Salinity exerted a distinctly differential effect on fructose-1,6-bisphosphatase (EC. 184.108.40.206) isolated from salt-sensitive and salt-tolerant rice (Oryza sativa) varieties. Cytosolic and chloroplastic isoforms of the enzyme from salt-sensitive rice seedlings exhibited decreased catalytic activity during growth in the presence of salt. Furthermore, chloroplastic fructose 1,6-bisphosphatase purified from salt-sensitive (O. sativa cv. IR26) and from the wild halophytic rice Porteresia coarctata differed in their in vitro salt tolerance property although they exhibited otherwise identical biochemical and immunological properties. This decline in enzyme activity was not correlated with de novo synthesis of the chloroplastic fructose-1,6-bisphosphatase protein in the presence of salt. The inhibitory effect of increasing concentration of NaCl on in vitro enzymatic activity could be prevented by preincubation of the enzyme with a number of osmolytes with an effectiveness in the order polyol>sugars. Further, the intrinsic tryptophan fluorescence of the purified rice enzyme is altered in vitro with increasing NaCl concentration which could be prevented by preincubation with inositol. Purified chloroplastic fructose-1.6-bisphosphatase from P. coarctata however, exhibits no such inhibition of enzyme activity in vitro or alteration in tryptophan fluorescence with increasing NaCl concentration.
The location of the phloem within a plant, and its vulnerability to disruption, make it a difficult tissue to study and therefore non-invasive studies of phloem functionality are important. Here we compare, phloem transport, measured non-invasively, in wild type Arabidopsis thaliana, and transposon-insertion mutants for AtSUC1 or AtSUC2, giving in vivo information on the importance of these sucrose transporters for phloem transport. The suc2 mutant showed an increase in both phloem leakage and transport time, consistent with reduced sucrose uptake into both transport and collection phloem. The results are consistent with the AtSUC2 transporter being important for retrieval of leaked sucrose in the transport phloem of Arabidopsis. There was no difference in phloem transport properties between the wild type and the suc1 mutants, implying that the AtSUC1 transporter does not play a significant role within the transport phloem of Arabidopsis under the conditions of our study.
Analysis of a sugarcane (Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG)(n) trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5' untranslated region. No primer pairs producing a polymorphic product were found in the 3' untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera (Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.
AtFBS1 is an F-box protein whose transcript accumulates in response to biotic and abiotic stresses. Previous evidence suggests that a postranscriptional event regulates AtFBS1 expression . We now found that AtFBS1 interacts with 14-3-3 proteins through its amino-terminus and the F-box motif. Deletion of any of these regions abolishes the interaction between AtFBS1 and 14-3-3 proteins. On the other hand, the treatment with the proteasome inhibitor MG132 or the deletion of the F-box from AtFBS1 increases β-glucuronidase (GUS) activity in plants containing a translational fusion of AtFBS1 with the GUS reporter gene, indicating that AtFBS1 is degraded by the 26S proteasome. MG132 treatment of seedlings containing a gene fusion between AtFBS1 and the TAP (Tandem Affinity Purification) cassette causes an increase in the half-life of the protein. In an attempt to understand the role of 14-3-3 interactions, we treated Arabidopsis seedlings with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5'-monophosphate (AICAR), an inhibitor of 14-3-3 client interactions. We observed an increase in AtFBS1-TAP stability as a consequence of AICAR treatment. Based on these data we propose that 14-3-3 proteins promote the dimerization of SCF(AtFBS1). This also may enhance the AtFBS1 autoubiquitination activity and its degradation by the 26S proteasome. AICAR also affects Cullin1 (CUL1) modification by RUB1, which would provide an additional element to the effect of this compound on AtFBS1 stability.
14-3-3 proteins are highly conserved among eukaryotes and perform diverse biochemical activities. We isolated five types of Arabidopsis 14-3-3 cDNAs in a screen for clones that could block ectopic meiosis driven by the pat1 mutation in fission yeast. Overexpression of fission yeast rad24, which encodes a 14-3-3 protein, also suppressed pat1. All Arabidopsis clones isolated could rescue the deformed morphology and elevated UV sensitivity of the rad24 mutant. Thus, it appears that Arabidopsis 14-3-3 proteins can generally substitute for their fission yeast counterpart in function. Expression of an Arabidopsis 14-3-3 clone, GF14µ, was shown to be rather ubiquitous among plant organs.
Selaginella bryopteris is a spike-moss lycophyte species with resurrection capability. These plants have small sized stomata that occur in higher density than in other fern species. The diurnal gas-exchange studies under natural conditions showed a bell shaped net photosynthesis curve. The effective quantum yield of PSII (ΔF/F(m')) showed an inverse relationship with light and recovered to its maximum at sunset. This suggests that there was a complete recovery of PSII efficiency during the late evening hours. S. bryopteris displayed broad temperature optima for net photosynthesis from 28 °C to 37 °C. The stomatal sensitivity in response to vapor pressure deficit (VPD), was maximum at 25 °C temperature while at temperatures from 30 to 35 °C it was low. Our study demonstrates that S. bryopteris plants show a very poor mechanism for its stomatal regulation in response to high light, high temperature, high VPD, high CO₂ and to ABA treatment. At the same time they show a high stomatal conductance leading to unrestricted rates of transpiration and a lack of capacity to optimize water use efficiency (WUE).
Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has the ability to produce scopoletin endogenously and release some of it into the culture medium. We investigated the mechanism of scopoletin uptake following treatment of a tobacco culture with 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of [14C]-labeled scopoletin showed that scopoletin was taken up by 2,4-D-treated cells and converted to scopolin, a 7-O-glucoside of scopoletin. This uptake of scopoletin began 6 h after 2,4-D addition to the cells. Experiments using several inhibitors showed that this uptake was energy-dependent. The phenomenon of 2,4-D-stimulated uptake was observed only for 7-hydroxycoumarins, such as scopoletin, umbelliferone and esculetin. To further investigate the site for scopoletin accumulation, we separated the vacuoles from T-13 cells and quantified the coumarin contents in this fraction. Most of the scopoletin in the vacuoles was present as glucoconjugate, scopolin. Moreover, glucosylation activity was absent from isolated vacuoles and, therefore, is likely to be located in the cytosol. Therefore, we can state that 2,4-D treatment of tobacco cells stimulated scopoletin uptake. The scopoletin was converted into scopolin in the cytoplasm, and then transferred into the vacuoles.
