Development and characterization of marker-free and transgene insertion site-defined transgenic wheat with improved grain storability and fatty acid content.

Development of marker-free and transgene insertion site-defined (MFTID) transgenic plants is essential for safe application of transgenic crops. However, MFTID plants have not been reported for wheat (Triticum aestivum). Here, we prepared a RNAi cassette for suppressing lipoxygenase (LOX) gene expression in wheat grains using a double right border T-DNA vector. The resultant construct was introduced into wheat genome via Agrobacterium-mediated transformation, with four homozygous marker-free transgenic lines (namely GLRW-1, -3, -5 and -8) developed. Aided by the newly published wheat genome sequence, the T-DNA insertion sites in GLRW-3 and GLRW-8 were elucidated at base-pair resolution. While the T-DNA in GLRW-3 inserted in an intergenic region, that of GLRW-8 inactivated an endogenous gene, which was thus excluded from further analysis. Compared to wild -type (WT) control, GLRW-1, -3 and -5 showed decreased LOX gene expression, lower LOX activity and less lipid peroxidation in the grains; they also exhibited significantly higher germination rates and better seedling growth after artificial ageing treatment. Interestingly, the three GLRW lines also had substantially increased contents of several fatty acids (e.g., linoleic acid and linolenic acid) in their grain and flour samples than WT control. Collectively, our data suggest that suppression of grain LOX activity can be employed to improve the storability and fatty acid content of wheat seeds and that the MFTID line GLRW-3 is likely of commercial value. Our approach may also be useful for developing the MFTID transgenic lines of other crops with enhanced grain storability and fatty acid content.

The TuMYB46L-TuACO3 module regulates ethylene biosynthesis in einkorn wheat defense to powdery mildew.

Powdery mildew disease, elicited by the obligate fungal pathogen Blumeria graminis f.sp. tritici (Bgt), causes widespread yield losses in global wheat crop. However, the molecular mechanisms governing wheat defense to Bgt are still not well understood. Here we found that TuACO3, encoding the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase functioning in ethylene (ET) biosynthesis, was induced by Bgt infection of the einkorn wheat Triticum urartu, which was accompanied by increased ET content. Silencing TuACO3 decreased ET production and compromised wheat defense to Bgt, whereas both processes were enhanced in the transgenic wheat overexpressing TuACO3. TuMYB46L, phylogenetically related to Arabidopsis MYB transcription factor AtMYB46, was found to bind to the TuACO3 promoter region in yeast-one-hybrid and EMSA experiments. TuMYB46L expression decreased rapidly following Bgt infection. Silencing TuMYB46L promoted ET content and Bgt defense, but the reverse was observed when TuMYB46L was overexpressed. Hence, decreased expression of TuMYB46L permits elevated function of TuACO3 in ET biosynthesis in Bgt-infected wheat. The TuMYB46L-TuACO3 module regulates ET biosynthesis to promote einkorn wheat defense against Bgt. Furthermore, we found four chitinase genes acting downstream of the TuMYB46L-TuACO3 module. Collectively, our data shed a new light on the molecular mechanisms underlying wheat defense to Bgt.

Development of a new set of molecular markers for examining Glu-A1 variants in common wheat and ancestral species.

In common wheat (Triticum aestivum L.), allelic variations of Glu-A1 locus have important influences on grain end-use quality. Among the three Glu-A1 alleles, Glu-A1a and -A1b encode the high-molecular-weight glutenin subunits (HMW-GSs) 1Ax1 and 1Ax2*, respectively, whereas Glu-A1c does not specify any subunit. Here, we detected a total of 11 Glu-A1 locus haplotypes (H1 to H11) in three wheat species, by developing and using a new set of DNA markers (Xrj5, Xid3, Xrj6, Xid4 and Xrj7). The main haplotypes found in the diploid wheat T. urartu were H1, H4, H5 and H6, with H1 and H4 expressing both 1Ax and 1Ay subunits. The major haplotypes revealed for tetraploid wheat (T. turgidum) were H1, H8 and H9, with the lines expressing both 1Ax and 1Ay belonging to H1, H4 or H7. Four major haplotypes (H1, H9, H10 and H11) were discovered in common wheat, with Glu-A1a associated with H1 and H8, Glu-A1b with H10 or H11, and Glu-A1c with H9. The Glu-A1 locus haplotypes and the new set of DNA markers have potential to be used for more effectively studying and utilizing the molecular variations of Glu-A1 to improve the end-use quality of common wheat are discussed.

