separated by chromatography on DEAE-cellulose. One of these was isolated in the Fe(3+) form and accounted for two-thirds of the total leghaemoglobin. When it was examined in the analytical ultracentrifuge and by amino acid analysis, this major component did not vary with the inoculant rhizobial strain. The molecule had an s(20,w) of 1.88S, a diffusion coefficient of 10.7x10(-7)cm(2).s(-1) and a mol. wt. of 16700. 4. These results strongly support the hypothesis that the mRNA for leghaemoglobin is transcribed from plant DNA.
A vector molecule for the efficient transformation of higher plants has been constructed with several features that make it efficient to use. It utilizes the trans acting functions of the vir region of a co-resident Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by left and right T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene that confers high levels of resistance to kanamycin, and a lac alpha-complementing region from M13mp19 that contains several unique restriction sites for the positive selection of inserted DNA.
The expression of a soybean leghemoglobin 5'lbc3-GUS-3'nos chimeric gene was analyzed in Lotus corniculatus after site-specific mutagenesis of the nodulin consensus sequences, AAAGAT and CTCTT, present in the organ-specific element (OSE) (-139 to -102). Full-length promoters (-1956, +46) carrying clustered point mutations in the CTCTT sequence or in both the AAAGAT and the CTCTT sequences were inactive. Point mutations in the AAAGAT sequence had only minor effects on the expression level. Substitution of the ATTG sequence between the AAAGAT and the CTCTT sequences in the OSE reduced the activity in nodules to 10%. This, together with the conservation of the ATTGT sequence in the same position of leghemoglobin genes from other legumes, indicates that these sequences, in addition to the nodulin consensus sequences of the OSE, are important for high-level nodule-specific expression. Substitution of the CTCTT sequences outside the OSE (-44, -40 and -79, -75) results in promoter activities of approximately 50%.
A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean. The VN-35 cDNA was inserted in the antisense orientation downstream of the caMV-35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes. Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot. Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing beta-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.
Phosphoenolpyruvate carboxylase (PEPC) kinase was partially purified about 3000-fold from soybean root nodules by a fast-protein liquid chromatography protocol. This protein-serine kinase has an apparent native molecular mass of about 30,000 as estimated by size-exclusion chromatography. Following electrophoresis of this partially purified PEPC-kinase preparation in a denaturing gel containing dephospho maize leaf PEPC as substrate, the in situ renaturation and assay of protein kinase activity revealed two, PEPC-dependent kinase polypeptides with molecular masses of about 32 and 37 kDa. The approximately 32-kDa polypeptide was significantly more active than the approximately 37-kDa catalytic subunit. The activity of this partially purified PEPC kinase, and a less purified sample, was Ca2+-insensitive. This protein kinase preparation was able to phosphorylate purified PEPCs from soybean nodules, maize leaves, and a sorghum recombinant C4 PEPC. In contrast, this PEPC kinase was unable to phosphorylate a phosphorylation-site mutant form of sorghum C4 PEPC (S8Y), two other soybean nodule phosphoproteins [nodulin-26 and nodulin-100 (sucrose synthase)], bovine serum albumin, and histone III-S. Following in vitro phosphorylation of purified dephospho soybean nodule PEPC from stem-girdled plants by the partially purified nodule PEPC kinase, the former's activity and sensitivity to L-malate inhibition increased and decreased, respectively. Notably, the Ca2+-independent PEPC kinase activity in nodules from illuminated plants was markedly greater than that in nodules harvested from plants subjected to stem girdling or prolonged darkness. Furthermore, the kinase activity in nodules was controlled reversibly by illumination and extended darkness pretreatments of the parent plants, suggesting that photosynthate supply from the shoots is likely responsible for these striking changes in PEPC kinase activity observed in planta in the legume nodule.
The effects of ultraviolet-B (UV-B) radiation on water relations, leaf development, and gas-exchange characteristics in pea (Pisum sativum L. cv Meteor) plants subjected to drought were investigated. Plants grown throughout their development under a high irradiance of UV-B radiation (0.63 W m-2) were compared with those grown without UV-B radiation, and after 12 d one-half of the plants were subjected to 24 d of drought that resulted in mild water stress. UV-B radiation resulted in a decrease of adaxial stomatal conductance by approximately 65%, increasing stomatal limitation of CO2 uptake by 10 to 15%. However, there was no loss of mesophyll light-saturated photosynthetic activity. Growth in UV-B radiation resulted in large reductions of leaf area and plant biomass, which were associated with a decline in leaf cell numbers and cell division. UV-B radiation also inhibited epidermal cell expansion of the exposed surface of leaves. There was an interaction between UV-B radiation and drought treatments: UV-B radiation both delayed and reduced the severity of drought stress through reductions in plant water-loss rates, stomatal conductance, and leaf area.
Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine. In plants GS is an octameric enzyme and is located either in the cytoplasm (GS1) or in the chloroplast (GS2). Two distinct classes of GS1 genes with unique 3'-untranslated region (3'UTR) have been identified in alfalfa. We have demonstrated that the two classes exhibit differential expression pattern in the different plant organs suggesting different functional roles for the different isozymes. To determine the functional significance of the two classes of GS1 genes in alfalfa, we have utilized antisense gene constructs aimed specifically at the 3'UTR of the two GS1 genes and introduced them individually into alfalfa. Our data show that the gene constructs are effective in lowering the corresponding transcript level very effectively though there were organ-specific differences in the level of reduction. No transcript corresponding to the antisense gene construct was detected in any of the alfalfa transformants though they accumulated to significant levels in transgenic tobacco containing the same construct. This suggests that the antisense transcript was not stable in the presence of the homologous target sequence. Transgenic alfalfa with up to 80% reduction in the transcript level corresponding to each gene class, however, showed no reduction in GS activity or GS1 polypeptide level. The results suggest that GS1 mRNA levels are not rate-limiting for GS1 polypeptide synthesis and that GS levels are controlled both at the transcriptional and translational/post-translational level.