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Over-expression of the Bacterial Phytase US417 in Arabidopsis Reduces the Concentration of Phytic Acid and Reveals Its Involvement in the Regulation of Sulfate and Phosphate Homeostasis and Signaling
Abstract
Phytic acid (PA) is the main phosphorus storage form in plant seeds. It is recognized as an anti-nutrient for humans and non-ruminant
animals, as well as one of the major sources of phosphorus that contributes to eutrophication. Therefore, engineering plants
with low PA content without affecting plant growth capacity has become a major focus in plant breeding. Nevertheless, lack
of knowledge on the role of PA seed reserves in regulating plant growth and in maintaining ion homeostasis hinders such an
agronomical application. In this context, we report here that the over-expression of the bacterial phytase PHY-US417 in Arabidopsis leads to a significant decrease in seed PA, without any effect on the seed germination potential. Interestingly, this over-expression
also induced a higher remobilization of free iron during germination. Moreover, the PHY-over-expressor lines show an increase
in inorganic phosphate and sulfate contents, and a higher biomass production after phosphate starvation. Finally, phosphate
sensing was altered because of the changes in the expression of genes induced by phosphate starvation or involved in phosphate
or sulfate transport. Together, these results show that the over-expression of PHY-US417 reduces PA concentration, and provide the first evidence for the involvement of PA in the regulation of sulfate and phosphate
homeostasis and signaling.
... Seeds of Arabidopsis thaliana ecotype Columbia (Col-0), transgenic lines expressing PHY-US417 gene (L7 and L9) (Belgaroui et al., 2014) and the ipk1-1 mutant (Stevenson-Paulik et al., 2005) were used in these experiments. Seeds were surface sterilized with 70% ethanol for 10 min and rinsed with sterile ultrapure water three times. ...
... In order to investigate an eventual role of PA in this Pi/Zn crosstalk and its functional relationship to plant growth, we were interested here to evaluate the effect of the decrease of endogenous PA contents on plant responses to individual or combined Pi and Zn deficiencies. For this purpose, two Arabidopsis transgenic lines (L7 and L9) overexpressing the bacterial phytase PHY-US417 which contain less than 40% of PA compared to the WT (Belgaroui et al., 2014), were grown along with the ipk1-1 mutant (with 83% decrease of PA) (Stevenson-Paulik et al., 2005), under zinc deficiency (-Zn; 0 μM Zn), low Pi supply (-Pi; 5 μM Pi) or combined low supplies of Zn and Pi (-Pi-Zn). Under -Pi conditions, Col-0, the transgenic lines and ipk1-1 mutant showed similar reduced growth of primary roots (Fig. 1A). ...
... Under standard conditions (MS medium), the Pi contents in the transgenic lines and the ipk1-1 mutant were approximately 2-fold higher than in the control plants ( Fig. 2A). On a -P or -P/-Zn media, the Pi contents sharply decreased in the tested genotypes compared with standard MS medium and tended to be higher in the shoots of the transgenic plants and ipk1-1 mutant (up to 45% increase) compared to control plants as previously reported (Belgaroui et al., 2014). When grown on Zn-deficient medium, WT plants displayed a higher shoot Pi accumulation (up to 23% increase) compared to MS medium, which is consistent with previous reports showing that Zn deficiency has been accompanied by an overaccumulation of Pi in shoots . ...
Inorganic phosphate (Pi) and zinc (Zn) are two essential nutrients for plant growth. Crosstalk between these two elements to control their uptake and homeostasis in plants has been previously demonstrated. However, the signaling molecule(s) required for the mechanisms underlying this interaction remain unknown.
Phytic acid (PA), the main P storage form in plants, serves also as a signalling molecule in processes controlling plant growth and development as well as responses to different stimuli. In this study, we investigated the involvement of PA in the control of Zn-Pi homeostasis interaction in Arabidopsis. For this purpose, we used two classes of low phytic acid (lpa) lines: the inositol polyphosphate kinase 1 gene (ipk1-1) mutant and two transgenic lines expressing the bacterial phytase PHY-US417. The transgenic lines exhibit an enhanced root growth under Zn-deficiency compared to wild type (WT) and ipk1-1. In addition, higher Pi and Zn contents were detected in the lpa lines under standard and also deficient conditions (-Pi and -Zn). However, the activation of shoot Pi accumulation which occurs in WT in response to Zn depletion was not observed in the lpa lines. Finally, we noticed that the changes in Pi and Zn accumulation seem to be correlated with a tight regulation of Pi and Zn transporters in the lpa lines. All these findings underline a regulatory role of PA in the control of the Zn-Pi crosstalk but also open the door to possible involvement of additional unknown signaling molecules in this process.
... The lipid-dependent pathway involves continuous addition of phosphates and subsequent phosphorylation events by the enzyme phosphatidylinositol kinases by the action of phospholipase C. The reaction starts with the conversion of Myo-inositol-3-phosphate also called as Ins in to phosphatidylinositol (PtdIns) which undergoes sequential phosphorylation via phosphatidylinositol kinases and produce phosphatidyl inositol (4,5) (1,2,3,4,5,6)P6] is formed from Ins(1,3,4,5,6)P5. (Fig. 2). ...
... The lipid-dependent pathway involves continuous addition of phosphates and subsequent phosphorylation events by the enzyme phosphatidylinositol kinases by the action of phospholipase C. The reaction starts with the conversion of Myo-inositol-3-phosphate also called as Ins in to phosphatidylinositol (PtdIns) which undergoes sequential phosphorylation via phosphatidylinositol kinases and produce phosphatidyl inositol (4,5) (1,2,3,4,5,6)P6] is formed from Ins(1,3,4,5,6)P5. (Fig. 2). ...
... Probably, some biosynthetic gene expression is hampered or the degradation pathway is hyperactive. So, a better understanding of these networks and key transcription factors involved in the phytic acid metabolism will help in the development of low phytic acid mutants [5]. Promoter mining, tilling and eco-tilling, global gene expression profiling, proteome and interactome analysis along with ionomics approach are some of the strategies to decipher these regulatory networks. ...
Phytic acid is considered as one of the most abundant storage forms of phosphorous in the plant seed tissues. It accumulates throughout seed development until maturity and accounts for 60–70% of the total phosphorus in legume seed. Its amount ranges from 0.40 to 2.06% in legumes. It is a strong cationic chelator and negatively impacts the bioavailability of essential micronutrients like iron (Fe) and Zinc (Zn). It has very limited digestibility in humans and other non-ruminants which lack phytases. Besides, it also acts as an inhibitor of salivary and gastric enzymes during digestion. On the contrary, the beneficial effects of phytic acid include its role as an antioxidant, anti-carcinogen, and regulator of blood glucose and cholesterol levels. Several biotechnological strategies have been employed to reduce the phytic acid content in plants especially legumes for better absorption of essential micronutrients and in turn for bio-fortification to address the malnutrition challenges across the world. This review focuses primarily on the mechanisms governing phytic acid biosynthesis and its regulation/ reduction using various biotechnological, mutation breeding and computational approaches. This will help us in understanding the molecularity of the phytic acid development and will also contribute towards developing newer and more efficient strategies aimed at regulating phytic acid content in legumes.
... tabacum), rape (Brassica napus), soybean (Glycine max), maize (Zea mays), and alfalfa (M. sativa) [21,24,26,27]. Note that plants were often transformed by genes encoding two classes of phytases, histidine acid (HAP) and beta-propeller (BPP) phytases [21,[26][27][28]. ...
... sativa) [21,24,26,27]. Note that plants were often transformed by genes encoding two classes of phytases, histidine acid (HAP) and beta-propeller (BPP) phytases [21,[26][27][28]. For example, rape (B. ...
... In the case of a plant growth on phytase-containing medium, the phytase activity is associated with the fraction of cell-wall proteins, whereas the corresponding activity of the soluble protein fraction is reduced, though it still exceeds that in the plants of control lines. The obtained data correlate with the results of constitutive expression of bacillar phytase in A. thaliana plants [27]. However, in contrast to the data obtained in our study, recombinant phytase described in the above-mentioned study had an intracellular localization that was rather inefficient in relation to the phytate acquisition from the medium and negatively influenced on the metabolic status of cells. ...
Transgenic plants containing genes of bacterial phytases represent one of the promising ways to solve the problem of phosphorus deficiency in the nutrition of plants and monogastric animals. Histidine acid phytase PaPhyC from Pantoea agglomerans has a high activity and represents a promising basis for the biotechnology of plants. In this study, the analysis of morphological characteristics, phytase activity, and phosphorus content in tissues of the earlier obtained, genetically modified Arabidopsis thaliana (L.) Heynh. plants producing extracellular phytase (PaPhyC) has been carried out. According to the obtained results, modified plants are able to grow on a medium supplemented with phytate as the sole source of phosphorus. Exterior characteristics (rosette diameter and area) of phytase-expressing plants grown on media containing phytate or inorganic phosphorus do not differ, which confirms that the plants use phytate as the phosphorus source. In the case of plant cultivation on a phytate-containing medium, a high phytase activity is observed in the cell walls of modified plants. The content of inorganic phosphorus in tissues of modified plants does not change in the case of their cultivation on the medium containing phytate as the sole source of phosphorus.
... Similarly, expression of B. subtilis 168phyA phytase in A. thaliana led to a higher shoot dry weight and an increase in phosphorus content by 100% compared to the wild type (Lung et al., 2005). Similar results have more recently been obtained with a related BPP phytase PHY-US417 expressed in A. thaliana (Belgaroui et al., 2014(Belgaroui et al., , 2016. ...
... Most of such phytases used in transgenic research are members of the HAP type of phytases (Pen et al., 1993;Verwoerd et al., 1995;Brinch-Pedersen et al., 2000;Ponstein et al., 2002;Hong et al., 2004;Bilyeu et al., 2008;Wang et al., 2013). In addition, a few studies reported successful use of BPP phytases (Yip et al., 2003;Lung et al., 2005;Chan et al., 2006;Belgaroui et al., 2014;Belgaroui et al., 2016) and PAP enzymes (Xiao et al., 2005;Ma et al., 2009Ma et al., , 2012. TTP phytases, which represent a relatively rare family of phytases found mostly in ruminant bacteria (Yanke et al., 1998), have also been utilized (Hong et al., 2004). ...
... 168phyA phytase has previously been expressed in tobacco and A. thaliana, though under different promoters (Yip et al., 2003;Lung et al., 2005;Chan et al., 2006). A closely related phytase from B. subtilis strain 417 (72% amino acid identity and 84% similarity to 168phyA phytase) has also recently been used for this purpose (Belgaroui et al., 2014(Belgaroui et al., , 2016. When grown on phytate, plants expressing these BPP-type phytases displayed twofold higher biomass levels (Belgaroui et al., 2016), as well as a significant increase in shoot P concentration compared to control plants (Lung et al., 2005;Belgaroui et al., 2016). ...
Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2. The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology.
... Alternative approaches to generate lpa mutants were successfully established by engineering plants to produce heterologous phytases (for review 17 ). In most cases, the reduction of PA levels following its hydrolysis by expression of intracellular phytase genes is associated to an increase in free inorganic phosphate (Pi) and also several inositol phosphate derivates in plant tissues [49][50][51] . For instance, the over-expression of the bacterial phytase PHY-US417 in Arabidopsis led to 40% decrease in PA content and increase in foliar Pi concentration, associated to an improved plant growth capacity under P-limited conditions 51 . ...
... In most cases, the reduction of PA levels following its hydrolysis by expression of intracellular phytase genes is associated to an increase in free inorganic phosphate (Pi) and also several inositol phosphate derivates in plant tissues [49][50][51] . For instance, the over-expression of the bacterial phytase PHY-US417 in Arabidopsis led to 40% decrease in PA content and increase in foliar Pi concentration, associated to an improved plant growth capacity under P-limited conditions 51 . ...
... Higher decrease of PA concentration (80%) such as observed in ipk1-1 mutant is rather detrimental. In combination with our previous results 51 showing that the overexpression of PHY-US417 improves the growth of plants under Pi deficiency, we demonstrate here that the PHY-US417 over-expression not only increases antioxidant activities and osmotic stress tolerance (individual stress), but also improves the plant capacity to tolerate combined osmotic stress and Pi deficiency. This constitutes a new and desired trait for biotechnological application in agriculture. ...
Engineering osmotolerant plants is a challenge for modern agriculture. An interaction between osmotic stress response and phosphate homeostasis has been reported in plants, but the identity of molecules involved in this interaction remains unknown. In this study we assessed the role of phytic acid (PA) in response to osmotic stress and/or phosphate deficiency in Arabidopsis thaliana. For this purpose, we used Arabidopsis lines (L7 and L9) expressing a bacterial beta-propeller phytase PHY-US417, and a mutant in inositol polyphosphate kinase 1 gene (ipk1-1), which were characterized by low PA content, 40% (L7 and L9) and 83% (ipk1-1) of the wild-type (WT) plants level. We show that the PHY-overexpressor lines have higher osmotolerance and lower sensitivity to abscisic acid than ipk1-1 and WT. Furthermore, PHY-overexpressors showed an increase by more than 50% in foliar ascorbic acid levels and antioxidant enzyme activities compared to ipk1-1 and WT plants. Finally, PHY-overexpressors are more tolerant to combined mannitol stresses and phosphate deficiency than WT plants. Overall, our results demonstrate that the modulation of PA improves plant growth under osmotic stress, likely via stimulation of enzymatic and non-enzymatic antioxidant systems, and that beside its regulatory role in phosphate homeostasis, PA may be also involved in fine tuning osmotic stress response in plants.
... It is well established that Pi is released from PA following the exogenous application of commercial bacterial phytases in the growth media (Belgaroui et al., 2014;Hayes et al., 2000;Idriss et al., 2002;Richardson et al., 2000). Moreover, expression of the intracellular form of b-propeller phytases from Bacillus subtilis in Arabidopsis and tobacco was tested and showed an enhancement of the growth performance of these transgenic plants under Pi-limited conditions (Belgaroui et al., 2014;Lung et al., 2005). ...
... It is well established that Pi is released from PA following the exogenous application of commercial bacterial phytases in the growth media (Belgaroui et al., 2014;Hayes et al., 2000;Idriss et al., 2002;Richardson et al., 2000). Moreover, expression of the intracellular form of b-propeller phytases from Bacillus subtilis in Arabidopsis and tobacco was tested and showed an enhancement of the growth performance of these transgenic plants under Pi-limited conditions (Belgaroui et al., 2014;Lung et al., 2005). Nevertheless, these approaches had only limited success for implementing strategies to increase crop production under P limitation. ...
... In a recent study (Belgaroui et al., 2014), we have established that Arabidopsis plants are able to produce an active intracellular form of the phytase PHY-US417, leading to changes in the response to Pi signalling pathway. As aforementioned, there is a growing interest for the use of secreted phytases not only to better utilize extracellular PA but also for intercropping practices. ...
Phytic acid (PA) is a major source of inorganic phosphate (Pi) in the soil, however the plant lacks the capacity to utilize it for Pi nutrition and growth. Microbial phytases constitute a group of enzymes that are able to remobilize Pi from PA. Thus, the use of these phytases to increase the capacity of higher plants to remobilize Pi from PA is of agronomical interest. In the current study, we generate transgenic Arabidopsis lines (ePHY) overexpressing an extracellular form of the phytase PHY-US417 of Bacillus subtilis, which are characterized by high levels of secreted phytase activity. In presence of PA as sole source of Pi, while the wild-type plants shows hallmark of Pi deficiency phenotypes, including the induction of the expression of Pi starvation induced genes (PSI, e.g PHT1;4) and the inhibition of growth capacity, the ePHYover-expressing lines show a higher biomass production and no PSI induction. Interestingly, when co-cultived with ePHY over-expressors, wild type Arabidopsis plants (or tobacco) show repression of the PSI genes, improvement of Pi content and increases in biomass production. In line with these results, mutants in the high affinity Pi transporters, namely pht1;1 and pht1;1-1;4, both fail to accumulate Pi and to grow when co-cultured with ePHY overexpressors. Taken together, these data demonstrate the potential of secreted phytases in improving the Pi content and enhancing growth of not only the transgenic lines but also the neighbouring plants. This article is protected by copyright. All rights reserved.
... In most cases, fungal (from Aspergillus species) or bacterial (from Bacillus subtilis strains) phytases were directly used as an additive growth factor in the medium or expressed in transgenic plants as intracellular or secreted forms [121,125,[127][128][129]. Although the exogenous application of bacterial phytases in the growth media is efficient in releasing Pi from PA [116,120,130,131], in planta heterologous expression of microbial phytases is more attractive and was successfully demonstrated on several plants species. Several transgenic plant species overexpressing HAP or BPP encoding genes have been reported in the literature. ...
... Likely, other similar bacterial phytases can be expressed in planta without any apparent negative traits, suggesting that lowering the PA content (perhaps to a certain threshold) does not affect systematically plant development. Indeed, the overexpression of PHY-US417 (a BPP from Bacillus subtilis) in Arabidopsis lead to a significant decrease in seed PA, without any effect on the seed germination potential [120]. ...
Phosphorus (P) is an essential macronutrient for all living organisms. In plants, P is taken up from the rhizosphere by the roots mainly as inorganic phosphate (Pi), which is required in large and sufficient quantities to maximize crop yields. In today’s agricultural society, crop yield is mostly ensured by the excessive use of Pi fertilizers, a costly practice neither eco-friendly or sustainable. Therefore, generating plants with improved P use efficiency (PUE) is of major interest. Among the various strategies employed to date, attempts to engineer genetically modified crops with improved capacity to utilize phytate (PA), the largest soil P form and unfortunately not taken up by plants, remains a key challenge. To meet these challenges, we need a better understanding of the mechanisms regulating Pi sensing, signaling, transport and storage in plants. In this review, we summarize the current knowledge on these aspects, which are mainly gained from investigations conducted in Arabidopsis thaliana, and we extended it to those available on an economically important crop, wheat. Strategies to enhance the PA use, through the use of bacterial or fungal phytases and other attempts of reducing seed PA levels, are also discussed. We critically review these data in terms of their potential for use as a technology for genetic manipulation of PUE in wheat, which would be both economically and environmentally beneficial.
... Our analysis showed that the promoter of OsSULTR3;3 also contains a P1BS-like sequence (GCATATTC), indicating that a cross-talk between sulfate and phosphate homeostasis via the rice PHR1 ortholog OsPHR2 may also involve OsSULTR3;3. Belgaroui et al. (2014) have demonstrated recently that the introduction of a bacterial phytase gene in A. thaliana not only reduces PA, but also alters sulfate homeostasis, thus suggesting that PA is involved in sulfate and phosphate homeostasis and signaling. In the present study, we have demonstrated that mutations of OsSULTR3;3 result in simultaneous concentration changes in phosphate and sulfate in both vegetative tissue (Table 3) and grain (Tables 1, 2), thus providing new evidence supporting the cross-talk between sulfate and phosphate homeostasis and/or signaling. ...
... In the present study, we have demonstrated that mutations of OsSULTR3;3 result in simultaneous concentration changes in phosphate and sulfate in both vegetative tissue (Table 3) and grain (Tables 1, 2), thus providing new evidence supporting the cross-talk between sulfate and phosphate homeostasis and/or signaling. However, although PA is also reduced in the ossultr3;3 mutants, we would argue that OsSULTR3;3 (directly or indirectly) plays a role in the cross-talk rather than the reduction in PA, as suggested by Belgaroui et al. (2014), because we did not observe similar metabolite changes in other lpa rice mutants (Frank et al., 2007). ...
Two low phytic acid ( lpa ) mutants have been developed previously with the aim to improve the nutritional value of rice ( Oryza sativa ) grains. In the present study, the impacts of lpa mutations on grain composition and underlying molecular mechanisms were investigated.
Comparative compositional analyses and metabolite profiling demonstrated that concentrations of both phytic acid ( PA ) and total phosphorus (P) were significantly reduced in lpa brown rice, accompanied by changes in other metabolites and increased concentrations of nutritionally relevant compounds. The lpa mutations modified the expression of a number of genes involved in PA metabolism, as well as in sulfate and phosphate homeostasis and metabolism.
Map‐based cloning and complementation identified the underlying lpa gene to be Os SULTR 3;3 . The promoter of Os SULTR 3;3 is highly active in the vascular bundles of leaves, stems and seeds, and its protein is localized in the endoplasmic reticulum. No activity of Os SULTR 3;3 was revealed for the transport of phosphate, sulfate, inositol or inositol 1,4,5 triphosphate by heterologous expression in either yeast or Xenopus oocytes.
The findings reveal that Os SULTR 3;3 plays an important role in grain metabolism, pointing to a new route to generate value‐added grains in rice and other cereal crops.
... The transporter ABCC4 plays a vital role in the detoxification process in both animals and plants, and it has also been implicated in other cellular processes, such as the regulation of stomatal aperture [44]. In soybean, the ABCC5 was identified as a transporter of phytate [45], a main phosphorus-containing compound that can increase osmotic tolerance through the stimulation of antioxidant systems [46]. The ABC proteins in the pleiotropic drug resistance (PDR) family have unique domain organizations and are implicated in responses to abiotic and biotic stress, in the latter case, by pumping antimicrobial compounds out of the cell [47]. ...