New insight into the function of wheat glutenin proteins as investigated with two series of genetic mutants.

Among the three major food crops (rice, wheat and maize), wheat is unique in accumulating gluten proteins in its grains. Of these proteins, the high and low molecular weight glutenin subunits (HMW-GSs and LMW-GSs) form glutenin macropolymers that are vital for the diverse end-uses of wheat grains. In this work, we developed a new series of deletion mutants lacking one or two of the three Glu-1 loci (Glu-A1, -B1 and -D1) specifying HMW-GSs. Comparative analysis of single and double deletion mutants reinforced the suggestion that Glu-D1 (encoding the HMW-GSs 1Dx2 and 1Dy12) has the largest effects on the parameters related to gluten and dough functionalities and breadmaking quality. Consistent with this suggestion, the deletion mutants lacking Glu-D1 or its combination with Glu-A1 or Glu-B1 generally exhibited strong decreases in functional glutenin macropolymers (FGMPs) and in the incorporation of HMW-GSs and LMW-GSs into FGMPs. Further examination of two knockout mutants missing 1Dx2 or 1Dy12 showed that 1Dx2 was clearly more effective than 1Dy12 in promoting FGMPs by enabling the incorporation of more HMW-GSs and LMW-GSs into FGMPs. The new insight obtained and the mutants developed by us may aid further research on the control of wheat end-use quality by glutenin proteins.

Genome-wide analysis of complex wheat gliadins, the dominant carriers of celiac disease epitopes.

Gliadins, specified by six compound chromosomal loci (Gli-A1/B1/D1 and Gli-A2/B2/D2) in hexaploid bread wheat, are the dominant carriers of celiac disease (CD) epitopes. Because of their complexity, genome-wide characterization of gliadins is a strong challenge. Here, we approached this challenge by combining transcriptomic, proteomic and bioinformatic investigations. Through third-generation RNA sequencing, full-length transcripts were identified for 52 gliadin genes in the bread wheat cultivar Xiaoyan 81. Of them, 42 were active and predicted to encode 25 α-, 11 γ-, one δ- and five ω-gliadins. Comparative proteomic analysis between Xiaoyan 81 and six newly-developed mutants each lacking one Gli locus indicated the accumulation of 38 gliadins in the mature grains. A novel group of α-gliadins (the CSTT group) was recognized to contain very few or no CD epitopes. The δ-gliadins identified here or previously did not carry CD epitopes. Finally, the mutant lacking Gli-D2 showed significant reductions in the most celiac-toxic α-gliadins and derivative CD epitopes. The insights and resources generated here should aid further studies on gliadin functions in CD and the breeding of healthier wheat.

High-throughput mining of E-genome-specific SNPs for characterizing Thinopyrum elongatum introgressions in common wheat.

Diploid Thinopyrum elongatum (EE, 2n = 2x = 14) and related polyploid species constitute an important gene pool for improving Triticeae grain and forage crops. However, the genomic and molecular marker resources are generally poor for these species. To aid the genetic, molecular, breeding and ecological studies involving Thinopyrum species, we developed a strategy for mining and validating E-genome-specific SNPs using Th. elongatum and common wheat (Triticum aestivum, AABBDD, 2n = 6x = 42) as experimental materials. By comparing the transcriptomes between Chinese Spring (CS, a common wheat variety) and the CS-Th. elongatum octoploid, 35,193 candidate SNPs between E genome genes and their common wheat orthologs were computed. Through comparative genomic analysis, these SNPs were putatively assigned to the seven individual E genome chromosomes. Among 420 randomly selected SNPs, 373 could be validated. Thus, approximately 89% of the mined SNPs may be authentic with respect to their polymorphism and chromosomal location. Using 14 such SNPs as molecular markers, complex E genome introgressions were reliably identified in 78 common wheat-Th. elongatum hybrids, and the structural feature of a novel recombinant chromosome formed by 6E and 7E was revealed. Finally, based on testing 33 SNPs assigned to chromosome 3E in multiple genotypes of Th. elongatum, Pseudoroegneria stipifolia (carrying the St genome related to E) and common wheat, we suggest that some of the SNP markers may also be applicable for genetic studies within and among the Thinopyrum species (populations) carrying E and/or St genomes in the future.