We investigated the transcript levels of 13 proteasome subunit genes, the protein levels of proteasomes, and the activities of the 26S proteasome in ANAC078-overexpressing Arabidopsis plants (Ox-ANAC078) and knockout ANAC078 (KO-ANAC078) mutants. The transcript levels and the protein levels of proteasomes were increased in the Ox-ANAC078 plants compared with the wild-type plants and KO-ANAC078 mutants under normal conditions and high-light (HL) stress. Although the activities of the 26S proteasome were decreased in all the plants under HL stress, they were higher in the Ox-ANAC078 plants than wild-type plants and KO-ANAC078 mutants under normal conditions and HL stress. These findings suggest that ANAC078 regulates the levels of proteasomes. To explore the function of the increased levels of proteasomes to HL stress, we assessed the tolerance to HL stress of the Ox-ANAC078 plants and KO-ANAC078 mutants. The photosystem II activities of Ox-ANAC078 remained high compared with those of the wild-type plants and KO-ANAC078 mutants under HL stress, suggesting that ANAC078 may play an important role in the response and adaptation to HL stress.
A set of 60 cDNAs were isolated by subtractive hybridization screening of a phage library using radioactively-labeled probes generated from root mRNAs isolated from tobacco (Nicotiana tabacum cv Burley 21) plants before and 3 days after topping. Among the differentially expressed gene products were full-length and partial cDNAs encoding arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and S-adenosylmethionine synthetase (SAMS), enzymes involved in polyamine and alkaloid biosynthesis. The other cDNAs isolated were placed into one of several categories and encode metabolic enzymes, proteins involved in transcription and translation, components of signal transduction pathways, and homologs of genes whose expression has been shown to be regulated by phytohormones (i.e. auxin, ABA), wounding or other stress responses. RNA gel blot analysis showed that the ADC and ODC transcripts were preferentially expressed in the roots and floral tissues of mature tobacco plants, whereas SAMS transcripts were detected in all tissues examined. The steady-state levels of the ADC and ODC mRNAs increased in the roots of wild-type tobacco plants during the 24 h period after topping, whereas little change was observed in the abundance of the SAMS transcripts in these tissues. The possible factors associated with the regulation of expression of these genes are discussed.
In plant cells, the Na(+)/H(+) antiport at the tonoplast provides a biochemical pathway to transport cytoplasmic Na(+) into the vacuole. Recently it was shown that overexpression of a vacuolar Na(+)/H(+) promotes sustained plant growth at high NaCl levels (Apse et al. Science 285, 1256, 1999). The sequestration of Na(+) ions into the vacuole can be followed using 31P and 23Na NMR spectroscopy. Suspension cell cultures are very suitable for this purpose and allow rapid and accurate assessment of the activity of the Na(+)/H(+) antiport and therefore potentially of salt tolerance. Perfusion experiments with maize cells that are not particularly salt (NaCl) tolerant showed that during salt stress the cytoplasmic pH remains unchanged while the vacuolar pH significantly increased. During Na(+) sequestration into the vacuole, the cytoplasmic pH equilibrates faster than that of the vacuole. Both vacuolar pH and the cellular Na(+) uptake rate were dependent on extracellular Na(+) for concentrations up to approximately 300 mM. For Na(+) concentrations >/=300 mM, both vacuolar pH and cellular Na(+) uptake became independent of the extracellular concentration. This indicates either a saturation of Na(+) uptake at the cell surface or a saturation of the Na(+)/H(+) transporter at the tonoplast. Na(+) uptake into the cell is accompanied by a rapid increase in vacuolar PO(4)(3-), broadening of the 31P resonances and a reduction in glucose monophosphate and UDPG.
Flowering on time is a critically important for successful reproduction of plants. Here we report an early-flowering mutant in Arabidopsis thaliana, accelerated flowering 1-1D (afl1-1D) that exhibited pleiotropic developmental defects including semi-dwarfism, curly leaf, and increased branching. Genetic analysis showed that afl1-1D mutant is a single, dominant mutant. Chromosomal mapping indicates that AFL1 resides at the middle of chromosome 4, around which no known flowering-related genes have been characterized. Expression analysis and double mutant studies with late flowering mutants in various floral pathways indicated that elevated FT is responsible for the early-flowering of afl1-1D mutant. Interestingly, not only flowering-related genes, but also several floral homeotic genes were ectopically overexpressed in the afl1-1D mutants in both FT-dependent and -independent manner. The degree of histone H3 Lys27-trimethylation (H3K27me3) was reduced in several chromatin including FT, FLC, AG and SEP3 in the afl1-1D, suggesting that afl1-1D might be involved in chromatin modification. In support, double mutant analysis of afl1-1D and lhp1-4 revealed epistatic interaction between afl1-1D and lhp1-4 in regard to flowering control. Taken together, we propose that AFL1 regulate various aspect of development through chromatin modification, particularly associated with H3K27me3 in A. thaliana.
A field study was conducted to investigate the impact of ambient solar UV on the various growth, physiological and yield parameters of four sorghum (Sorghum bicolor L.) varieties-Indore-12, Indore-26, CSV-23 and Indore-27 by excluding either UV-B (<315nm) or UV-A/B (<400nm) components of solar spectrum. Exclusion of UV significantly enhanced plant height, area and specific leaf weight of flag leaf, biomass accumulation, yield parameters and harvest index in all the sorghum varieties. Chlorophyll b was significantly enhanced and chlorophyll a increased to a lesser extent, UV-B absorbing substances and chlorophyll a/b ratio were significantly decreased by the exclusion of solar UV. The enhancement in the vegetative growth and yield by UV exclusion might be linked to the remarkable increase in rate of photosynthesis in sorghum varieties. The magnitude of the response was high in I-26 and I-27 as compared to CSV-23 and I-12 after exclusion of solar UV. All the varieties of sorghum had a negative cumulative stress response index (CSRI), the sensitivity of the sorghum varieties was in the following sequence I-12>CSV-23>I-26>I-27. Thus I-27 was the most sensitive and I-12 the least sensitive variety to present level of solar UV radiation. The differences in UV sensitivity identified among sorghum varieties might be useful in breeding programs for increased tolerance to UV-B radiation.