Ethephon (ET) is an ethylene-releasing plant growth regulator (PGR) that can delay the bloom time in Prunus, thus reducing the risk of spring frost, which is exacerbated by global climate change. However, the adoption of ET is hindered by its detrimental effects on tree health. Little knowledge is available regarding the mechanism of how ET shifts dormancy and flowering phenology in peach. This study aimed to further characterize the dormancy regulation network at the transcriptional level by profiling the gene expression of dormant peach buds from ET-treated and untreated trees using RNA-Seq data. The results revealed that ET triggered stress responses during endodormancy, delaying biological processes related to cell division and intercellular transportation, which are essential for the floral organ development. During ecodormancy, ET mainly impeded pathways related to antioxidants and cell wall formation, both of which are closely associated with dormancy release and budburst. In contrast, the expression of dormancy-associated MADS (DAM) genes remained relatively unaffected by ET, suggesting their conserved nature. The findings of this study signify the importance of floral organogenesis during dormancy and shed light on several key processes that are subject to the influence of ET, therefore opening up new avenues for the development of effective strategies to mitigate frost risks.
... Plants such as cereals, nuts, and seeds store phosphorus (P) in the form of phytic acid (C6H18O24P6 or Ip6) (Belgaroui et al., 2014, Singh et al., 2018. Phytic acid retains 60 % to 90 % of inorganic phosphorus in legumes, oil seeds, and cereals, rendering it is un available for metabolic reactions in living systems (Corrêa dan Arajo, 2020). ...
Phytase enzymes are applied to animal feed to help animals absorb more nutrients. The use of feed raw materials containing phytase enzymes is expected to reduce the cost of animal feed production. Efforts to increase the phytase content in maize were carried out by improving genetics, in the way of assembling transgenic plants containing high phytase content. The 27-kDa γ Zein promoter is a specific promoter that expresses genes in caryopsis, and promoter CaMV 35S is a constitutive promoter that controls gene expression in all tissues and generally does not depend on the growth phase. Transgenic maize was transformed using Agrobacterium tumefacien infection method on maize B104. The reverse transcriptase polymerase chain reaction (RT-PCR) approach was used to examine the expression of phytase genes in leaves, roots, and caryopsis was done 10, 20, and 30 days after pollination (DAP). The phytase enzyme activity test was also carried out by using the colorimetric phosphomolybdate analysis method to see the phytase enzyme activity in unit µg-1. The results showed that the phytase gene in transgenic plants with the 27-kDa γ Zein promoter was highly expressed in maize caryopsis, but in line Z6.10 was also expressed in leaves, while in the CaMV 35S promoter the phytase gene was only expressed on the leaves. Phytase enzyme activity showed that transgenic maize was higher than non-transgenic maize.
... Phytate is the most abundant myoinositol phosphate. The potential of phytate to form very stable complexes with minerals and proteins confers this molecule with its notorious antinutritional properties, which indicates its ability to chelate mineral cations to compromise mineral absorption (Belgaroui et al., 2014;Humer et al., 2015). Degradation of phytate is achieved by a group of enzymes called phytases that are capable of initiating stepwise release of phosphate from phytate. ...
Osteoporosis (OP) is a chronic disease in the elderly, and China is entering an aging demographic trend. In recent years, increasing evidence has demonstrated that probiotics can treat osteoporosis. This study aimed to explore the relevant mechanisms and to validate the beneficial effect on osteoporosis by high-throughput metagenome-wide gene sequencing in humans. In this study, compared with controls, several species had altered abundances, and specific functional pathways were found in the OP group. At the species level, the species that had increased in OP individuals were positively correlated to bone resorption markers and negatively correlated to 25-OH-D3 and bone formation markers, with Streptococcus sanguinis showing the strongest relevance, followed by Streptococcus gordonii , Actinomyces odontolyticus , and Olsenella unclassified. Additionally, Actinomyces graevenitzii , enriched in the OP group, was positively correlated to inflammation indicators that included white blood cell (WBC), neutrophil count (NEC), and the neutrophil-to-lymphocyte ratio (NLR) ( p < 0.05). Conversely, the levels of Akkermansia muciniphila , Bacteroides eggerthii , Bacteroides fragilis , Bacteroides uniformis , and Butyricimonas synergistic were increased in the control group, which had a negative correlation with bone resorption markers and positive correlation with bone formation markers and 25-OH-D3. Additionally, Bacteroides fragilis had a negative correlation with inflammation indicators (WBC, NEC, and NLR) and the above pathways ( p < 0.05). Functional prediction revealed that 106 metabolic pathways, enriched in the OP group, were significantly higher than in the control group ( p < 0.05). In particular, pathways related to LPS biosynthesis, phytate degradation, lactate acid, and ethanol fermentation were more abundant in the OP group than in the control and were positively related to WBC and NEC. Taken together, several species with altered abundances and specific functional pathways were found in OP individuals. The role of phytases in OP provides novel epidemiological evidence to elucidate the underlying microbiota-relevant mechanisms in bone mineralization and should be explored further.
... Phytate content is one of the key factors in Fe remobilization during germination and seedling establishment. It has been shown that transgenic plants overexpressing bacterial phytase, an enzyme that degrades phytate, remobilize Fe faster during germination than wild type plants (Belgaroui et al., 2014). From a dietary point of view for humans, phytate limits Fe, and also Zn, absorption in the intestinal tract, thereby contributing to mineral deficiencies (Gibson et al., 2018). ...
In plants, iron (Fe) transport and homeostasis are highly regulated processes. Fe deficiency or excess dramatically limit plant and algae productivity. Interestingly, complex and unexpected interconnections between Fe and various macro- and micronutrient homeostatic networks, supposedly maintaining general ionic equilibrium and balanced nutrition, are currently uncovered. Although these phenomena have profound consequences on our understanding of Fe homeostasis and its regulation, the molecular bases and biological significance of these interactions remain poorly understood. Here, we review recent knowledge gained on how Fe interacts with micronutrient (e.g. zinc, manganese) and macronutrient (e.g. sulfur, phosphate) homeostasis, and on how these interactions affect Fe uptake and trafficking. We finally highlight the importance of developing an improved model of how Fe signaling pathways are integrated into functional networks to control plant growth and development in response to fluctuating environments.
... Unfortunately, no other evidence is available to support this extra regulatory circuit further and to fully appreciate its possible physiological impact on S metabolism in P deficient plants. Finally, the observation that Arabidopsis lines engineered for low PA content show alterations in SO 4 2− distribution and changes in expression of some SULTRs suggests the existence of another level of complexity in the cross-talk between S and P, which directly involves PA [56]. ...
A few new papers report that mutations in some genes belonging to the group 3 of plant sulfate transporter family result in low phytic acid phenotypes, drawing novel strategies and approaches for engineering the low-phytate trait in cereal grains. Here, we shortly review the current knowledge on phosphorus/sulfur interplay and sulfate transport regulation in plants, to critically discuss some hypotheses that could help in unveiling the physiological links between sulfate transport and phosphorus accumulation in seeds.
... Interestingly, two genes necessary for the replacement of phospholipids by sulfolipids in Pi-deficient plants, SQD1 and SQD2, contain a PHR1 binding sequence (P1BS) in their promoter, and are up-regulated by Pi deficiency in a PHR1-dependant manner ( Franco-Zorrilla et al., 2004;Stefanovic et al., 2007). The accumulation of SO 4 and Pi was affected in Arabidopsis lines characterized by a very low inositol-6-phosphate (phytic acid, PA) content ( Belgaroui et al., 2014). The expression of genes involved in the SO 4 and Pi transport or signaling was altered in these low PA mutants. ...
Le fer (Fe) est un élément indispensable à la vie. Sa capacité à perdre ou à gagner un électron lui permet d’être un cofacteur de choix pour de nombreuses réactions enzymatiques telles que la photosynthèse, la synthèse d’ADN ou la respiration. Cependant, le fer est très réactif et potentiellement toxique pour la cellule. Les plantes doivent donc strictement réguler leur homéostasie en fer afin d’éviter toute carence ou tout excès préjudiciable pour leur organisme. Parmi les acteurs du maintien de l’équilibre ferrique, les ferritines jouent un rôle majeur. Chez les végétaux, elles sont principalement régulées transcriptionnellement. Le gène modèle des ferritines, AtFER1, est régulé par au moins trois voies indépendantes (l’excès de fer, la carence en phosphate, et l’alternance jour/nuit). Toutefois, la façon dont ces signaux s’intègrent au niveau de son promoteur n’est pas formellement établie. Mon travail a consisté à mettre en place une étude fonctionnelle du promoteur d’AtFER1 en caractérisant des lignées stables d’Arabidopsis thaliana exprimant le gène rapporteur GUS (β-glucuronidase) sous le contrôle de différentes versions du promoteur d’AtFER1 (délétions en 5’ et en 3’, mutagenèse dirigée) selon différents traitements (e.g. disponibilité en fer). Cette approche a mis en évidence le rôle clef de certains éléments cis du promoteur. Des cribles simple hybride chez la levure sur ces éléments ont permis l’identification du facteur de transcription bHLH105/ILR3 comme régulateur potentiel d’AtFER1. Une caractérisation moléculaire et physiologique des mutants ilr3 a démontré l’implication de ce facteur dans la réponse des plantes à l’excès de fer. Elle a aussi mis en évidence qu’ILR3 avait un rôle central d’intégrateur dans l’homéostasie du fer chez les plantes. D’autre part, des données suggéraient qu’un long ARN non codant (At5g01595) pouvait potentiellement réguler AtFER1. Une caractérisation des mécanismes potentiellement impliqués a démontré que cette régulation n’était pas avérée.Les mécanismes moléculaires et physiologiques mis en place par les végétaux en réponse à une carence en fer sont relativement bien décrits. A l’inverse, peu d’informations sur la réponse des plantes à un « excès » de fer sont disponibles. Dans ce contexte, une expérience visant à décrypter, au niveau du transcriptome (puces à ADN), la dynamique de la réponse précoce (de quelques minutes à 2 heures) à un excès de fer a été mise en place. Une analyse de variance a été réalisée sur les données d’expression générées afin d’identifier les gènes dont l’expression est affectée par le traitement. Nous nous sommes plus particulièrement focalisés sur l’identification de facteurs de transcription, acteurs majeurs du maintien de l’homéostasie du fer. Parmi eux, WRKY33, WRKY40, ZAT10 et MYB51, tous liés à la réponse au ROS, semblent avoir un rôle clé dans la réponse précoce au fer.D’autre part, un mécanisme clé de l’homéostasie du fer est le prélèvement. Une précédente étude a montré que la nutrition en fer était facilitée par la synthèse et la sécrétion de composés phénoliques via le transporteur PDR9. Une caractérisation des mutants pdr9 a permis d’établir que d’une part (i) ses composés pouvaient être stockés dans les vacuoles des cellules racinaires, et d’autre part (ii) qu’ils permettaient l’entrée de fer via le système de prélèvement gouverné par le mécanisme FRO2/IRT1.Mes travaux de thèse ont permis d’apporter des éléments nouveaux sur les mécanismes moléculaires et physiologiques impliqués dans le contrôle de l’homéostasie du fer chez Arabidopsis.