A novel allele of L-galactono-1,4-lactone dehydrogenase is associated with enhanced drought tolerance through affecting stomatal aperture in common wheat.

In higher plants, L-galactono-1,4-lactone dehydrogenase (GLDH) plays important roles in ascorbic acid (AsA) biosynthesis and assembly of respiration complex I. Here we report three homoeologous genes (TaGLDH-A1, -B1 and -D1) encoding common wheat GLDH isozymes and a unique allelic variant (TaGLDH-A1b) associated with enhanced drought tolerance. TaGLDH-A1, -B1 and -D1 were located on chromosomes 5A, 5B and 5D, respectively, and their transcripts were found in multiple organs. The three homoeologs each conferred increased GLDH activity when ectopically expressed in tobacco. Decreasing TaGLDH expression in wheat significantly reduced GLDH activity and AsA content. TaGLDH-A1b differed from wild type allele TaGLDH-A1a by an in-frame deletion of three nucleotides. TaGLDH-A1b was biochemically less active than TaGLDH-A1a, and the total GLDH activity levels were generally lower in the cultivars carrying TaGLDH-A1b relative to those with TaGLDH-A1a. Interestingly, TaGLDH-A1b cultivars showed stronger water deficiency tolerance than TaGLDH-A1a cultivars, and TaGLDH-A1b co-segregated with decreased leaf water loss in a F2 population. Finally, TaGLDH-A1b cultivars generally exhibited smaller leaf stomatal aperture than TaGLDH-A1a varieties in control or water deficiency environments. Our work provides new information on GLDH genes and function in higher plants. TaGLDH-A1b is likely useful for further studying and improving wheat tolerance to drought stress.

Coexpression network analysis of the genes regulated by two types of resistance responses to powdery mildew in wheat.

Powdery mildew disease caused by Blumeria graminis f. sp. tritici (Bgt) inflicts severe economic losses in wheat crops. A systematic understanding of the molecular mechanisms involved in wheat resistance to Bgt is essential for effectively controlling the disease. Here, using the diploid wheat Triticum urartu as a host, the genes regulated by immune (IM) and hypersensitive reaction (HR) resistance responses to Bgt were investigated through transcriptome sequencing. Four gene coexpression networks (GCNs) were developed using transcriptomic data generated for 20 T. urartu accessions showing IM, HR or susceptible responses. The powdery mildew resistance regulated (PMRR) genes whose expression was significantly correlated with Bgt resistance were identified, and they tended to be hubs and enriched in six major modules. A wide occurrence of negative regulation of PMRR genes was observed. Three new candidate immune receptor genes (TRIUR3_13045, TRIUR3_01037 and TRIUR3_06195) positively associated with Bgt resistance were discovered. Finally, the involvement of TRIUR3_01037 in Bgt resistance was tentatively verified through cosegregation analysis in a F2 population and functional expression assay in Bgt susceptible leaf cells. This research provides insights into the global network properties of PMRR genes. Potential molecular differences between IM and HR resistance responses to Bgt are discussed.

Genetic analysis of chromosomal loci affecting the content of insoluble glutenin in common wheat.