To improve the rooting ability, the dwarfing apple rootstock M.9/29 was transformed with the rolB gene by Agrobacterium-mediated gene transfer. The use of sorbitol in the induction medium resulted in a successful transformation, while the use of sucrose failed to give any transformants. Totally 14 putative clones, named ARB1-14, were obtained from ten different leaves. Polymerase chain reaction (PCR) and Southern analyses confirmed that all the clones contained the nptII and rolB genes, while only four of them contained the intact gus gene. The in vitro rooting test showed that all the tested clones rooted to 83-100% on the hormone free rooting medium, while only 1% for the control plants. The root number of the transgenic clones ranged from 3.5 to 9, while the control plants produced only one root. Growth analysis showed that the clone ARB9 and ARB10 had a significant reduced node number and stem length compared with the control plants. However, the relative growth rate (RGR) of the tested clones was similar to that of the control plants, indicating that RGR is not directly related to dwarfism of a plant. The clone ARB10 also showed a significant reduced internode length compared with the control plants. The root length and root morphology did not differ between the transgenic clones and the untransformed control plants.
Powdery mildew caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a destructive foliar disease on wheat in many regions of the world. Triticum turgidum ssp. dicoccum (2n=4x=28) shows particular promises as a donor source of useful genetic variation for several traits, including disease resistances that could be introgressed to cultivated wheats. Accession MG5323, resistant to powdery mildew, was crossed to the susceptible durum cultivar Latino and a set of 122 recombinant inbred lines (RILs) was produced. F(1) and F(2) progenies and the RIL population were tested with one isolate of Blumeria graminis and data obtained indicated that a single dominant gene, temporarily designated Ml5323, controlled resistance at the seedling stage. Molecular markers were used to characterize and map the powdery mildew resistance gene. Twelve microsatellite markers were linked to the resistance gene, and among them, EST-SSR CA695634 was tightly linked to the resistance gene, which was assigned to chromosome arm 2BS and physically mapped to the gene rich region of fragment length (FL) 0.84-1.00. An allelism test showed that the Ml5323 gene and the resistant gene Pm26 of ssp. dicoccoides localized in the same bin, are not allelic and tightly linked.
Biotic signaling molecules including abscisic acid (ABA) serve as an integrator of abiotic stress including high salinity and drought. Recent studies have led to the identification of an ABA signaling pathway from the ABA receptor to stomatal closure in response to abiotic stress. ABA is linked to ABA receptors and protein phosphatase 2C (PP2C) members. In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction between the RCAR type receptor and AIP1, which is one of the group A PP2C member. Several ABA receptors interact with AIP1 in an ABA dependent or independent manner. aip1 null mutant plants exhibited reduced sensitivity to ABA and glucose during the seed germination and seedling stage. Taken together, these results demonstrated that AIP1 is associated with ABA-mediated cell signaling and function as positive regulators of ABA.
A metabolic depletion syndrome was discovered at early vegetative stages in roots of salt sensitive rice cultivars after prolonged exposure to 100mM NaCl. Metabolite profiling analyses demonstrate that this syndrome is part of the terminal stages of the rice salt response. The phenotype encompasses depletion of at least 30 primary metabolites including sucrose, glucose, fructose, glucose-6-P, fructose-6P, organic- and amino-acids. Based on these observations we reason that sucrose allocation to the root may modify the rice response to high salt. This hypothesis was tested using antisense lines of the salt responsive OsSUT1 gene in the salt sensitive Taipei 309 cultivar. Contrary to our expectations of a plant system impaired in one component of sucrose transport, we find improved gas exchange and photosynthetic performance as well as maintenance of sucrose levels in the root under high salinity. Two independent OsSUT1 lines with an antisense inhibition similar to the naturally occurring salt induced reduction of OsSUT1 gene expression showed these phenomena but not a more extreme antisense inhibition line. We investigated the metabolic depletion syndrome by metabolomic and physiological approaches and discuss our results with regard to the potential role of sucrose transporters and sucrose transport for rice salt acclimation.
Our previous studies found strict gene silencing associated with CaMV-35S promoter-specific de novo methylation in transgenic gentian plants. To dissect the de novo methylation machinery, especially in association with histone modification, 35S-driven sGFP-expressing and -silenced gentian cultured cell lines that originated from a single transformation event were produced and used for epigenetic analyses. A sGFP-expressing primarily induced cell suspension culture (PS) was hypomethylated in the 35S promoter region, although a low level of de novo methylation at the 35S enhancer region (-148 to -85) was detected. In contrast, a sGFP-silenced re-induced cell suspension culture (RS), which originated from leaf tissues of a transgenic plant, was hypermethylated in the 35S promoter region. Chromatin immunoprecipitation analysis showed that in RS, histone H3 of the silenced 35S promoter region was deacetylated and also dimethylated on lysine 9. Interestingly, in the silenced 35S promoter 3' region, dimethylation of histone H3 lysine 4 was also observed. When hypomethylation and histone H3 acetylation of the 35S region occurred in PS, de novo methylation at the 35S enhancer region had already taken place. The de novo methylation status was also resistant to 5-aza-2'-deoxycytidine treatment. These results suggest that de novo methylation of the enhancer region is a primitive process of 35S silencing that triggers histone H3 deacetylation.
A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5mg/L 2,4-D and 1.0mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD(600)=0.8) were co-cultured on LS30-AsPC medium for 2 days at 25±2°C in the dark. Based on GUS expression analysis, 10min co-cultivation time with 100μM acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10g/L PVP, 10% coconut water and 250mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T(1)) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T(0) and T(1)).