... In contrast, phytic acid (PA), the organic form of P in seeds, has increased following global changes (e.g., elevated atmospheric CO 2 ) in various plant species including wheat [84]. Since PA is considered an anti-nutrient, increasing Pi content in grain while decreasing PA has become a trait of great interest [85]. ...
Phosphorus (P) is an essential macronutrient for plants to complete their life cycle. P taken up from the soil by the roots is transported to the rest of the plant and ultimately stored in seeds. This stored P is used during germination to sustain the nutritional demands of the growing seedling in the absence of a developed root system. Nevertheless, P deficiency, an increasing global issue, greatly decreases the vigour of afflicted seeds. To combat P deficiency, current crop production methods rely on heavy P fertilizer application, an unsustainable practice in light of a speculated decrease in worldwide P stocks. Therefore, the overall goal in optimizing P usage for agricultural purposes is both to decrease our dependency on P fertilizers and enhance the P-use efficiency in plants. Achieving this goal requires a robust understanding of how plants regulate inorganic phosphate (Pi) transport, during vegetative growth as well as the reproductive stages of development. In this short review, we present the current knowledge on Pi transport in the model plant Arabidopsis thaliana and apply the information towards the economically important cereal crop wheat. We highlight the importance of developing our knowledge on the regulation of these plants’ P transport systems and P accumulation in seeds due to its involvement in maintaining their vigour and nutritional quality. We additionally discuss further discoveries in the subjects this review discusses substantiate this importance in their practical applications for practical food security and geopolitical applications.
... The acquired soil P, is vital for plant development that supports the germination of wheat seedling and vegetative growth till maturity. The high accumulation of PA in grains also suggests the presence of a controlled regulatory mechanism for Pi-PA homeostasis that could be active during seed development stages 27,28 . In order to develop Pi-efficient wheat, it is therefore important to understand the process that involves the Pi homeostasis and allocation in the developing grains of wheat. ...
Approaches enabling efficient phosphorus utilization in crops are of great importance. In cereal crop like wheat, utilization of inorganic phosphate (Pi) is high and mature grains are the major sink for Pi utilization and storage. Research that addresses the importance of the Pi homeostasis in developing grains is limited. In an attempt to understand the Pi homeostasis in developing wheat grains, we identified twelve new phosphate transporters (PHT), these are phyologentically well distributed along with the members reported from Arabidopsis and rice. Enhanced expression of PHT1-subfamily genes was observed in roots subjected to the Pi starvation suggesting their active role in Pi homeostasis. Differential expression patterns of all the PHT genes during grain filling stages suggested their importance in the filial tissues. Additionally, high accumulation of Pi and total P in aleurone correlates well with the expression of TaPHTs and other phosphate starvation related genes. Tissue specific transcript accumulation of TaPHT1.1, TaPHT1.2, TaPHT1.4 in aleurone; TaPHT3.1 in embryo and TaPHT4.2 in the endosperm was observed. Furthermore, their transcript abundance was affected in low phytate wheat grains. Altogether, this study helps in expanding the knowledge and prioritize the candidate wheat Pi-transporters to modulate the Pi homeostasis in cereal grains.
... Although it has never been unambiguously demonstrated, several lines of evidence point to an association of Fe with phytates in these globoids. Indeed, Fe and phosphorus (P) do co-localize (Mary et al., 2015) and the overexpression of a bacterial phytase in Arabidopsis accelerates the process of remobilization of Fe from vacuoles after germination (Belgaroui et al., 2014). Fe complexes with phytate are insoluble and notoriously poorly available for animal nutrition (Clemens, 2014). ...
Plant iron (Fe) uptake relies to a large extent on the capacity of cells to control and extract Fe pools safely conserved in extracytoplasmic environments such as the apoplast and vacuoles, at least as much as on the transport machinery nested in plasma membranes. Recent studies on root and embryo Fe nutrition support this assertion and show that the root Fe‐deficiency response also includes the dynamic use of a large Fe reservoir bound to cell wall components in the root apoplast, secretion in the apoplast of phenolic compounds of the coumarin family, which solubilize Fe in calcareous soils, and inhibition of suberization of endodermal cells in order to allow apoplastic and transcellular radial transport of Fe. All of these responses are regulated by the stress hormones ethylene and abscisic acid ( ABA ), suggesting an integrated strategy within the root to adapt to Fe shortage. For its nutrition, the embryo has developed both an original uptake mechanism, in which ascorbate is effluxed to chemically reduce Fe ³⁺ to the transport‐competent Fe ²⁺ form, and an efficient strategy to control utilization of a large Fe pool in vacuoles. This review will attempt to summarize exciting new insights into the diverse routes that Fe takes to feed plant tissues.
Contents
Summary
521
I.
Introduction
521
II.
How does the root extracellular space contribute to Fe nutrition?
522
III.
Fe in the seed: acquisition and distribution strategies
523
IV.
Conclusions and open questions
524
Acknowledgements
525
References
525
... A BPP from B. subtilis (168 phyA) was constitutively expressed in tobacco under the control of the CaMV 35S promoter, and carrot extensin signal peptides were attached for root-specific secretion of 0.4% of total soluble leaf protein [172]. The bacterial phytase US417 was overexpressed in Arabidopsis resulting in a decrease in seed phytic acid content as well as an increase in sulphate and inorganic phosphate content, while also significantly remobilizing free iron without affecting seed germination [173]. ...
Phytases release inorganic phosphorus from phytic acid or its salts, which are the major forms of organic phosphorus in plant-derived food and feed ingredients. These enzymes have been widely used in animal food/feed to improve nutritional value and to reduce phosphorus pollution in animal waste. The effectiveness of phytases as food/feed additive is dependent on their in vitro properties such as pH/temperature optima as well as stability and resistance to the action of proteases. Furthermore, all commercial phytases certified for use as feed additives are produced by recombinant microorganisms with similar properties. The microbial sources for phytases span from fungi to bacteria. The bacterial phytases differ from fungal phytases in that their pH optima range from acidic to alkaline, and they are Ca2+ dependent and highly substrate specific. Furthermore, bacterial phytases are resistant to the action of proteases found in the gastrointestinal tract of monogastric animals. Bacterial phytases have a molecular mass of 37–55 kDa, and are usually active within pH and temperature ranges of 4.5–8.5 and 25–70 °C, respectively. Bacterial phytases are used in food and feed industries for improving nutritional quality, bread making, promotion of plant growth promotion and reduction of phosphorus pollutant levels in the environment.
... Perturbation of Pi and sulfate (SO4) homeostasis and signaling have been observed in lpa plants obtained by over-expressing a bacterial phytase in Arabidopsis [152]. These plants (PHY-US417) showed up to 50% and 45% increases in shoot Pi and SO4 concentrations, respectively, and improved plant growth with enhanced root growth capacity in Pi deficiency. ...
Most of the phosphorus in seeds is accumulated in the form of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, InsP6). This molecule is a strong chelator of cations important for nutrition, such as iron, zinc, magnesium, and calcium. For this reason, InsP6 is considered an antinutritional factor. In recent years, efforts to biofortify seeds through the generation of low phytic acid (lpa) mutants have been noteworthy. Moreover, genes involved in the biosynthesis and accumulation of this molecule have been isolated and characterized in different species. Beyond its role in phosphorus storage, phytic acid is a very important signaling molecule involved in different regulatory processes during plant development and responses to different stimuli. Consequently, many lpa mutants show different negative pleitotropic effects. The strength of these pleiotropic effects depends on the specific mutated gene, possible functional redundancy, the nature of the mutation, and the spatio-temporal expression of the gene. Breeding programs or transgenic approaches aimed at development of new lpa mutants must take into consideration these different aspects in order to maximize the utility of these mutants.
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which are required for photosynthetic processes and plant growth. How plants ensure enhanced growth despite meager P status remains enigmatic. In this study, we utilize genome-wide association analysis in Arabidopsis thaliana to identify a P transporter, PHT4;3, which mediates the reduction of P in chloroplasts at high CO2. Decreasing chloroplastic P fine-tunes the accumulation of a sugar-P metabolite, phytic acid, to support plant growth. Furthermore, we demonstrate that this adaptive mechanism is conserved in rice. Our results establish a mechanistic framework for sustainable food production against the backdrop of soaring CO2 levels across the world.
Phytases belong to the class of phosphohydrolases that begin the step-wise hydrolysis of phosphates from phytates. Phytates are a derivative of myo-inositol, which is the primary storage form of organic phosphorus in plant cells. Phytase has been used globally to diminish phosphorus pollution and to enhance nutrition in monogastrics. In this review, the classification, sources, and diversity of microbial phytases, and their practical applications, as well as supplementation of the soil with transgenic and wild types of microbial strains, which can release phytase to enhance phosphorus availability for plant uptake and reduce the need for fertilizers, are discussed. The overexpressed microbial phytases in transgenic plants enhance the growth capacity of co-cultivated plants and can therefore be employed in agricultural and biotechnological practices, such as intercropping. The introduction of phytases into the soil for improved plant growth and enhanced crop yield can be accomplished without extra cost. A diverse group of photoautotrophic microalgae can synthesize phytase and will likely be useful in many human food and animal industries.
To ensure the success of the new generation in annual species, the mother plant transfers a large proportion of the nutrients it has accumulated during its vegetative life to the next generation through its seeds. Iron (Fe) is required in large amounts to provide the energy and redox power to sustain seedling growth. However, free Fe is highly toxic as it leads to the generation of reactive oxygen species. Fe must, therefore, be tightly bound to chelating molecules to allow seed survival for long periods of time without oxidative damage. Nevertheless, when conditions are favorable, the seed's Fe stores have to be readily remobilized to achieve the transition toward active photosynthesis before the seedling becomes able to take up Fe from the environment. This is likely critical for the vigor of the young plant. Seeds constitute an important dietary source of Fe, which is essential for human health. Understanding the mechanisms of Fe storage in seeds is a key to improve their Fe content and availability in order to fight Fe deficiency. Seed longevity, germination efficiency and seedling vigor are also important traits that may be affected by the chemical form under which Fe is stored. In this review, we summarize the current knowledge on seed Fe loading during development, long-term storage and remobilization upon germination. We highlight how this knowledge may help seed Fe biofortification and discuss how Fe storage may affect the seed quality and germination efficiency.