In common wheat, insoluble glutenin (IG) is an important fraction of flour glutenin macropolymers, and insoluble glutenin content (IGC) is positively associated with key end-use quality parameters. Here, we present a genetic analysis of the chromosomal loci affecting IGC with the data collected from 90 common wheat varieties cultivated in four environments. Statistical analysis showed that IGC was controlled mainly genetically and influenced by the environment. Among the major genetic components known to affect end-use quality, 1BL/1RS translocation had a significantly negative effect on IGC across all four environments. As to the different alleles of Glu-A1, -B1 and -D1 loci, Glu-A1a, Glu-B1b and Glu-D1d exhibited relatively strong positive effects on IGC in all environments. To identify new loci affecting IGC, association mapping with 1355 DArT markers was conducted. A total of 133 markers were found associated with IGC in two or more environments (P < 0.05), ten of which consistently affected IGC in all four environments. The phenotypic variance explained by the ten markers varied from 4.66% to 8.03%, and their elite alleles performed significantly better than the inferior counterparts in enhancing IGC. Among the ten markers, wPt-3743 and wPt-733835 reflected the action of Glu-D1, and wPt-664972 probably indicated the effect of Glu-A1. The other seven markers, forming three clusters on 2AL, 3BL or 7BL chromosome arms, represented newly identified genetic determinants of IGC. Our work provided novel insights into the genetic control of IGC, which may facilitate wheat end-use quality improvement through molecular breeding in the future.

Efficient isolation of ion beam-induced mutants for homoeologous loci in common wheat and comparison of the contributions of Glu-1 loci to gluten functionality.

KEY MESSAGE: Ion beam mutations can be efficiently isolated and deployed for functional comparison of homoeologous loci in polyploid plants, and Glu - 1 loci differ substantially in their contribution to wheat gluten functionality. To efficiently conduct genetic analysis, it is beneficial to have multiple types of mutants for the genes under investigation. Here, we demonstrate that ion beam-induced deletion mutants can be efficiently isolated for comparing the function of homoeologous loci of common wheat (Triticum aestivum). Through fragment analysis of PCR products from M2 plants, ion beam mutants lacking homoeologous Glu-A1, Glu-B1 or Glu-D1 loci, which encode high molecular weight glutenin subunits (HMW-GSs) and affect gluten functionality and end-use quality of common wheat, could be isolated simultaneously. Three deletion lines missing Glu-A1, Glu-B1 or Glu-D1 were developed from the original mutants, with the Glu-1 genomic regions deleted in these lines estimated using newly developed DNA markers. Apart from lacking the target HMW-GSs, the three lines all showed decreased accumulation of low molecular weight glutenin subunits (LMW-GSs) and increased amounts of gliadins. Based on the test data of five gluten and glutenin macropolymer (GMP) parameters obtained with grain samples harvested from two environments, we conclude that the genetic effects of Glu-1 loci on gluten functionality can be ranked as Glu-D1 > Glu-B1 > Glu-A1. Furthermore, it is suggested that Glu-1 loci contribute to gluten functionality both directly (by promoting the formation of GMP) and indirectly (through keeping the balance among HMW-GSs, LMW-GSs and gliadins). Finally, the efficient isolation of ion beam mutations for functional comparison of homoeologous loci in polyploid plants and the usefulness of Glu-1 deletion lines for further studying the contribution of Glu-1 loci to gluten functionality are discussed.

Haplotype variation of Glu-D1 locus and the origin of Glu-D1d allele conferring superior end-use qualities in common wheat.

In higher plants, seed storage proteins (SSPs) are frequently expressed from complex gene families, and allelic variation of SSP genes often affects the quality traits of crops. In common wheat, the Glu-D1 locus, encoding 1Dx and 1Dy SSPs, has multiple alleles. The Glu-D1d allele frequently confers superior end-use qualities to commercial wheat varieties. Here, we studied the haplotype structure of Glu-D1 genomic region and the origin of Glu-D1d. Using seven diagnostic DNA markers, 12 Glu-D1 haplotypes were detected among common wheat, European spelt wheat (T. spelta, a primitive hexaploid relative of common wheat), and Aegilops tauschii (the D genome donor of hexaploid wheat). By comparatively analyzing Glu-D1 haplotypes and their associated 1Dx and 1Dy genes, we deduce that the haplotype carrying Glu-D1d was likely differentiated in the ancestral hexaploid wheat around 10,000 years ago, and was subsequently transmitted to domesticated common wheat and T. spelta. A group of relatively ancient Glu-D1 haplotypes was discovered in Ae. tauschii, which may serve for the evolution of other haplotypes. Moreover, a number of new Glu-D1d variants were found in T. spelta. The main steps in Glu-D1d differentiation are proposed. The implications of our work for enhancing the utility of Glu-D1d in wheat quality improvement and studying the SSP alleles in other crop species are discussed.