The study evaluated effects of drought on some agro-morphological traits of 60 rice genotypes comprising 54 introgression lines with their parents, IR 64 (Oryza sativa) and TOG 5681 (Oryza glaberrima) and four NERICA-L varieties developed from the same parents for comparison. The genotypes were subjected either to full irrigation from sowing to maturity (control) or to 21-day drought applied by stopping irrigation from the 45th day after sowing (DAS) onward (drought) in the dry seasons of 2006 and 2007-2008. Plant height, spikelet fertility, grain yield and leaf area at harvesting were consistently reduced by drought in both seasons. Values of leaf temperature, leaf rolling, leaf tip drying, leaf blast, days from seeding to flowering and maturity were higher under drought. The results on SPAD and number of tillers were not consistent. Significant relationship (P<0.05) was observed between all traits evaluated and grain yield under drought. Introgression lines, SEN-L13-2, MPL-15-3, SEN-L10-1, SEN-L26-3 and SEN-L21-2 showed significantly higher yield than the highest yield NERICA-L variety (all of them had higher yield than the parents). Among them, SEN-L13-2 showed the lowest yield loss by drought and MPL-15-3 had high yield potential and considerably low yield loss by drought.
Dynamin family proteins in eukaryotic cells assemble into rings or spirals on the surface of membranes and pinch the membranes. We found 21 dynamin-related protein (DRP) genes in the Physcomitrella patens genome. Phylogenetic analysis indicated that three of them (PpDRP5B-1, PpDRP5B-2, and PpDRP5B-3) showed robust monophyly with Arabidopsis thaliana DRP5B and Cyanidioschyzon merolae CmDnm2, both of which are related to plastid division. Quantitative RT-PCR analysis showed that the amounts of DRP5B-3 transcripts were 14-fold and 8-fold higher than those of DRP5B-1 and DRP5B-2, respectively. We generated PpDRP5B knockout transformants for each of these genes. Subapical protonemata cells in wild-type plants had an average of 47 chloroplasts. The cells in the PpDRP5B-3 knockout transformant had slightly enlarged chloroplasts, with an average chloroplast number of 28, whereas the PpDRP5B-1 and 5B-2 knockout lines had no effect on chloroplast number in P. patens. To analyze function of each PpDRP5B gene, we generated double- and triple-knockout lines. Whereas there were 32 chloroplasts in a cell of the PpDRP5B-1/5B-2 double-knockout lines, the triple-knockout line had only a few macrochloroplasts. A transient expression assay with the triple-knockout line demonstrated that the PpDRP5B-3 gene could recover the normal chloroplast phenotype.
The genome of the cyanobacterium Synechocystis sp. PCC 6803 (hereafter, Synechocystis) contains an aqpZ gene (slr2057) which encodes an aquaporin (SsAqpZ), a membrane channel protein that might play a role in osmotic water transport and therefore the growth of Synechocystis. Structural characterization of SsAqpZ by protein sequence analysis and homology modelling revealed that it was more similar to bacterial aquaporin Z than the glycerol facilitator. To understand the functional role of SsAqpZ, the aqpZ knockout (KO) and myc-tagged aqpZ knockin (KI) Synechocystis were constructed. Water channel activity assays showed that SsAqpZ facilitated water transportation. SsAqpZ-mediated changes in cell volume were observed in wild-type (WT) and KI Synechocystis. Expression of SsAqpZ in KI Synechocystis was induced by extracellular hyperosmolarity. In the absence of hyperosmolarity, WT, KO and KI Synechocystis showed the same pattern of growth and no morphological or phenotypical perturbations. Under hyperosmotic condition, while the WT and also KI cells maintained a similar growth rate throughout the entire exponential phase, KO cells grew significantly slower. These results indicate that SsAqpZ has water channel activity and is involved in the adaptation and maintenance of growth of Synechocystis in a hyperosmotic environment.
A DNA repair synthesis assay monitoring nucleotide excision repair (NER) was established in cell-free extracts of unicellular alga Chlorella pyrenoidosa using cisplatin- or mitomycin C-damaged plasmid DNA as the repair substrate. The algal extracts promoted a damage-dependent increase in 32P-dATP incorporation after normalization against an internal control. To identify the proteins responsible for NER, a biotin-labeled duplex 27 mer (2 µg) irradiated with or without UV (27 kJ m(-2)) was immobilized on streptavidin-conjugated agarose beads and incubated with C. pyrenoidosa extracts (50 µg) to pull down repair proteins. The extracts post incubation with beads carrying unirradiated 27 mer promoted an eightfold increase in repair synthesis in plasmid DNA (1 µg) damaged by 16.8 pmol of cisplatin. The extracts obtained after affinity adsorption with UV-damaged DNA ligand, however, failed to repair plasmid DNA treated with cisplatin, reflecting that some proteins crucial to NER had been sequestered by the damaged 27 mer. A polypeptide approximately 70-72 kDa in molecular mass was found to bind much more strongly to the damaged DNA than to the control DNA after analyzing the proteins bound to the beads by SDS-PAGE, and this polypeptide is believed to play a role in NER in C. pyrenoidosa.
To understand the molecular mechanism of the plant vacuolar H(+)-ATPase in endocytic trafficking and adaptation to high salinity, yeast two-hybrid assay, IP-western hybridization, trafficking assay, RT- and qRT-PCR analyses and growth assay were performed here. To confirm the interaction between OsVHA-a1 and OsGAP1, pull-down assay and Co-IP were performed in vitro and in vivo, respectively. qRT-PCR analysis revealed that the transcription of OsVHA-a1, OsGAP1 and OsRab11 was induced under high salinity. Through the protoplast-based trafficking assay, OsVHA-a1 localized predominantly from the TGN to the PVC under stressed conditions. In addition, both OsGAP1 (R385A) and OsRab11 (S28N) mutants did not interact with OsVHA-a1, and blocked the vesicular trafficking of OsVHA-a1 to the PVC. In a seedling growth assay using the dominant negative OsRab11 (S28N), this mutant was much more sensitive to high salinity than the wild-type. Furthermore, the trafficking assay using isolated vacuoles demonstrated directly that OsGAP1 targeted to the tonoplast of the central vacuole under high salinity. Taken together, it is suggested that OsGAP1 and OsRab11 are essential for the vesicle trafficking of OsVHA-a1 to the PVC and/or the central vacuole under high salinity.