In plants, while it is clear the homeostasis of different nutrients is highly dependent on each other, they are usually studied independent of each other. Given the paucity of past studies assessing the biological significance of mineral nutrient homeostasis interaction, very little is known about the genetic and molecular basis of such interactions. During my thesis, we made significant progress in going towards a more integrative comprehension of the problem and identify the molecular and genetic bases for a highly important and conserved nutrients interaction: the interaction of zinc and phosphate. First, using the phosphate transporter PHO1;H3 as entry molecular point, and by combining system biology and functional genomics approaches we have identified the functional module (four transcription factors) that regulates the expression and activity of PHO1;H3 under Zn deficiency leading to control Pi accumulation in shoots. Second, following our discovery of Lyso PhosphatidylCholine (PC) AcylTransferase 1 (LPCAT1) using genome-wide association studies (GWAS), we determined complete molecular pathway controlling the expression of this gene. We further uncovered a fundamental link between phospholipid metabolism and Pi-Zn homeostasis interaction via LPCAT1, which lays the foundations to explore a new role for Lyso-PC and PC in control of macro- and micronutrients homeostasis interaction. Taken together, our discoveries offer a new perspective on how to improve Pi content in plants, as our findings suggests that modulating the Zn-deficiency signalling pathway might be a good and simple approach for that.
Phosphate and sulfate are essential macro-elements for plant growth and development, and deficiencies in these mineral elements alter many metabolic functions. Nutritional constraints are not restricted to macro-elements. Essential metals such as zinc and iron have their homeostasis strictly genetically controlled, and deficiency or excess of these micro-elements can generate major physiological disorders, also impacting plant growth and development. Phosphate and sulfate on one hand, and zinc and iron on the other hand, are known to interact. These interactions have been partly described at the molecular and physiological levels, and are reviewed here. Furthermore the two macro-elements phosphate and sulfate not only interact between themselves but also influence zinc and iron nutrition. These intricated nutritional cross-talks are presented. The responses of plants to phosphorus, sulfur, zinc, or iron deficiencies have been widely studied considering each element separately, and some molecular actors of these regulations have been characterized in detail. Although some scarce reports have started to examine the interaction of these mineral elements two by two, a more complex analysis of the interactions and cross-talks between the signaling pathways integrating the homeostasis of these various elements is still lacking. However, a MYB-like transcription factor, PHOSPHATE STARVATION RESPONSE 1, emerges as a common regulator of phosphate, sulfate, zinc, and iron homeostasis, and its role as a potential general integrator for the control of mineral nutrition is discussed.
Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13 % over a temperature range of 40–75 °C at pH 7.0, respectively. The k
cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.
Phytic acid (PA) is poorly digested by humans and monogastric animals and negatively affects human/animal nutrition and the environment. Rice mutants with reduced PA content have been developed but are often associated with reduced seed weight and viability, lacking breeding value. In the present study, a new approach was explored to reduce seed PA while attaining competitive yield. The OsMRP5 gene, of which mutations are known to reduce seed PA as well as seed yield and viability, was down-regulated specifically in rice seeds by using an artificial microRNA driven by the rice seed specific promoter Ole18. Seed PA contents were reduced by 35.8-71.9 % in brown rice grains of transgenic plants compared to their respective null plants (non-transgenic plants derived from the same event). No consistent significant differences of plant height or number of tillers per plant were observed, but significantly lower seed weights (up to 17.8 % reduction) were detected in all transgenic lines compared to null plants, accompanied by reductions of seed germination and seedling emergence. It was observed that the silencing of the OsMRP5 gene increased the inorganic P (Pi) levels (up to 7.5 times) in amounts more than the reduction of PA-P in brown rice. This indicates a reduction in P content in other cellular compounds, such as lipids and nucleic acids, which may affect overall seed development. Put together, the present study demonstrated that seed specific silencing of OsMRP5 could significantly reduce the PA content and increase Pi levels in seeds; however, it also significantly lowers seed weight in rice. Discussions were made regarding future directions towards producing agronomically competitive and nutritionally valuable low PA rice.
Phosphate (Pi) is an essential nutrient for plants but is normally fixed in soil, which limits plant growth and reproduction. In response
to low availability of Pi, shoots and roots react differently but cooperatively to improve Pi acquisition from the rhizosphere and adjust Pi distribution and metabolism within plants. Shoot and root responses are coordinated by the trafficking of various kinds of
systemic signals through the vasculature. Mutual communication between different tissues is necessary to integrate the environmental
stimuli with the internal cues at the whole-plant level. Different approaches have been used to monitor or manipulate components
in the vascular stream to reveal several candidates of systemic signals from roots or shoots, including photosynthates, phytohormones,
microRNAs, and Pi. In addition, the downstream signalling pathways mediated by these signals have been discovered. The crosstalk among different
signalling pathways has been revealed, showing the complexity of the Pi signalling network. In this review, we summarize the approaches used for studying systemic signalling and discuss recent
progress and challenges in investigating the systemic signalling pathway that integrates Pi starvation responses to maintain Pi at physiological concentrations. Knowledge gained from this study may help improve the phosphorus use efficiency of crops.
Background
Phytic acid (InsP6) is considered as the major source of phosphorus and inositol phosphates in cereal grains. Reduction of phytic acid level in cereal grains is desirable in view of its antinutrient properties to maximize mineral bioavailability and minimize the load of phosphorus waste management. We report here RNAi mediated seed-specific silencing of myo-inositol-3-phosphate synthase (MIPS) gene catalyzing the first step of phytic acid biosynthesis in rice. Moreover, we also studied the possible implications of MIPS silencing on myo-inositol and related metabolism, since, first step of phytic acid biosynthesis is also the rate limiting step of myo-inositol synthesis, catalyzed by MIPS.
Results
The resulting transgenic rice plants (T3) showed a 4.59 fold down regulation in MIPS gene expression, which corresponds to a significant decrease in phytate levels and a simultaneous increment in the amount of inorganic phosphate in the seeds. A diminution in the myo-inositol content of transgenic plants was also observed due to disruption of the first step of phytic acid biosynthetic pathway, which further reduced the level of ascorbate and altered abscisic acid (ABA) sensitivity of the transgenic plants. In addition, our results shows that in the transgenic plants, the lower phytate levels has led to an increment of divalent cations, of which a 1.6 fold increase in the iron concentration in milled rice seeds was noteworthy. This increase could be attributed to reduced chelation of divalent metal (iron) cations, which may correlate to higher iron bioavailability in the endosperm of rice grains.
Conclusion
The present study evidently suggests that seed-specific silencing of MIPS in transgenic rice plants can yield substantial reduction in levels of phytic acid along with an increase in inorganic phosphate content. However, it was also demonstrated that the low phytate seeds had an undesirable diminution in levels of myo-inositol and ascorbate, which probably led to sensitiveness of seeds to abscisic acid during germination. Therefore, it is suggested that though MIPS is the prime target for generation of low phytate transgenic plants, down-regulation of MIPS can have detrimental effect on myo-inositol synthesis and related pathways which are involved in key plant metabolism.
Phytic acid (InsP6) is considered to be the major source of phosphorus and inositol phosphates in most cereal grains. However, InsP6 is not utilized efficiently by monogastric animals due to lack of phytase enzyme. Furthermore, due to its ability to chelate mineral cations, phytic acid is considered to be an antinutrient that renders these minerals unavailable for absorption. In view of these facts, reducing the phytic acid content in cereal grains is a desired goal for the genetic improvement of several crops. In the present study, we report the RNAi-mediated seed-specific silencing (using the Oleosin18 promoter) of the IPK1 gene, which catalyzes the last step of phytic acid biosynthesis in rice. The presence of the transgene cassette in the resulting transgenic plants was confirmed by molecular analysis, indicating the stable integration of the transgene. The subsequent T4 transgenic seeds revealed 3.85-fold down-regulation in IPK1 transcripts, which correlated to a significant reduction in phytate levels and a concomitant increase in the amount of inorganic phosphate (Pi). The low-phytate rice seeds also accumulated 1.8-fold more iron in the endosperm due to the decreased phytic acid levels. No negative effects were observed on seed germination or in any of the agronomic traits examined. The results provide evidence that silencing of IPK1 gene can mediate a substantial reduction in seed phytate levels without hampering the growth and development of transgenic rice plants.
A yeast one-hybrid screening allowed the selection of PHR1 as a factor that interacted with the AtFer1 ferritin gene promoter. In mobility shift assays, PHR1 and its close homologue PHL1 (PHR1-like 1) interact with Element 2 of the AtFer1 promoter, containing a P1BS (PHR1 binding site). In a loss of function mutant for genes encoding PHR1 and PHL1 (phr1 phl1 mutant), the response of AtFer1 to phosphate starvation was completely lost, showing that the two transcription factors regulate AtFer1 expression upon phosphate starvation. This regulation does not involve the IDRS (iron-dependent regulatory sequence) present in the AtFer1 promoter and involved in the iron-dependent regulation. The phosphate starvation response of AtFer1 is not linked to the iron status of plants and is specifically initiated by phosphate deficiency. Histochemical localization of iron, visualized by Perls DAB staining, was strongly altered in a phr1 phl1 mutant, revealing that both PHR1 and PHL1 are major factors involved in the regulation of iron homeostasis.
Background: Physiological evidences have linked phosphate and iron nutrition in plants.
Results: Both PHR1 and PHL1 interact with AtFer1 promoter region and regulate its expression in an iron-independent manner.
Conclusion: A molecular link exists between the control of iron and of phosphate homeostasis.
Significance: PHR1 and PHL1 play a critical role in the regulation of both phosphate and iron homeostasis.
Comparison of wild type and mutants of Arabidopsis thaliana offers an opportunity to study the genetic control of nutrient storage in seeds. We used energy dispersive X-ray analysis to determine the elements present and their relative amounts in globoids of dry wild-type seeds, as well as seeds of a reduced total P uptake mutant (pho1), a phosphate accumulator (pho2), and a metal accumulator (man1). Globoids are spherical inclusions, rich in phytate that function as a store for inositol, P, K, Mg, Ca, Fe, and Zn. Key findings of this study were the following: (i) globoids in protein bodies from nine different tissues and (or) organs in dry Arabidopsis thaliana seeds contained P, K, Mg, and Ca, and sometimes traces of Fe and Zn; (ii) globoids contained higher Ca and lower Mg amounts than occur in globoids in seeds of most other plant species; (iii) globoids in comparable tissue and (or) organ regions of seeds were very similar in elemental composition for wild type and all mutant plants.Key words: Arabidopsis, dry seeds, phytate, mineral nutrient mutants, phosphorus, globoids.