Association analysis of genomic loci important for grain weight control in elite common wheat varieties cultivated with variable water and fertiliser supply.

Grain weight, an essential yield component, is under strong genetic control and markedly influenced by the environment. Here, by genome-wide association analysis with a panel of 94 elite common wheat varieties, 37 loci were found significantly associated with thousand-grain weight (TGW) in one or more environments differing in water and fertiliser levels. Five loci were stably associated with TGW under all 12 environments examined. Their elite alleles had positive effects on TGW. Four, two, three, and two loci were consistently associated with TGW in the irrigated and fertilised (IF), rainfed (RF), reduced nitrogen (RN), and reduced phosphorus (RP) environments. The elite alleles of the IF-specific loci enhanced TGW under well-resourced conditions, whereas those of the RF-, RN-, or RP-specific loci conferred tolerance to the TGW decrease when irrigation, nitrogen, or phosphorus were reduced. Moreover, the elite alleles of the environment-independent and -specific loci often acted additively to enhance TGW. Four additional loci were found associated with TGW in specific locations, one of which was shown to contribute to the TGW difference between two experimental sites. Further analysis of 14 associated loci revealed that nine affected both grain length and width, whereas the remaining loci influenced either grain length or width, indicating that these loci control grain weight by regulating kernel size. Finally, the elite allele of Xpsp3152 frequently co-segregated with the larger grain haplotype of TaGW2-6A, suggesting probable genetic and functional linkages between Xpsp3152 and GW2 that are important for grain weight control in cereal plants. Our study provides new knowledge on TGW control in elite common wheat lines, which may aid the improvement of wheat grain weight trait in further research.

HRS1 acts as a negative regulator of abscisic acid signaling to promote timely germination of Arabidopsis seeds.

In this work, we conducted functional analysis of Arabidopsis HRS1 gene in order to provide new insights into the mechanisms governing seed germination. Compared with wild type (WT) control, HRS1 knockout mutant (hrs1-1) exhibited significant germination delays on either normal medium or those supplemented with abscisic acid (ABA) or sodium chloride (NaCl), with the magnitude of the delay being substantially larger on the latter media. The hypersensitivity of hrs1-1 germination to ABA and NaCl required ABI3, ABI4 and ABI5, and was aggravated in the double mutant hrs1-1abi1-2 and triple mutant hrs1-1hab1-1abi1-2, indicating that HRS1 acts as a negative regulator of ABA signaling during seed germination. Consistent with this notion, HRS1 expression was found in the embryo axis, and was regulated both temporally and spatially, during seed germination. Further analysis showed that the delay of hrs1-1 germination under normal conditions was associated with reduction in the elongation of the cells located in the lower hypocotyl (LH) and transition zone (TZ) of embryo axis. Interestingly, the germination rate of hrs1-1 was more severely reduced by the inhibitor of cell elongation, and more significantly decreased by the suppressors of plasmalemma H(+)-ATPase activity, than that of WT control. The plasmalemma H(+)-ATPase activity in the germinating seeds of hrs1-1 was substantially lower than that exhibited by WT control, and fusicoccin, an activator of this pump, corrected the transient germination delay of hrs1-1. Together, our data suggest that HRS1 may be needed for suppressing ABA signaling in germinating embryo axis, which promotes the timely germination of Arabidopsis seeds probably by facilitating the proper function of plasmalemma H(+)-ATPase and the efficient elongation of LH and TZ cells.

Molecular analysis of phosphomannomutase (PMM) genes reveals a unique PMM duplication event in diverse Triticeae species and the main PMM isozymes in bread wheat tissues.