Metallothioneins (MT) play an important role in heavy metal detoxification and homeostasis of intracellular metal ions in plant. In this study, two transgenic lines expressing MT type 2 gene (PsMT(A1)) from Pisum sativum, a regenerated non transformed line NT and clone AL22, selected as heavy metal tolerant, were characterized in presence of the heavy metals for the ability to accumulate zinc and copper and to activate antioxidative enzyme defences: superoxide dismutase, catalase, ascorbate peroxidase. The levels of expression of MT type 2 gene assessed by RT-qPCR confirmed the gene over-expression in transgenic lines and evidenced in NT and AL22 the up-regulation of gene transcription by zinc and copper. Transgenic poplar lines during heavy metal stress displayed increased ability to translocate and accumulate zinc and copper compared with NT and AL22. The antioxidant enzyme defence was differently activated in response to metals in the transgenic lines without a significant increase of ROS. These results suggested that PsMT(A1) could play a role in ROS scavenging leading to enhanced metal tolerance and increased zinc and copper sequestration in root and leaf.
The activity of mitochondrial phospholipase A(2) (PLA(2)) was shown for the first time in plants. It was observed in etiolated seedlings from durum wheat, barley, tomato, spelt and green seedlings of maize, but not in potato and topinambur tubers and lentil etiolated seedlings. This result was achieved by a novel spectrophotometric assay based on the coupled PLA(2)/lipoxygenase reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine as substrate; the mitochondrial localisation was assessed by checking recovery of marker enzymes. Durum wheat mitochondrial PLA(2) (DWM-PLA(2)) showed maximal activity at pH 9.0 and 1mM Ca(2+), hyperbolic kinetics (K(m)=90±6μM, V(max)=29±1nmolmin(-1)mg(-1) of protein) and inhibition by methyl arachidonyl fluorophosphonate, 5-(4-benzyloxyphenyl)-4S-(7-phenylheptanoylamino)pentanoic acid and palmityl trifluoromethyl ketone. Reactive oxygen species had no effect on DWM-PLA(2), that instead was activated by about 50% and 95%, respectively, under salt (0.21M NaCl) and osmotic (0.42M mannitol) stress imposed during germination. Contrarily, a secondary Ca(2+)-independent activity, having optimum at pH 7.0, was stress-insensitive. We propose that the activation of DWM-PLA(2) is responsible for the strong increase of free fatty acids recently measured in mitochondria under the same stress conditions [Laus, et al., J. Exp. Bot. 62 (2011) 141-154] that, in turn, activate potassium channel and uncoupling protein, able to counteract hyperosmotic stress.
Plant heat shock transcription factors (HSFs) regulate transcription of heat shock (HS) genes. In Arabidopsis thaliana, 21 HSFs have been classified into groups A-C. Members of class A act as typical transcriptional activators, whereas B HSFs function as coactivators or repressors depending on promoter context. The function of class C HSFs is still unclear. Here, we present the isolation and characterization of the first HSF from alfalfa (Medicago sativa L.) and designate it MsHSFA4 based on amino acid sequence analysis. The MsHSFA4 gene was determined to be single copy and was detected at two separate genetic loci in the tetraploid Medicago sativa. Overexpression of MsHSFA4 in tobacco mesophyll protoplasts resulted in weak transcriptional activity, similar to that exhibited by Arabidopsis AtHSFA4a. The MsHSFA4 proximal promoter contains three putative HSE elements, and the gene itself is activated both by heat and cold stress.
The phytohormone abscisic acid (ABA) plays an important role in developmental processes in addition to mediating plant adaptation to stress. In the current study, transcriptional response of 17 genes involved in ABA metabolism and transport has been examined in vegetative and reproductive organs exposed to cold and heat stress. Temperature stress activated numerous genes involved in ABA biosynthesis, catabolism and transport; however, several ABA biosynthesis genes (ABA1, ABA2, ABA4, AAO3, NCED3) were differentially expressed (up- or down-regulated) in an organ-specific manner. Key genes (CYP707As) involved in ABA catabolism responded differentially to temperature stress. Cold stress strongly activated ABA catabolism in all organs examined, whereas heat stress triggered more subtle activation and repression of select CYP707A genes. Genes involved in conjugation (UGT71B6), hydrolysis (AtBG1), and transport (ABCG25, ABCG40) of ABA or ABA glucose ester responded to temperature stress and displayed unique organ-specific expression patterns. Comparing the transcriptional response of vegetative and reproductive organs revealed ABA homeostasis is differentially regulated at the whole plant level. Taken together our findings indicate organs in close physical proximity undergo vastly different transcriptional programs in response to abiotic stress and developmental cues.
We have identified an abscisic acid (ABA) and stress-responsive GRAM (Glucosyltransferases, Rab-like GTPase activators and Myotubularins) domain protein GER5 (GEm-Related 5) closely related to GEM (GLABRA2 Expression Modulator), a novel regulator of cell division and cell fate determination in epidermal cells. A loss-of-function T-DNA line (ger5-2) and transgenic lines silencing (GER5(RNAi)) or overexpressing (GER5(OE)) GER5 displayed several defects in reproductive development affecting seed and embryo development. RNA in situ studies revealed GER5 and related GRAM genes (GEM and GEm-Related 1 (GER1)) have both overlapping and unique expression domains in male and female reproductive organs. Hormone immunolocalization experiments further indicate GER5 transcripts preferentially localize to reproductive tissues which accumulate ABA. Expression analysis revealed members of the GRAM family (GER5, GER1, GEM) display tissue-specific expression patterns and are responsive to phytohormones and abiotic stress, in addition to genetic lesions (aba1, aba2, ctr1) affecting ABA biosynthesis or ethylene signalling. Mature seeds of ger5-2 mutants also exhibit reduced sensitivity to ABA during seed germination assays. Microarray analysis of aborting and developing seeds isolated from ger5-2 mutants revealed underlying transcriptional changes in carbohydrate metabolism, hormone signalling and catabolic processes (e.g. protein degradation, autophagy). Taken together, our results indicate ABA-responsive GRAM genes play a novel role in regulating the reproductive development of plants, and raise intriguing questions regarding the functional relationship between members of the GRAM gene family.
Abscisic acid (ABA) and nitric oxide (NO) are both extremely important signalling molecules employed by plants to control many aspects of physiology. ABA has been extensively studied in the mechanisms which control stomatal movement as well as in seed dormancy and germination and plant development. The addition of either ABA or NO to plant cells is known to instigate the actions of many signal transduction components. Both may have an influence on the phosphorylation of proteins in cells mediated by effects on protein kinases and phosphatases, as well as recruiting a wide range of other signal transduction molecules to mediate the final effects. Both ABA and NO may also lead to the regulation of gene expression. However, it is becoming more apparent that NO may be acting downstream of ABA, with such action being mediated by reactive oxygen species such as hydrogen peroxide in some cases. However not all ABA responses require the action of NO. Here, examples of where ABA and NO have been put together into the same signal transduction pathways are discussed.
The plant hormone abscisic acid (ABA) regulates many key processes in plants including the response to abiotic stress. ABA signal transduction consists of a double-negative regulatory mechanism, whereby ABA-bound PYR/RCARs inhibit PP2C activity, and PP2Cs inactivate SnRK2s. We studied and analyzed the various genes participating in the ABA signaling cascade of the grape (Vitis vinifera). The grape ABA signal transduction consists of at least six SnRK2s. Yeast two-hybrid system was used to test direct interactions between core components of grape ABA signal transduction. We found that a total of forty eight interactions can occur between the various components. Exogenous abscisic acid (ABA) and abiotic stresses such as drought, high salt concentration and cold, were applied to vines growing in a hydroponic system. These stresses regulated the expression of various grape SnRK2s as well as ABFs in leaves and roots. Based on the interactions between SnRK2s and its targets and the expression pattern, we suggest that VvSnRK2.1 and VvSnRK2.6, can be considered the major VvSnRK2 candidates involved in the stomata response to abiotic stress. Furthermore, we found that the expression pattern of the two grape ABF genes indicates organ specificity of these genes. The key role of ABA signaling in response to abiotic stresses makes the genes involve in this signaling potential candidates for manipulation in programs designed to improve fruit tree performance in extreme environments.
DWD (DDB1 binding WD40) proteins have been reported as substrate receptors for cullin-RING ubiquitin ligase 4 complexes (CRL4), a family of E3 ligases. Abscisic acid (ABA) plays an important role in plant responses to abiotic stresses, such as drought and salinity. Upon screening T-DNA mutants of DWD genes for ABA responses we obtained several candidates that exhibited ABA-hypersensitivity, and one was named DWA3 (DWD hypersensitive to ABA 3). DWA3 interacted with DDB1, which has been proposed to be an adaptor protein of the CUL4 E3 ligase complex, in yeast two-hybrid assays. Also, association between DWA3 and the DDB1-CUL4 machinery was detected in vivo by co-immunoprecipitation assays, indicating that DWA3 may function as a substrate receptor for CRL4. ABA-inducible transcription factors (ABI5 and AtMYC2) and their downstream genes were hyper-induced by ABA in dwa3. Taken together, we suggest that DWA3 is a negative regulator of ABA responses and may be involved in protein degradation mediated by CRL4.
Abscisic acid (ABA) plays an essential function in plant physiology since it is required for biotic and abiotic stress responses as well as control of plant growth and development. A new family of soluble ABA receptors, named PYR/PYL/RCAR, has emerged as ABA sensors able to inhibit the activity of specific protein phosphatases type-2C (PP2Cs) in an ABA-dependent manner. The structural and functional mechanism by which ABA is perceived by these receptors and consequently leads to inhibition of the PP2Cs has been recently elucidated. The module PYR/PYL/RCAR-ABA-PP2C offers an elegant and unprecedented mechanism to control phosphorylation signaling cascades in a ligand-dependent manner. The knowledge of their three-dimensional structures paves the way to the design of ABA agonists able to modulate the plant stress response.
Plants are continuously challenged by abiotic and biotic stress factors and need to mount appropriate responses to ensure optimal growth and survival. We have identified ERD15 as a central component in several stress responses in Arabidopsis thaliana. Comparative genomics demonstrates that ERD15 is a member of a small but highly conserved protein family ubiquitous but specific to the plant kingdom. The origin of ERD15 family of proteins can be traced to the time of emergence of land plants. The presence of the conserved PAM2 motif in ERD15 proteins is indicative of a possible interaction with poly(A) binding proteins and could suggest a role in posttranscriptional regulation of gene expression. The function of the other highly conserved motifs in ERD15 remains to be elucidated. The biological role of all ERD15 family members studied so far appears associated to stress responses and stress adaptation. Studies in Arabidopsis demonstrate a role in abiotic stress tolerance where ERD15 is a negative regulator of ABA signaling. The role in ABA signaling may also explain how ERD15 regulates stomatal aperture and consequently controls plant water relations.
The characterization of yield trait mutants is important for understanding the regulation of grain yield formation in staple food crops. Meh0239 is a yield trait-related mutant identified from a mutant library of the common wheat cultivar Wangshuibai created by ethylmethyl sulfide (EMS) treatment of dry seeds. To shed some light on the nature of this mutation, it was investigated morphologically, physiologically, anatomically and genetically. The mutant plant showed obvious phenotypic differences in comparison with the wild type, starting at the seedling stage, including reduced plant height, wider and shorter leaves, shortened spikes, spikelets and grains and a more compact spikelet distribution. Also, seeds produced in the mutant germinated more slowly. Meh0239 contained a significantly higher level of abscisic acid (ABA) but lower levels of indole-3-acetic acid (IAA), methyl jasmonate (MeJA) and zeatin riboside (ZR) in flag leaves. Cells of all types in the leaf epidermis appeared shorter along the axial direction. The bulliform cells and long cells on the adaxial leaf surface were abnormal in shape. A genetic analysis using two F₂ segregating populations indicated that a single recessive mutation in wheat chromosome 7DS, about 3.1cM distal from Xwmc506, caused these variations. Because of the pleiotropic nature of this gene and its relation with yield trait formation, we named it Yt1 for yield trait related 1.
Cultivars of rice (Oryza sativa L.), especially the large-spikelet-type, often fail to achieve the high yield potential due to poor grain-filling of their inferior (late-flowering) spikelets. The superior (early-flowering) spikelets normally contain more abscisic acid (ABA) than the inferior spikelets. It was speculated that ABA might play a pivotal role in the grain-filling of inferior spikelets. To understand the molecular regulation involved in this process, we employed the 2-D gel-based comparative proteomic and phosphoproteomic analyses to search for differentially expressed proteins in the inferior spikelets under exogenous ABA treatment. A total of 111 significantly differential proteins and 31 phosphoproteins were found in the inferior spikelets after treatment. Among them, 100 proteins and 23 phosphoproteins were identified by using MALDI-TOF/TOF MS. In addition, the gene expression patterns of the inferior spikelets were confirmed with RT-PCR. These differentially expressed proteins are active in defense response, carbohydrate, protein, amino acid, energy and secondary metabolisms, as well as cell development and photosynthesis. The results suggest that the grain-filling of rice inferior spikelets is regulated by ABA through some proteins and phosphoproteins participating in carbon, nitrogen and energy metabolisms.
Drought is a major environmental stress factor that affects growth and development of plants. Abscisic acid (ABA), osmotically active compounds, and synthesis of specific proteins, such as proteins that scavenge oxygen radicals, are crucial for plants to adapt to water deficit. LOS5/ABA3 (LOS5) encodes molybdenum-cofactor sulfurase, which is a key regulator of ABA biosynthesis. We overexpressed LOS5 in tobacco using Agrobacterium-mediated transformation. Detached leaves of LOS5-overexpressing seedlings showed lower transpirational water loss than that of nontransgenic seedlings in the same period under normal conditions. When subjected to water-deficit stress, transgenic plants showed less wilting, maintained higher water content and better cellular membrane integrity, accumulated higher quantities of ABA and proline, and exhibited higher activities of antioxidant enzymes, i.e., superoxide dismutase, catalase, peroxidase and ascorbate peroxidase, as compared with control plants. Furthermore, LOS5-overexpressing plants treated with 30% polyethylene glycol showed similar performance in cellular membrane protection, ABA and proline accumulation, and activities of catalase and peroxidase to those under drought stress. Thus, overexpression of LOS5 in transgenic tobacco can enhance drought tolerance.
We generated transgenic rice plants overexpressing OsHAP3E which encodes a subunit of a CCAAT-motif binding HAP complex. The OsHAP3E-overexpressing plants showed various abnormal morphologies both in their vegetative and reproductive phases. The OsHAP3E-overexpressing plants were dwarf with erected leaves and similar to brassinosteroid mutants in the vegetative phase. In the reproductive phase, dense panicle was developed, and occasionally successive generation of lateral rachises and formation of double flowers were observed. These phenotypes indicate association of OsHAP3E with determination of floral meristem identity. On the other hand, repression of OsHAP3E by RNAi or by overexpressing chimeric repressor fusion constructs brought about lethality to transformed cells, and almost no transformant was obtained. This suggests that the OsHAP3E function is essential for rice cells. Altogether, our loss-of-function and gain-of-function analyses suggest that OsHAP3E plays important pleiotropic roles in vegetative and reproductive development or basic cellular processes in rice.
Cytological abnormalities were observed in transgenic oat (Avena sativa L. cv. GAF/Park-1) produced by microprojectile bombardment of mature seed-derived highly regenerative tissues. Of the plants from 48 independent transgenic lines examined, plants from only 20 lines (42%) were karyotypically normal (2n=6x=42) without detectable chromosomal aberrations; plants from 28 lines (58%) had chromosomal variation, i.e. aneuploids and structural changes. No significant difference in cytological aberration was observed between the two different culturing systems used for transformation: 57% chromosomal abnormalities in plants derived from D'BC2 medium (2.0 mg/l 2,4-D, 0.1 mg/l BAP and 5.0 &mgr;M cupric sulfate) used for tissue initiation and maintenance and 60% in plants from tissue initiated on D'BC2 and maintained on DBC3 (1.0 mg/l 2,4-D, 0.5 mg/l BAP and 5.0 &mgr;M cupric sulfate). Comparative differences in chromosomal status frequently occurred among plants regenerated from the same T(0) line. The most common cytological aberration in transgenic plants was aneuploidy, followed by deletion of chromosomal segments; no change in ploidy level was observed. In contrast, nontransgenic plants, regenerated from tissues comparable in age and culture media to that used for transgenic tissues, had a much lower percentage of karyotypic abnormality (0-14%). Our data indicate that some stress(es) imposed by the transformation process, e.g. osmotic treatment, bombardment and selection, leads to cytological variation in transgenic oat plants, an observation similar to that observed in our recent studies with transgenic barley plants.
Norway spruce (Picea abies L. Karst) grown under ambient (365-377 μmol(CO(2)) mol(-1); AC) and elevated (700 μmol(CO(2)) mol(-1); EC) CO(2) concentrations within glass domes with automatically adjustable windows and on an open-air control site were studied after 8 years of treatment. The effect of EC on photosynthesis, mesophyll structure and phenolics accumulation in sun and shade needles was examined. Photosynthetic assimilation and dark respiration rates were measured gasometrically; the structural parameters of mesophyll were determined using confocal microscopy and stereological methods. The contents of total soluble phenolics and lignin were assessed spectrophotometrically, and localizations of different phenolic groups were detected histochemically on needle cross-sections. EC enhanced the light-saturated CO(2) assimilation rate and reduced dark respiration in the current-year needles. No effects of CO(2) enrichment on mesophyll structural parameters were observed. Similarly, the accumulation and localization of phenolics and lignin remained unaffected by EC treatment. Needles differentiated into sun and shade ecotypes in the same manner and to the same extent irrespective of CO(2) treatment. Based on these results, it is apparent that the EC-induced enhancement of photosynthesis is not related to changes in the examined structural parameters of mesophyll and accumulation of phenolic compounds.
The function of selenium independent glutathione peroxidase (GPx) in response to biotic and abiotic stresses was investigated in transgenic tomato plants overexpressing an exogenous GPx and exhibiting a 50% increase in total GPx activity. GPx-overexpressing and control plants were challenged either by a mechanical stress or by infection with the biotrophic parasite Oidium neolycopersici or the necrotrophic parasite Botrytis cinerea. In mechanically stressed plants, internode growth was significantly less modified in GPx-overexpressing plants compared to controls. This stress resistant phenotype was not accompanied with any change in the global antioxidant response of the plants other than their increased GPx activity. Following infection by O. neolycopersici or by B. cinerea, lesion extension was increased in GPx-overexpressing plants compared with controls. These results showed that GPx overexpression provoked opposite effects in situations of biotic and abiotic challenges, suggesting a key role for this scavenger enzyme in controlling biotic and abiotic stress responses.
Plants protect against pathogen infections by a combination of constitutive and induced strategies. The induction of plant defense involves the recognition of compounds derived from the pathogen or the plant itself, called elicitors. Looking for new genes involved in plant defense responses, we isolated a cDNA clone corresponding to an elicitor-induced mRNA from Phaseolus vulgaris cell suspension cultures. This clone, PvFBS1, encodes a protein with an F-box, therefore a putative component of an SCF ubiquitin ligase complex. PvFBS1 mRNA accumulates in leaves of whole plants in response to wounding or osmotic stress, as well as, following the application of methyl jasmonate (MeJA). salicylic acid (SA) or abscisic acid (ABA). Several sequences related to PvFBS1 were found in the GenBank. In Arabidopsis thaliana there are 4 genomic sequences coding for proteins with similarity to PvFBS1. One of them, AtFBS1, displays a pattern of induction analogous to the one observed for PvFBS1. A yeast two-hybrid assay proved that AtFBS1 was able to interact with ASK1, the component of the SCF complex that binds the F-box. A deletion of the F-box in AtFBS1 abolishes the ability of this protein to interact with ASK1. This demonstrates the functionality of the F-box contained in AtFBS1. Gene fusions to the GUS reporter gene revealed a complex regulation for AtFBS1 expression.
Grain number is the only yield component that is directly associated with increased grain yield in important cereal crops like wheat. Historical yield studies show that increases in grain yield are always accompanied by an increase in grain number. Adverse weather conditions can cause severe fluctuations in grain yield and substantial yield losses in cereal crops. The problem is global and despite its impact on world food production breeding and selection approaches have only met with limited success. A specific period during early reproductive development, the young microspore stage of pollen development, is extremely vulnerable to abiotic stress in self-fertilising cereals (wheat, rice, barley, sorghum). A better understanding of the physiological and molecular processes that lead to stress-induced pollen abortion may provide us with the key to finding solutions for maintaining grain number under abiotic stress conditions. Due to the complexity of the problem, stress-proofing our main cereal crops will be a challenging task and will require joint input from different research disciplines.
Rapid production of doubled haploids (DHs) through androgenesis is an important and promising method for genetic improvement of crop plants. Through androgenesis complete homozygous plants can be produced within a year compared to long inbreeding methods that may take several years and costly. Significant advantage of androgenesis is that it not only speeds up the process to achieve homozygosity, but also increases the selection efficiency. Though success in androgenesis has been achieved in many crop plants, yet there are certain limitations especially, low frequency of embryogenesis and regeneration in few species. In fact in many cereals, induction of embryos and regeneration of green plants is still a hurdle that one needs to overcome to improve the efficiency of androgenesis. Efficient androgenesis is usually induced by the successful application of different stress pretreatment. Since so many stress factors can trigger the reprogramming of microspores and that have been co-related to change the ultrastuctural changes of cells to embryos and finally haploid plants. It has been shown that certain pretreatment such as (i) physical stresses as cold, heat shock, starvation, drought stress, osmotic pressure, gamma irradiation, oxidative stress, reduced atmospheric pressure, and (ii) chemical treatments such as colchicine, heavy metal, ABA, CGA, AEC, Azetidine, 2-NHA, either individual or combined effect of more than one stress factors may positively influence androgenetic efficiency. This review highlights the recent and past work on uses of various abiotic stresses and pretreatments and their impact on enhancing the efficiency of androgenesis on some major crop species for the development of doubled haploid plants.
Nitric oxide (NO), polyamines (PAs), diamine oxidases (DAO) and polyamine oxidases (PAO) play important roles in wide spectrum of physiological processes such as germination, root development, flowering and senescence and in defence responses against abiotic and biotic stress conditions. This functional overlapping suggests interaction of NO and PA in signalling cascades. Exogenous application of PAs putrescine, spermidine and spermine to Arabidopsis seedlings induced NO production as observed by fluorimetry and fluorescence microscopy using the NO-binding fluorophores DAF-2 and DAR-4M. The observed NO release induced by 1 mM spermine treatment in the Arabidopsis seedlings was very rapid without apparent lag phase. These observations pave a new insight into PA-mediated signalling and NO as a potential mediator of PA actions. When comparing the functions of NO and PA in plant development and abiotic and biotic stresses common to both signalling components it can be speculated that NO may be a link between PA-mediated stress responses filing a gap between many known physiological effects of PAs and amelioration of stresses. NO production indicated by PAs could be mediated either by H(2)O(2), one reaction product of oxidation of PAs by DAO and PAO, or by unknown mechanisms involving PAs, DAO and PAO.