Background: Sulfate and phosphate are both vital macronutrients required for plant growth and development. Despite evidence for interaction between sulfate and phosphate homeostasis, no transcriptional factor has yet been identified in higher plants that affects, at the gene expression and physiological levels, the response to both elements. This work was aimed at examining whether PHR1, a transcription factor previously shown to participate in the regulation of genes involved in phosphate homeostasis, also contributed to the regulation and activity of genes involved in sulfate inter-organ transport. Results: Among the genes implicated in sulfate transport in Arabidopsis thaliana, SULTR1;3 and SULTR3;4 showed up-regulation of transcripts in plants grown under phosphate-deficient conditions. The promoter of SULTR1;3 contains a motif that is potentially recognizable by PHR1. Using the phr1 mutant, we showed that SULTR1;3 up-regulation following phosphate deficiency was dependent on PHR1. Furthermore, transcript up-regulation was found in phosphate-deficient shoots of the phr1 mutant for SULTR2;1 and SULTR3;4, indicating that PHR1 played both a positive and negative role on the expression of genes encoding sulfate transporters. Importantly, both phr1 and sultr1;3 mutants displayed a reduction in their sulfate shoot-to-root transfer capacity compared to wild-type plants under phosphate-deficient conditions. Conclusions: This study reveals that PHR1 plays an important role in sulfate inter-organ transport, in particular on the regulation of the SULTR1;3 gene and its impact on shoot-to-root sulfate transport in phosphate-deficient plants. PHR1 thus contributes to the homeostasis of both sulfate and phosphate in plants under phosphate deficiency. Such a function is also conserved in Chlamydomonas reinhardtii via the PHR1 ortholog PSR1.
Iron deficiency is the most widespread micronutrient deficiency world-wide. A major cause is the poor absorption of iron from
cereal and legume-based diets high in phytic acid. We have explored three approaches for increasing the amount of iron absorbed
from rice-based meals. We first introduced a ferritin gene from Phaseolus vulgaris into rice grains, increasing their iron content up to two-fold. To increase iron bioavailability, we introduced a thermotolerant
phytase from Aspergillus fumigatus into the rice endosperm. In addition, as cysteine peptides are considered a major enhancer of iron absorption, we overexpressed
the endogenous cysteine-rich metallothionein-like protein. The content of cysteine residues increased about seven-fold and
the phytase level in the grains about 130-fold, giving a phytase activity sufficient to completely degrade phytic acid in
a simulated digestion experiment. High phytase rice, with an increased iron content and rich in cysteine-peptide, has the
potential to greatly improve iron nutrition in rice-eating populations.
In most plant seeds, phosphorus is stored primarily as myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP6; phytic acid). Reducing the phytic acid content of seeds is a major breeding target, both to increase the availability of mineral nutrients and to decrease the environmental load of phosphorus. The first step in phytic acid biosynthesis and inositol metabolism is catalyzed by 1d-myo-inositol 3-phosphate (Ins(3)P1) synthase. In this study, we aimed to reduce phytic acid levels in rice seeds by manipulating the expression of the rice Ins(3)P1 synthase gene RINO1 using transgenic methods. RINO1 cDNA was transformed into rice plants in the antisense orientation under the control of the rice major storage protein glutelin GluB-1 promoter. The T4 generation of a stable transgenic line that contained four copies of the transgene showed little morphological differences compared to non-transgenic rice. In the T5 seeds of this line, severe reductions in RINO1 protein levels were observed during the late maturing stages of ripening. Most of the T5 seeds contained higher amounts of inorganic phosphates (Pi), without a reduction in total phosphorus levels, compared to non-transgenic seeds. Ion chromatography analysis suggested that the increase in available Pi is accompanied by a molar-equivalent decrease in phytic acid P. The expression patterns of RINO1 and GluB-1 were examined by quantitative real-time reverse transcriptase-polymerase chain-reaction (RT-PCR). Potential strategies for further molecular breeding to reduce phytic acid levels in seeds are discussed.
In recent years, the increasing knowledge on the molecular mechanisms underlying mineral uptake, transport, homeostasis and
deposition within plants, has paved the way for a more targeted approach to improving the nutrient status of crop plants based
on biotechnology. In the present paper we will briefly review existing knowledge on the distribution and transport pathways
of iron in the two small grained cereals, barley and wheat, and focus on the efforts made to increase the iron content in
cereals in general. However, mineral content is not the only factor of relevance for improving the nutritional status of poor
populations. It is thus well documented that a number of plant components can act either as promoters or inhibitors of mineral
uptake in the human digestive system (Frossard et al. J Sci Food Agric 80, 817-879 2000; Brinch-Pedersen et al. J Cereal Sci 46, 308-326 2007). The nutritional impact of increasing mineral content accordingly has to be seen in the context of mineral bioavailability.
Finally, we will briefly report on recent data from barley, where laser capture microdissection of the different grain tissues
combined with gene expression profiling has provided some insight into metal transport and deposition (Tauris et al. 2009). In the present paper we will provide a tentative and preliminary roadmap for iron trafficking in the barley grain.
The main source of phosphorus for plants is inorganic phosphate (Pi), which is characterized by its poor availability and low mobility. Uptake of this element from the soil relies heavily upon the PHT1 transporters, a specific family of plant plasma membrane proteins that were identified by homology with the yeast PHO84 Pi transporter. Since the discovery of PHT1 transporters in 1996, various studies have revealed that their function is controlled by a highly complex network of regulation. This review will summarize the current state of research on plant PHT1 multigenic families, including physiological, biochemical, molecular, cellular, and genetics studies.
Sulfate and phosphate are both vital macronutrients required for plant growth and development. Despite evidence for interaction between sulfate and phosphate homeostasis, no transcriptional factor has yet been identified in higher plants that affects, at the gene expression and physiological levels, the response to both elements. This work was aimed at examining whether PHR1, a transcription factor previously shown to participate in the regulation of genes involved in phosphate homeostasis, also contributed to the regulation and activity of genes involved in sulfate inter-organ transport.
Among the genes implicated in sulfate transport in Arabidopsis thaliana, SULTR1;3 and SULTR3;4 showed up-regulation of transcripts in plants grown under phosphate-deficient conditions. The promoter of SULTR1;3 contains a motif that is potentially recognizable by PHR1. Using the phr1 mutant, we showed that SULTR1;3 up-regulation following phosphate deficiency was dependent on PHR1. Furthermore, transcript up-regulation was found in phosphate-deficient shoots of the phr1 mutant for SULTR2;1 and SULTR3;4, indicating that PHR1 played both a positive and negative role on the expression of genes encoding sulfate transporters. Importantly, both phr1 and sultr1;3 mutants displayed a reduction in their sulfate shoot-to-root transfer capacity compared to wild-type plants under phosphate-deficient conditions.
This study reveals that PHR1 plays an important role in sulfate inter-organ transport, in particular on the regulation of the SULTR1;3 gene and its impact on shoot-to-root sulfate transport in phosphate-deficient plants. PHR1 thus contributes to the homeostasis of both sulfate and phosphate in plants under phosphate deficiency. Such a function is also conserved in Chlamydomonas reinhardtii via the PHR1 ortholog PSR1.
Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.
Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, or Ins P6) is the most abundant storage form of P in seeds, yet indigestible by humans and nonruminant livestock. A wheat (Triticum aestivum L.) mutant is described herein with greatly reduced seed phytic acid P but little change in seed total P, similar to lpa1-type mutants described in other grain species. One nonlethal mutant from 562 ethyl-methanesulfonate (EMS) mutagenized M2 lines was identified with a high inorganic phosphate (HIP) phenotype and designated Js-12-LPA. Js-12-LPA homozygotes produced seed in which phytic acid P represented 48.2% of seed total P, in contrast to 74.7% of seed total P in nonmutant or wild-type control, Js-12-WT. The inorganic portion of seed P was increased from 9.1% in Js-12-WT to 50.1% in Js-12-LPA, with little effect on total seed P. Weight distributions among milling fractions were similar for the Js-12-LPA and Js-12-WT genotypes. The low phytic acid trait altered the distribution of total P within the kernel, increasing the P content of the central endosperm and decreasing the P content of the bran. The low phytic acid trait decreased the phytic acid concentration in the bran by 43% and increased the inorganic P concentration in the bran nearly four-fold. Inheritance data of F2 and F(4:6) families was inconsistent with a single-gene mutation and suggests the involvement of two or more genes. This low phytic acid wheat mutant is a genetic resource for studying the biology of seed phytic acid metabolism and wheat quality improvement.
Deciphering how cellular iron (Fe) pools are formed, where they are localized, and which ones are remobilized represents an important challenge to better understand Fe homeostasis. The recent development of imaging techniques, adapted to plants, has helped gain insight into these events. We have analyzed the localization of Fe during embryo development in Arabidopsis (Arabidopsis thaliana) with an improved histochemical staining based on Perls coloration intensified by a second reaction with diaminobenzidine and hydrogen peroxide. The procedure, quick to set up and specific for Fe, was applied directly on histological sections, which dramatically increased its subcellular resolution. We have thus unambiguously shown that in dry seeds Fe is primarily stored in the endodermis cell layer, within the vacuoles, from which it is remobilized during germination. In the vit1-1 mutant, in which the Fe pattern is disturbed, Fe is stored in vacuoles of cortex cells of the hypocotyl/radicle axis and in a single subepidermal cell layer in the cotyledons. During the early stages of embryo development, Fe is evenly distributed in the cells of both wild-type and vit1-1 mutants. Fe eventually accumulates in endodermal cells as the vascular system develops, a process that is impaired in vit1-1. Our results have uncovered a new role for the endodermis in Fe storage in the embryo and have established that the Perls/diaminobenzidine staining is a method of choice to detect Fe in plant tissues and cells.
Fifty crossbred barrows with an average initial age of 31 d and BW of 9.94 kg were used in a 28-d experiment to evaluate the effect of a low-phytic acid (LPA) barley mutant (M) M955, a near-isogenic progeny of the normal barley (NB) cultivar Harrington with about 90% less phytate than NB, to increase the utilization of Fe, Zn, and Cu compared with diets containing NB. The response criteria were growth performance, hematocrit volume, metacarpal bone characteristics, and the apparent absorption, retention, and excretion of Zn and Cu. The 2 barley cultivars (NB and M955) and the 5 trace mineral (TM) treatment concentrations of Fe and Zn (0, 25, 50, 75, and 100% of the requirement as FeSO(4) and ZnSO(4)) and Cu (0, 40, 80, 120, and 160% of the requirement as CuSO(4)) made 10 treatments in a factorial arrangement. Available P was equalized at 0.33% in all diets by adding monosodium phosphate to the basal diet containing NB, and all diets contained 0.65% Ca. Diets were adequate in all other nutrients. Barley and soybean meal were the only sources of phytate in the practical diets that also contained spray-dried whey. The barrows were fed the diets to appetite in meal form twice daily in individual metabolism crates. There were no barley cultivar x TM treatment interactions, and there were no differences between the NB and M955 barley cultivars for any of the response criteria measured. However, for the TM treatments, there were linear increases (P < or = 0.05) in ADFI, ADG, hematocrit volume, metacarpal bone breaking strength and ash weight, and the apparent absorption, retention, and excretion (mg/d) of Zn and Cu. In conclusion, the LPA barley had no effect on the response criteria in this experiment, apparently because of the small increase in the availability of the endogenous trace minerals in the practical diets containing M955 compared with NB. However, increasing the supplementation of Fe and Zn from 0 to 100% (160% for Cu) of the requirement resulted in linear increases in growth performance, hematocrit volume, metacarpal bone strength and ash weight, and the apparent absorption, retention, and excretion of Zn and Cu. Therefore, these results indicate that the inorganic trace mineral supplementation of practical diets for young pigs should not be less than the National Research Council requirements for swine.
Phytic acid is considered as one of the major antinutritional compounds in cereal and legume seeds. The development of lpa (low phytic acid) grains, resulting in increased mineral cation availability, is considered a major goal in the improvement of the nutritional quality of seed crops, especially those largely consumed in developing countries. From a mutagenized population of common bean we isolated a homozygous lpa mutant line (lpa-280-10) showing, compared to wild type, a 90% reduction of phytic acid, a 25% reduction of raffinosaccharides and a much higher amount of free or weakly bound iron cations in the seed. Genetic analysis showed that the lpa character is due to a recessive mutation that segregates in a monogenic, Mendelian fashion. Germination tests performed using varying ageing or stress conditions, clearly showed that the bean line lpa-280-10 has a better germination response than the wild type. These data, together with those obtained from 2 years of agronomic trials showing that the mutant seed yield is close to that of its parents and other evidence, indicate that the new lpa-280-10 mutation might be the first devoid of visible macroscopic negative effects in plants, pods and seeds.
A maize mutant defective in the synthesis of phytic acid during seed maturation was used as a tool to study the consequences
of the lack of this important reserve substance on seed survival. Data on germinability, free iron level, free radical relative
abundance, protein carbonylation level, damage to DNA, degree of lipid peroxidation, α- and γ-tocopherol amount and antioxidant
capacity were recorded on seeds of maize B73 and of an isogenic low phytic acid mutant (lpa1-241), either unaged or incubated for 7 d in accelerated ageing conditions (46 °C and 100% relative humidity). The lpa1-241 mutant, compared to wild type (wt), showed a lower germination capacity, which decreased further after accelerated ageing.
Whole lpa1-241 mutant kernels contained about 50% more free or weakly bound iron than wt ones and showed a higher content of free radicals,
mainly concentrated in embryos; in addition, upon accelerated ageing, lpa1-241 seed proteins were more carbonylated and DNA was more damaged, whereas lipids did not appear to be more peroxidated, but
the γ-tocopherol content was decreased by about 50%. These findings can be interpreted in terms of previously reported but
never proven antioxidant activity of phytic acid through iron complexation. Therefore, a novel role in plant seed physiology
can be assigned to phytic acid, that is, protection against oxidative stress during the seed's life span. As in maize kernels
the greater part of phytic acid (and thus of metal ions) is concentrated in the embryo, its antioxidant action may be of particular
relevance in this crop.
*INRA, Centre de Jouy-en-Josas (FRA) Diffusion du document : INRA, Centre de Jouy-en-Josas (FRA)
An understanding of the mineral nutrition of plants is of fundamental importance in both basic and applied plant sciences. The Second Edition of this book retains the aim of the first in presenting the principles of mineral nutrition in the light of current advances. This volume retains the structure of the first edition, being divided into two parts: Nutritional Physiology and Soil-Plant Relationships. In Part I, more emphasis has been placed on root-shoot interactions, stress physiology, water relations, and functions of micronutrients. In view of the worldwide increasing interest in plant-soil interactions, Part II has been considerably altered and extended, particularly on the effects of external and interal factors on root growth and chapter 15 on the root-soil interface. The second edition will be invaluable to both advanced students and researchers.
A single solution reagent is described for the determination of phosphorus in sea water. It consists of an acidified solution of ammonium molybdate containing ascorbic acid and a small amount of antimony. This reagent reacts rapidly with phosphate ion yielding a blue-purple compound which contains antimony and phosphorus in a 1:1 atomic ratio. The complex is very stable and obeys Beer's law up to a phosphate concentration of at least 2 μg/ml.The sensitivity of the procedure is comparable with that of the stannous chloride method. The salt error is less than 1 %.
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.
Real-time reverse transcription followed by polymerase chain reaction (RT–PCR) is the most suitable method for the detection
and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative
expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene.
Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and
the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the
calculation of relative expression ratio allow only for the determination of a single transcription difference between one
control and one sample. Therefore a new software tool was established, named REST© (relative expression software tool), which
compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target
genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample
and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance
by a randomisation test. Herein, development and application of REST© is explained and the usefulness of relative expression
in real-time PCR using REST© is discussed. The latest software version of REST© and examples for the correct use can be downloaded
at http://www.wzw.tum.de/gene-quantification/.
Phytic acid (myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate or Ins P6) typically represents approximately 75% to 80% of maize (Zea mays) seed total P. Here we describe the origin, inheritance, and seed phenotype of two non-lethal maize low phytic acid mutants, lpa1-1 and lpa2-1. The loci map to two sites on chromosome 1S. Seed phytic acid P is reduced in these mutants by 50% to 66% but seed total P is unaltered. The decrease in phytic acid P in mature lpa1-1 seeds is accompanied by a corresponding increase in inorganic phosphate (Pi). In mature lpa2-1 seed it is accompanied by increases in Pi and at least three othermyo-inositol (Ins) phosphates (and/or their respective enantiomers): d-Ins(1,2,4,5,6) P5;d-Ins (1,4,5,6) P4; andd-Ins(1,2,6) P3. In both cases the sum of seed Pi and Ins phosphates (including phytic acid) is constant and similar to that observed in normal seeds. In both mutants P chemistry appears to be perturbed throughout seed development. Homozygosity for either mutant results in a seed dry weight loss, ranging from 4% to 23%. These results indicate that phytic acid metabolism during seed development is not solely responsible for P homeostasis and indicate that the phytic acid concentration typical of a normal maize seed is not essential to seed function.
Inorganic phosphate (Pi) and zinc (Zn) are two essential nutrients for plant growth. In soils, these two minerals are either
present in low amounts or are poorly available to plants. Consequently, worldwide agriculture has become dependent on external
sources of Pi and Zn fertilizers to increase crop yields. However, this strategy is neither economically nor ecologically
sustainable in the long term, particularly for Pi, which is a non-renewable resource. To date, research has emphasized the
analysis of mineral nutrition considering each nutrient individually, and showed that Pi and Zn homeostasis is highly regulated
in a complex process. Interestingly, numerous observations point to an unexpected interconnection between the homeostasis
of the two nutrients. Nevertheless, despite their fundamental importance, the molecular bases and biological significance
of these interactions remain largely unknown. Such interconnections can account for shortcomings of current agronomic models
that typically focus on improving the assimilation of individual elements. Here, current knowledge on the regulation of the
transport and signalling of Pi and Zn individually is reviewed, and then insights are provided on the recent progress made
towards a better understanding of the Zn–Pi homeostasis interaction in plants.
The rice low phytic acid (lpa) mutant Os-lpa-XS110-1(XS-lpa) has ~45 % reduction in seed phytic acid (PA) compared with the wild-type cultivar Xiushui 110. Previously, a single recessive gene mutation was shown to be responsible for the lpa phenotype and was mapped to a region of chromosome 3 near OsMIK (LOC_Os03g52760) and OsIPK1 (LOC_Os03g51610), two genes involved in PA biosynthesis. Here, we report the identification of a large insert in the intron of OsMIK in the XS-lpa mutant. Sequencing of fragments amplified through TAIL-PCRs revealed that the insert was a derivative of the LINE retrotransposon gene LOC_Os03g56910. Further analyses revealed the following characteristics of the insert and its impacts: (1) the inserted sequence of LOC_Os03g56910 was split at its third exon and rejoined inversely, with its 5' and 3' flanking sequences inward and the split third exon segments outward; (2) the LOC_Os03g56910 remained in its original locus in XS-lpa, and the insertion probably resulted from homologous recombination repair of a DNA double strand break; (3) while the OsMIK transcripts of XS-lpa and Xiushui 110 were identical, substantial reductions of the transcript abundance (~87 %) and the protein level (~60 %) were observed in XS-lpa, probably due to increased methylation in its promoter region. The above findings are discussed in the context of plant mutagenesis, epigenetics and lpa breeding.
Comparison of wild type and mutants of Arabidopsis thaliana offers an opportunity to study the genetic control of nutrient storage in seeds. We used energy dispersive X-ray analysis to determine the elements present and their relative amounts in globoids of dry wild-type seeds, as well as seeds of a reduced total P uptake mutant (pho1), a phosphate accumulator (pho2), and a metal accumulator (man1). Globoids are spherical inclusions, rich in phytate that function as a store for inositol, P, K, Mg, Ca, Fe, and Zn. Key findings of this study were the following: (i) globoids in protein bodies from nine different tissues and (or) organs in dry Arabidopsis thaliana seeds contained P, K, Mg, and Ca, and sometimes traces of Fe and Zn; (ii) globoids contained higher Ca and lower Mg amounts than occur in globoids in seeds of most other plant species; (iii) globoids in comparable tissue and (or) organ regions of seeds were very similar in elemental composition for wild type and all mutant plants.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
Phytases are phosphohydrolytic enzymes that initiate stepwise removal of phosphate from phytate. Simple-stomached species such as swine, poultry, and fish require extrinsic phytase to digest phytate, the major form of phosphorus in plant-based feeds. Consequently, this enzyme is supplemented in these species’ diets to decrease their phosphorus excretion, and it has emerged as one of the most effective and lucrative feed additives. This chapter provides a comprehensive review of the evolving course of phytase science and technology. It gives realistic estimates of the versatile roles of phytase in animal feeding, environmental protection, rock phosphorus preservation, human nutrition and health, and industrial applications. It elaborates on new biotechnology and existing issues related to developing novel microbial phytases as well as phytase-transgenic plants and animals. And it targets critical and integrated analyses on the global impact, novel application, and future demand of phytase in promoting anim...
myo-Inositol-1,2,3,4,5,6-hexakisphosphate (Ins P(6)) was first described as an abundant form of phosphorus in plant seeds and other plant tissues and dubbed "phytic acid". Subsequently it was found to be a common constituent in eukaryotic cells, its metabolism a basic component of cellular housekeeping. In addition to phosphate, myo-inositol (Ins) and mineral storage and retrieval in plant organs and tissues, other roles for Ins P(6) include service as a major metabolic pool in Ins phosphate and pyrophosphate pathways involved in signaling and regulation; possibly as an effector or ligand in these processes; as a form of energy currency and in ATP regeneration; in RNA export and DNA repair; and as an anti-oxidant. The relatively recent demonstration that pyrophosphate-containing derivatives of Ins P(6) can function as phosphate donors in the regeneration of ATP is reminiscent of the proposal, made four decades ago in studies of seed development, that Ins P(6) itself may serve in this function. Studies of Ins P(6) in non-plant systems rarely include the consideration that this compound might represent a significant fraction of cellular P; cellular phosphate nutrition has been viewed as either not interesting or of little importance. However, there may be few fundamental differences among diverse eukaryotes in both the metabolic pathways involving Ins P(6) and the spectrum of possible roles for it and its metabolites.
This review provides a current summary of the literature concerning various aspects of phytate. These include data relative to its chemical structure, its occurrence in numerous cereals and legumes, the role of phytase, and the influence of food-processing conditions on phytate/phytase activity. In addition, the nutritional significance of phytate with regard to mineral binding abilities and methods commonly used for the analysis of phytate are also discussed.
A rapid method is described for the colorimetric determination of 1.5–15 μg phytate phosphorus in concentrations as low as 3 μg ml−1 in extracts of cereal grains and cereal products. The phytic acid is precipitated with an acidic iron-III-solution of known iron content. The decrease of iron in the supernatant is a measure for the phyticacid content.
To investigate the uptake and long-distance translocation of sulphate in plants, we have characterized three cell-type-specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana. Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high-affinity sulphate transporter (Km for sulphate 3.6 ± 0.6 μm), whereas Sultr2;1 and Sultr2;2 encode low-affinity sulphate transporters (Km for sulphate 0.41 ± 0.07 m m and ≥ 1.2 m m, respectively). In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots. β-glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem. Expression of the Sultr2;2 promoter–GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells. Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves. The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short-term stress caused by withdrawal of sulphate. Addition of selenate in the sulphate-sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots. Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.
Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin- and ethylene-related mutants. In a previous study,Masucci & Schiefelbein (1996) showed that neither the auxin response mutationsaux1 and axr1nor the ethylene response mutationsetr1 and ein2have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild-type,axr1andetr1seedlings with the synthetic auxin, 2,4-D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore,axr1seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.
A large proportion of soil phosphorus (P) exists as organic compounds, of which phytic acid (IHP) is the dominant form. To generate transgenic plants capable of utilizing exogenous IHP, β-propeller phytase from Bacillus subtilis (168phyA) was constitutively expressed in tobacco and Arabidopsis, and was shown to be secreted from their roots. In tobacco, phytase activities in transgenic leaf and root extracts were seven to nine times higher than those in wild-type extracts; whereas, the extracellular phytase activities of transgenic plants were enhanced by four to six times. In sterile hydroponic culture using 1 mM Na-IHP as the sole P source, the transgenic tobacco lines accumulated 1.7–2.2 times more shoot biomass than the wild-type plants after 30 days of growth, concomitant with a 27–36% increase in shoot P concentration. Similar results were observed from the transgenic Arabidopsis. Our work on the exudation of recombinant Bacillus phytase from plant roots may offer a new perspective on mobilizing soil phytate into inorganic phosphate for plant uptake.
A single solution reagent is described for the determination of phosphorus in sea water. It consists of an acidified solution of ammonium molybdate containing ascorbic acid and a small amount of antimony. This reagent reacts rapidly with phosphate ion yielding a blue-purple compound which contains antimony and phosphorus in a 1:1 atomic ratio. The complex is very stable and obeys Beer's law up to a phosphate concentration of at least 2 μg/ml.The sensitivity of the procedure is comparable with that of the stannous chloride method. The salt error is less than 1 %.RésuméUne méthode spectrophotométrique est décrite pour le dosage du phosphate dans l'eau de mer, an moyen de molybdate d'ammonium, en présence d'acide ascorbique et d'antimoinc. Il se forme rapidement un composé violet bleu, renfermant antimoine et phosphore dans un rapport atomique de 1:1.ZusammenfassungBeschreibung einer Methode zur Bestimmung von Phosphat in Mecrwasser mit Hilfe von Ammoniummolybdat in Gegenwart von Ascorbinsäure und Antimon. Der gebildete blau-violette Komplex wird spektrophotometrisch gemessen.
About 75% of seed total phosphorus (P) is found in a single compound, phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate or InsP6). Phytic acid is not efficiently utilized by monogastric animals (poultry, swine, fish), which creates phosphorus management and environmental impact problems in animal production. Phytic acid also functions as an antinutrient when consumed in human and animal diets. These problems can be addressed via feed or food supplementation with P and other minerals or phytase, or more efficiently and sustainably at their source by crop breeding or bioengineering of low-phytic acid/high-available P crops. However, since phytic acid and its synthetic pathways are central to a number of metabolic, developmental and signaling pathways important to plant function and productivity, low-phytate can translate into low-yield or stress susceptibility. The biological functions of phytic acid and identification of genetic resources and strategies useful in engineering high-yielding, stress-tolerant low-phytate germplasm will be reviewed here. One promising approach that can avoid undesirable outcomes due to impacts on phytic acid metabolism is to engineer “high-phytase” seeds. In contrast to the issue of seed phytic acid, there has been relatively little interest in seed total P as a trait of agricultural importance. However, seed total P is very important to the long-term goal of sustainable and environmentally friendly agricultural production. Certain low-phytate genotypes are also “low-total P”, which might represent the ideal seed P trait for nearly all end-uses, including uses in ruminant and non-ruminant feeds and in biofuels production. Future research directions will include screening for additional genetic resources such as seed total P mutants.
Phytases are a group of enzymes capable of releasing phosphates from phytates, one of the major forms of phosphorus (P) in animal feeds of plant origin. These enzymes have been widely used in animal feed to improve phosphorus nutrition and to reduce phosphorus pollution in animal waste. This review covers the basic nomenclature and crystal structures of phytases and emphasizes both the protein engineering strategies used for the development of new, effective phytases with improved properties and the potential biotechnological applications of phytases.
The majority of phosphorus (P) in seeds is found in phytic acid (InsP6) which accumulates as the mixed salt phytate. InsP6 is generally considered to be an anti-nutrient and the development of low phytic acid (lpa) seed crops is of significant interest. We have employed a reverse genetics approach to examine the impact of disrupting genes involved in inositol phosphate metabolism on Arabidopsis seed InsP6 levels. Our analysis revealed that knockout mutations in three genes (AtITPK1, AtITPK4, and AtMIK/At5g58730) reduced seed InsP6 in addition to knockouts of four previously reported genes (AtIPK1, AtIPK2β, AtMRP5, and At5g60760). Seeds of these lpa mutants also exhibited reduced germination under various stress conditions. The greatest reduction in InsP6 (>70%) was observed in atmrp5 seeds which were also among the least sensitive to the stresses examined. Expression analysis of the lpa genes revealed three distinct patterns in developing siliques consistent with their presumed roles. Disruption of each lpa gene resulted in changes in the expression in some of the other lpa genes indicating that transcription of lpa genes is modulated by other constituents of InsP6 metabolism. While all the lpa genes represent possible targets for genetic engineering of low phytate seed crops, mutations in AtMRP5,
AtMIK, and At5g60760 may be most successful for conventional approaches such as mutation breeding.
Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.
Phytic acid (myo-inositol 1,2,3,4,5,6 hexakisphosphate) is the most abundant form of phosphorus (P) in seeds and is virtually indigestible by humans or non-ruminant livestock. It was hypothesized that one class of maize (Zea mays L.) and barley (Hordeum vulgare L.) low phytic acid mutations, designated lpa1, interrupt myo-inositol supply during seed development and may be mutations of the myo-inositol 1-phosphate synthase (MIPS) gene. This study describes the isolation, inheritance, and genetic mapping of the first rice lpa1 mutation and reexamines the MIPS/lpa1 candidate gene hypothesis in rice. Grain from 3632 rice M2 lines, derived from gamma-irradiated seed, was screened for the lpa phenotype. Two mutations, one lethal and one non-lethal, were identified. The non-lethal mutation is phenotypically similar to maize and barley lpa1 mutants and was designated rice lpa1-1. Homozygosity for rice lpa1-1 reduces the phytic acid portion of seed P from 71 to 39% and increases the inorganic portion of seed P from 5 to 32%, with little effect on total seed P. This rice lpa1 mutation was mapped to a 2.2-cM interval on chromosome 2L. A single-copy rice MIPS gene was mapped to a locus on rice chromosome 3 that is orthologous to MIPS loci on maize chromosome 1S (near maize lpa1) and barley chromosome 4H. Unlike maize lpa1-1, the rice and barley lpa1 mutations loci are clearly distinguishable from this canonical MIPS gene. No relationship can be inferred between the maize, barley, and rice lpa1 loci. Although this canonical MIPS gene may be an appropriate target for controlling seed phytic acid synthesis, modifications of other genes (e.g., maize lpa2, barley lpa1, barley lpa2, and rice lpa1) may also be useful in reducing grain phytic acid and improving the nutritional value of cereal grains and/or milling by-products.
Phosphate (Pi) availability is a major factor limiting growth, development, and productivity of plants. In both ecological and agricultural contexts, plants often grow in soils with low soluble phosphate content. Plants respond to this situation by a series of developmental and metabolic adaptations that are aimed at increasing the acquisition of this vital nutrient from the soil, as well as to sustain plant growth and survival. The development of a comprehensive understanding of how plants sense phosphate deficiency and coordinate the responses via signaling pathways has become of major interest, and a number of signaling players and networks have begun to surface for the regulation of the phosphate-deficiency response. In practice, application of such knowledge to improve plant Pi nutrition is hindered by complex cross-talks, which are emerging in the face of new data, such as the coordination of the phosphate-deficiency signaling networks with those involved with hormones, photo-assimilates (sugar), as well as with the homeostasis of other ions, such as iron. In this review, we focus on these cross-talks and on recent progress in discovering new signaling players involved in the Pi-starvation responses, such as proteins having SPX domains.
Phytic acid acts as the major storage form of phosphorus in plant seeds and is poorly digested by monogastric animals. The degradation of phytic acid in animal diets is necessary to overcome both environmental and nutritional issues. The enzyme 1D-myo-inositol 3-phosphate [Ins(3)P(1)] synthase (EC 5.5.1.4) catalyses the first step of myo-inositol biosynthesis and directs phytic acid biosynthesis in seeds. The rice Ins(3)P(1) synthase gene (RINO1) is highly expressed in developing seed embryos and in the aleurone layer, where phytic acid is synthesized and stored. In rice seeds, 18-kDa oleosin (Ole18) is expressed in a seed-specific manner, and its transcripts are restricted to the embryo and the aleurone layer. Therefore, to effectively suppress phytic acid biosynthesis, antisense RINO1 cDNA was expressed under the control of the Ole18 promoter, directing the same spatial pattern in seeds as RINO1 in transgenic rice plants. The generated transgenic rice plants showed strong 'low phytic acid' (lpa) phenotypes, in which seed phytic acid was reduced by 68% and free available phosphate was concomitantly increased. No negative effects on seed weight, germination or plant growth were observed. The available phosphate levels of the stable transgenic plants surpassed those of currently available rice lpa mutants.