BACKGROUND: Phosphomannomutase (PMM) is an essential enzyme in eukaryotes. However, little is known about PMM gene and function in crop plants. Here, we report molecular evolutionary and biochemical analysis of PMM genes in bread wheat and related Triticeae species. RESULTS: Two sets of homologous PMM genes (TaPMM-1 and 2) were found in bread wheat, and two corresponding PMM genes were identified in the diploid progenitors of bread wheat and many other diploid Triticeae species. The duplication event yielding PMM-1 and 2 occurred before the radiation of diploid Triticeae genomes. The PMM gene family in wheat and relatives may evolve largely under purifying selection. Among the six TaPMM genes, the transcript levels of PMM-1 members were comparatively high and their recombinant proteins were all enzymatically active. However, PMM-2 homologs exhibited lower transcript levels, two of which were also inactive. TaPMM-A1, B1 and D1 were probably the main active isozymes in bread wheat tissues. The three isozymes differed from their counterparts in barley and Brachypodium distachyon in being more tolerant to elevated test temperatures. CONCLUSION: Our work identified the genes encoding PMM isozymes in bread wheat and relatives, uncovered a unique PMM duplication event in diverse Triticeae species, and revealed the main active PMM isozymes in bread wheat tissues. The knowledge obtained here improves the understanding of PMM evolution in eukaryotic organisms, and may facilitate further investigations of PMM function in the temperature adaptability of bread wheat.

New insights into the organization, recombination, expression and functional mechanism of low molecular weight glutenin subunit genes in bread wheat.

The bread-making quality of wheat is strongly influenced by multiple low molecular weight glutenin subunit (LMW-GS) proteins expressed in the seeds. However, the organization, recombination and expression of LMW-GS genes and their functional mechanism in bread-making are not well understood. Here we report a systematic molecular analysis of LMW-GS genes located at the orthologous Glu-3 loci (Glu-A3, B3 and D3) of bread wheat using complementary approaches (genome wide characterization of gene members, expression profiling, proteomic analysis). Fourteen unique LMW-GS genes were identified for Xiaoyan 54 (with superior bread-making quality). Molecular mapping and recombination analyses revealed that the three Glu-3 loci of Xiaoyan 54 harbored dissimilar numbers of LMW-GS genes and covered different genetic distances. The number of expressed LMW-GS in the seeds was higher in Xiaoyan 54 than in Jing 411 (with relatively poor bread-making quality). This correlated with the finding of higher numbers of active LMW-GS genes at the A3 and D3 loci in Xiaoyan 54. Association analysis using recombinant inbred lines suggested that positive interactions, conferred by genetic combinations of the Glu-3 locus alleles with more numerous active LMW-GS genes, were generally important for the recombinant progenies to attain high Zeleny sedimentation value (ZSV), an important indicator of bread-making quality. A higher number of active LMW-GS genes tended to lead to a more elevated ZSV, although this tendency was influenced by genetic background. This work provides substantial new insights into the genomic organization and expression of LMW-GS genes, and molecular genetic evidence suggesting that these genes contribute quantitatively to bread-making quality in hexaploid wheat. Our analysis also indicates that selection for high numbers of active LMW-GS genes can be used for improvement of bread-making quality in wheat breeding.

Overexpressing HRS1 confers hypersensitivity to low phosphate-elicited inhibition of primary root growth in Arabidopsis thaliana.

Phosphate (Pi) deficiency causes dramatic root system architecture (RSA) changes in higher plants. Here we report that overexpression of HRS1 leads to enhanced sensitivity to low Pi-elicited inhibition of primary root growth in Arabidopsis thaliana seedlings. Bioinformatic investigations uncovered that HRS1 and its six homologs encode putative G2-like transcription factors in Arabidopsis. Analysis of promoter::GUS reporter lines revealed that HRS1 transcripts were present mainly in the root hair region and root hair cells under Pi-sufficient conditions. Pi deprivation increased HRS1 expression level and expanded its expression domain. Although HRS1 knockout mutant did not differ from wild type (WT) control irrespective of Pi status, its overexpression lines were significantly more susceptible to low Pi-elicited primary root shortening. In both WT and HRS1 overexpression seedlings, low Pi-induced primary root shortening was accompanied by enhanced root hair cell differentiation, but this enhancement occurred to a greater extent in the latter genotype. Collectively, our data suggest that HRS1 may be involved in the modulation of primary root and root hair growth in Pi-deprived Arabidopsis seedlings, and provide useful clues for further research into the function of HRS1 and its homologs and the mechanisms behind RSA changes under Pi-deficient conditions.

Transgenic expression of MYB15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana.

Abiotic stresses cause serious crop losses. Knowledge on genes functioning in plant responses to adverse growth conditions is essential for developing stress tolerant crops. Here we report that transgenic expression of MYB15, encoding a R2R3 MYB transcription factor in Arabidopsis thaliana, conferred hypersensitivity to exogenous abscisic acid (ABA) and improved tolerance to drought and salt stresses. The promoter of MYB15 was active in not only vegetative and reproductive organs but also the guard cells of stomata. Its transcript level was substantially upregulated by ABA, drought or salt treatments. Compared with wild type (WT) control, MYB15 overexpression lines were hypersensitive to ABA in germination assays, more susceptible to ABA-elicited inhibition of root elongation, and more sensitive to ABA-induced stomatal closure. In line with the above findings, the transcript levels of ABA biosynthesis (ABA1, ABA2), signaling (ABI3), and responsive genes (AtADH1, RD22, RD29B, AtEM6) were generally higher in MYB15 overexpression seedlings than in WT controls after treatment with ABA. MYB15 overexpression lines displayed improved survival and reduced water loss rates than WT control under water deficiency conditions. These overexpression lines also displayed higher tolerance to NaCl stress. Collectively, our data suggest that overexpression of MYB15 improves drought and salt tolerance in Arabidopsis possibly by enhancing the expression levels of the genes involved in ABA biosynthesis and signaling, and those encoding the stress-protective proteins.

Reduction of root flavonoid level and its potential involvement in lateral root emergence in Arabidopsis thaliana grown under low phosphate supply.

Although it is well known that phosphate (Pi) deficiency affects flavonoid accumulation in higher plants, knowledge on the regulation and potential function of flavonoids in the plants grown with low Pi supply is lacking. In this work, we found that low Pi treatment caused significant reduction of root flavonoid (e.g. quercetin, kaempferol and their derivatives) levels in both Columbia (Col-0) and Landsberg erecta (Ler) ecotypes of Arabidopsis thaliana (L.) Heynh. Further investigations revealed that the dysfunction of PHR1, PHO1, PHO2 and NPC4 did not affect the decrease of root flavonoid level by low Pi treatment. In contrast, pldζ2, a knockout mutant of the Arabidopsis phospholipase Dζ2, exhibited defects in the reduction of root flavonoid level and lateral root (LR) emergence under low Pi conditions. When grown under low Pi supply, the transport of auxin from the shoot apex into the root, expression of the auxin responsive DR5::GUS marker and induction of the auxin responsive genes were all significantly less efficient in pldζ2 than in wild-type (WT) control. This is the first report on the reduction of root flavonoid level and its likely contribution to increased LR emergence in Arabidopsis under Pi deficiency conditions, which may facilitate the adaptation of plants to the growth environments with poor Pi availability.

Molecular and functional analysis of hypoxanthine-guanine phosphoribosyltransferase from Arabidopsis thaliana.

Hypoxanthine-guanine phosphoribosyltransferase (HGPT) occurs in both eukaryotic and prokaryotic organisms. However, the molecular and functional properties of plant HGPT are not well understood. In this study, it was found that the putative HGPT proteins from dicot and monocot plant species exhibited significant identities to their homologs from other cellular organisms. Ectopic expression of the HGPTs from Arabidopsis, soybean or wheat complemented HGPT deficiency in the hpt1 mutant of Saccharomyces cerevisiae. Recombinant Arabidopsis HGPT (AtHGPT) catalyzed both forward and reverse reactions in in vitro biochemical assays. The relative catalytic efficiency for the synthesis of guanosine monophosphate (GMP) was significantly greater than that for the production of guanine from GMP. Further investigations led to identification of the candidate residues that may form the pyrophosphate (PPi) binding loop in AtHGPT. AtHGPT expression level was dynamically regulated in Arabidopsis organs and during leaf development and senescence and seed germination. AtHGPT knockout mutant germinated more slowly than wild type control, whereas its overexpression mutant exhibited accelerated germination. Collectively, the data suggest that functional HGPTs are expressed in higher plants. In Arabidopsis, HGPT plays an active role in the salvage of purine bases and its activity is required for efficient seed germination.

Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana.

Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine-tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose-1-phosphate into mannose-6-phosphate and glucose-1-phosphate into glucose-6-phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants.