Genome-wide association and targeted transcriptomic analyses reveal loci and candidate genes regulating preharvest sprouting in barley.

KEY MESSAGE: Genome-wide association study of diverse barley genotypes identified loci, single nucleotide polymorphisms and candidate genes that control seed dormancy and therefore enhance resistance to preharvest sprouting. Preharvest sprouting (PHS) causes significant yield and quality loss in barley and it is strongly associated with the level of seed dormancy. This study performed genome-wide association study using a collection of 255 diverse barley genotypes grown over four environments to identify loci controlling dormancy/PHS. Our phenotypic analysis revealed substantial variation in germination index/dormancy levels among the barley genotypes. Marker-trait association and linkage disequilibrium (LD) decay analyses identified 16 single nucleotide polymorphisms (SNPs) and two QTLs associated with dormancy/PHS, respectively, on chromosome 3H and 5H explaining 6.9% to 11.1% of the phenotypic variation. QTL.5H consist of 14 SNPs of which 12 SNPs satisfy the FDR threshold of α = 0.05, and it may represent the SD2 locus. The QTL on 3H consists of one SNP that doesn't satisfy FDR (α = 0.05). Genes harbouring the significant SNPs were analyzed for their expression pattern in the seeds of selected dormant and non-dormant genotypes. Of these genes, HvRCD1, HvPSRP1 and HvF3H exhibited differential expression between the dormant and non-dormant seed samples, suggesting their role in controlling seed dormancy/PHS. Three SNPs located within the differentially expressed genes residing in QTL.5H explained considerable phenotypic variation (≥ 8.6%), suggesting their importance in regulating PHS resistance. Analysis of the SNP marker data in QTL.5H identified a haplotype for PHS resistance. Overall, the study identified loci, SNPs and candidate genes that control dormancy and therefore play important roles in enhancing PHS resistance in barley through marker-assisted breeding.

Jasmonate regulates seed dormancy in wheat via modulating the balance between gibberellin and abscisic acid.

Jasmonate (JA) regulates seed dormancy and germination; however, the underlying mechanisms remain poorly understood. Furthermore, it is unclear if JA is an essential regulator of dormancy and germination. We investigated whether the role of JA in regulating seed dormancy in wheat (Triticum aestivum L.) is mediated by modulation of gibberellin (GA)/abscisic acid (ABA) balance and if the reciprocal modulation of JA level and sensitivity is required for GA-mediated dormancy loss using physiological, pharmacological, and targeted transcriptomic and metabolomic approaches. JA-induced dormancy release in wheat seeds was associated with no change in GA level but up-regulation of GA signaling and ABA catabolism genes, and reduction of the ABA level. Although JA did not affect the expression levels of ABA signaling genes, up-regulation of germination-associated genes indicates a contribution of reduced ABA sensitivity to dormancy release. After-ripening-mediated dormancy loss was also associated with JA-GA synergistic and JA-ABA antagonistic interplays. The prevalence of no effect of GA, which effectively broke dormancy, on the JA-Ile level and expression patterns of JA biosynthesis/signaling and responsive genes reflects that GA-mediated dormancy release occurs independently of JA. Our study concludes that JA induces seed dormancy release in wheat via modulating ABA/GA balance; however, JA is not an essential regulator of dormancy and germination.

Salicylic Acid Enhances Adventitious Root and Aerenchyma Formation in Wheat under Waterlogged Conditions.

The present study investigated the role of salicylic acid (SA) in regulating morpho-anatomical adaptive responses of a wheat plant to waterlogging. Our pharmacological study showed that treatment of waterlogged wheat plants with exogenous SA promotes the formation axile roots and surface adventitious roots that originate from basal stem nodes, but inhibits their elongation, leading to the formation of a shallow root system. The treatment also enhanced axile root formation in non-waterlogged plants but with only slight reductions in their length and branch root formation. Exogenous SA enhanced the formation of root aerenchyma, a key anatomical adaptive response of plants to waterlogging. Consistent with these results, waterlogging enhanced SA content in the root via expression of specific isochorismate synthase (ICS; ICS1 and ICS2) and phenylalanine ammonia lyase (PAL; PAL4, PAL5 and PAL6) genes and in the stem nodes via expression of specific PAL (PAL5 and PAL6) genes. Although not to the same level observed in waterlogged plants, exogenous SA also induced aerenchyma formation in non-waterlogged plants. The findings of this study furthermore indicated that inhibition of ethylene synthesis in SA treated non-waterlogged and waterlogged plants does not have any effect on SA-induced emergence of axile and/or surface adventitious roots but represses SA-mediated induction of aerenchyma formation. These results highlight that the role of SA in promoting the development of axile and surface adventitious roots in waterlogged wheat plants is ethylene independent while the induction of aerenchyma formation by SA requires the presence of ethylene.

A shift in abscisic acid/gibberellin balance underlies retention of dormancy induced by seed development temperature.

Through a combination of physiological, pharmacological, molecular and targeted metabolomics approaches, we showed that retention of wheat (Triticum aestivum L.) seed dormancy levels induced by low and high seed development temperatures during post-desiccation phases is associated with modulation of gibberellin (GA) level and seed responsiveness to abscisic acid (ABA) and GA via expression of TaABI5 and TaGAMYB, respectively. Dormancy retention during imbibition, however, is associated with modulations of both ABA level and responsiveness via expression of specific ABA metabolism (TaNCED2 and TaCYP707A1) and signalling (TaPYL2, TaSnRK2, TaABI3, TaABI4 and TaABI5) genes, and alterations of GA levels and responsiveness through expression of specific GA biosynthesis (TaGA20ox1, TaGA20ox2 and TaGA3ox2) and signalling (TaGID1 and TaGID2) genes, respectively. Expression patterns of GA signalling genes, TaRHT1 and TaGAMYB, lacked positive correlation with that of GA regulated genes and dormancy level observed in seeds developed at the two temperatures, implying their regulation at post-transcriptional level. Our results overall implicate that a shift in ABA/GA balance underlies retention of dormancy levels induced by seed development temperature during post-desiccation and imbibition phases. Consistently, genes regulated by ABA and GA during imbibition overlapped with those differentially expressed between imbibed seeds developed at the two temperatures and mediate different biological functions.

Identification of loci for pre-harvest sprouting resistance in the highly dormant spring wheat RL4137.

KEY MESSAGE: Combination of RL4137 alleles at three QTLs on chromosomes 4A, 6B and 6D, and 'Roblin' allele at a novel QTL on chromosome 1D increases pre-harvest sprouting resistance in 'Roblin'/RL4137 doubled haploid population. Pre-harvest sprouting (PHS) significantly reduces wheat grain yield and quality. Therefore, identifying quantitative trait loci (QTL) for PHS resistance is key to facilitate marker-assisted breeding. To this end, we studied PHS in a population of 330 doubled haploid (DH) lines derived from 'Roblin'/RL4137. The parental and DH lines were examined for their PHS phenotype based on speed of germination index in five environments and genotyped using the wheat Infinium 90 K SNP array. A total of five QTLs were detected on linkage groups 1D, 4A.2, 6B.1, 6D and 7A over the five environments. The QTL QPhs.umb-4A on linkage group 4A.2 was the most consistent across all environments and explained 40-50% of phenotypic variation. The QTL on 1D is a novel QTL and explained 1.99-2.33% of phenotypic variation. The QTLs on 6B.1 and 6D each explained 3.09-4.33% and 1.62-2.45% of phenotypic variation, respectively. A combination of four stable QTLs on linkage groups 1D, 4A.2, 6B.1 and 6D greatly increased PHS resistance. Allelic effects for the QTLs QPhs.umb-4A, QPhs.umb-6B and QPhs.umb-6D were contributed by RL4137, whereas 'Roblin' contributed the resistant allele for QPhs.umb-1D. QPhs.umb-4A was required for strong dormancy in the 'Roblin'/RL4137 DH population, and the presence of QTLs QPhs.umb-1D, QPhs.umb-6B and QPhs.umb-6D incrementally increased dormancy; DH lines carrying all four QTLs are considerably more dormant than those carrying only QPhs.umb-4A or none of the four QTLs. Thus, the QTLs identified in this study have the potential to improve PHS resistance in spring wheat.

Ethylene regulates post-germination seedling growth in wheat through spatial and temporal modulation of ABA/GA balance.

This study aimed to gain insights into the molecular mechanisms underlying the role of ethylene in regulating germination and seedling growth in wheat by combining pharmacological, molecular, and metabolomics approaches. Our study showed that ethylene does not affect radicle protrusion but controls post-germination endospermic starch degradation through transcriptional regulation of specific α-amylase and α-glucosidase genes, and this effect is mediated by alteration of endospermic bioactive gibberellin (GA) levels, and GA sensitivity via expression of the GA signaling gene, TaGAMYB. Our data implicated ethylene as a positive regulator of embryo axis and coleoptile growth through transcriptional regulation of specific TaEXPA genes. These effects were associated with modulation of GA levels and sensitivity, through expression of GA metabolism (TaGA20ox1, TaGA3ox2, and TaGA2ox6) and signaling (TaGAMYB) genes, respectively, and/or the abscisic acid (ABA) level and sensitivity, via expression of specific ABA metabolism (TaNCED2 or TaCYP707A1) and signaling (TaABI3) genes, respectively. Ethylene appeared to regulate the expression of TaEXPA3 and thereby root growth through its control of coleoptile ABA metabolism, and root ABA signaling via expression of TaABI3 and TaABI5. These results show that spatiotemporal modulation of ABA/GA balance mediates the role of ethylene in regulating post-germination storage starch degradation and seedling growth in wheat.

Phytoglobins regulate nitric oxide-dependent abscisic acid synthesis and ethylene-induced program cell death in developing maize somatic embryos.

MAIN CONCLUSION: During maize somatic embryogenesis, suppression of phytoglobins (Pgbs) reduced ABA levels leading to ethylene-induced programmed cell death in the developing embryos. These effects modulate embryonic yield depending on the cellular localization of specific phytoglobin gene expression. Suppression of Zea mays phytoglobins (ZmPgb1.1 or ZmPgb1.2) during somatic embryogenesis induces programmed cell death (PCD) by elevating nitric oxide (NO). While ZmPgb1.1 is expressed in many embryonic domains and its suppression results in embryo abortion, ZmPgb1.2 is expressed in the basal cells anchoring the embryos to the embryogenic tissue. Down-regulation of ZmPgb1.2 is required to induce PCD in these anchor cells allowing the embryos to develop further. Exogenous applications of ABA could reverse the effects caused by the suppression of either of the two ZmPgbs. A depletion of ABA, ascribed to a down-regulation of biosynthetic genes, was observed in those embryonic domains where the respective ZmPgbs were repressed. These effects were mediated by NO. Depletion in ABA content increased the transcription of genes participating in the synthesis and response of ethylene, as well as the accumulation of ethylene, which influenced embryogenesis. Somatic embryo number was reduced by high ethylene levels and increased with pharmacological treatments suppressing ethylene synthesis. The ethylene inhibition of embryogenesis was linked to the production of reactive oxygen species (ROS) and the execution of PCD. Integration of ABA and ethylene in the ZmPgb regulation of embryogenesis is proposed in a model where NO accumulates in ZmPgb-suppressing cells, decreasing the level of ABA. Abscisic acid inhibits ethylene biosynthesis and the NO-mediated depletion of ABA relieves this inhibition causing ethylene to accumulate. Elevated ethylene levels trigger production of ROS and induce PCD. Ethylene-induced PCD in the ZmPgb1.1-suppressing line [ZmPgb1.1 (A)] leads to embryo abortion, while PCD in the ZmPgb1.2-suppressing line [ZmPgb1.2 (A)] results in the elimination of the anchor cells and the successful development of the embryos.

Spatiotemporal modulation of abscisic acid and gibberellin metabolism and signalling mediates the effects of suboptimal and supraoptimal temperatures on seed germination in wheat (Triticum aestivum L.).

Seed germination is a complex process regulated by intrinsic hormonal cues such as abscisic acid (ABA) and gibberellin (GA), and environmental signals including temperature. Using pharmacological, molecular and metabolomics approaches, we show that supraoptimal temperature delays wheat seed germination through maintaining elevated embryonic ABA level via increased expression of ABA biosynthetic genes (TaNCED1 and TaNCED2), increasing embryo ABA sensitivity through upregulation of genes regulating ABA signalling positively (TaPYL5, TaSnRK2, ABI3 and ABI5) and decreasing embryo GA sensitivity via induction of TaRHT1 that regulates GA signalling negatively. Endospermic ABA and GA appeared to have minimal roles in regulating germination at supraoptimal temperature. Germination inhibition by suboptimal temperature is associated with elevated ABA level in the embryo and endosperm tissues, mediated by induction of TaNCEDs and decreased expression of endospermic ABA catabolic genes (TaCYP707As), and increased ABA sensitivity in both tissues via upregulation of TaPYL5, TaSnRK2, ABI3 and ABI5 in the embryo and TaSnRK2 and ABI5 in the endosperm. Furthermore, suboptimal temperature suppresses GA synthesis in both tissues and GA sensitivity in the embryo via repressing GA biosynthetic genes (TaGA20ox and TaGA3ox2) and inducing TaRHT1, respectively. These results highlight that spatiotemporal modulation of ABA and GA metabolism and signalling in wheat seeds underlies germination response to temperature.

Hormonal regulation in adventitious roots and during their emergence under waterlogged conditions in wheat.

To gain insights into the molecular mechanisms underlying hormonal regulation in adventitious roots and during their emergence under waterlogged conditions in wheat, the present study investigated transcriptional regulation of genes related to hormone metabolism and transport in the root and stem node tissues. Waterlogging-induced inhibition of axile root elongation and lateral root formation, and promotion of surface adventitious and axile root emergence and aerenchyma formation are associated with enhanced expression levels of ethylene biosynthesis genes, ACS7 and ACO2, in both tissues. Inhibition of axile root elongation is also related to increased root indole acetic acid (IAA) and jasmonate (JA) levels that are associated with up-regulation of specific IAA biosynthesis/transport (TDC, YUC1, and PIN9) and JA metabolism (LOX8, AOS1, AOC1, and JAR1) genes, and transcriptional alteration of gibberellin (GA) metabolism genes (GA3ox2 and GA2ox8). Adventitious root emergence from waterlogged stem nodes is associated with increased levels of IAA and GA but decreased levels of cytokinin and abscisic acid (ABA), which are regulated through the expression of specific IAA biosynthesis/transport (TDC, YUC1, and PIN9), cytokinin metabolism (IPT5-2, LOG1, CKX5, and ZOG2), ABA biosynthesis (NCED1 and NCED2), and GA metabolism (GA3ox2 and GA2ox8) genes. These results enhance our understanding of the molecular mechanisms underlying the adaptive response of wheat to waterlogging.

Seed maturation associated transcriptional programs and regulatory networks underlying genotypic difference in seed dormancy and size/weight in wheat (Triticum aestivum L.).

BACKGROUND: Maturation forms one of the critical seed developmental phases and it is characterized mainly by programmed cell death, dormancy and desiccation, however, the transcriptional programs and regulatory networks underlying acquisition of dormancy and deposition of storage reserves during the maturation phase of seed development are poorly understood in wheat. The present study performed comparative spatiotemporal transcriptomic analysis of seed maturation in two wheat genotypes with contrasting seed weight/size and dormancy phenotype. RESULTS: The embryo and endosperm tissues of maturing seeds appeared to exhibit genotype-specific temporal shifts in gene expression profile that might contribute to the seed phenotypic variations. Functional annotations of gene clusters suggest that the two tissues exhibit distinct but genotypically overlapping molecular functions. Motif enrichment predicts genotypically distinct abscisic acid (ABA) and gibberellin (GA) regulated transcriptional networks contribute to the contrasting seed weight/size and dormancy phenotypes between the two genotypes. While other ABA responsive element (ABRE) motifs are enriched in both genotypes, the prevalence of G-box-like motif specifically in tissues of the dormant genotype suggests distinct ABA mediated transcriptional mechanisms control the establishment of dormancy during seed maturation. In agreement with this, the bZIP transcription factors that co-express with ABRE enriched embryonic genes differ with genotype. The enrichment of SITEIIATCYTC motif specifically in embryo clusters of maturing seeds irrespective of genotype predicts a tissue specific role for the respective TCP transcription factors with no or minimal contribution to the variations in seed dormancy. CONCLUSION: The results of this study advance our understanding of the seed maturation associated molecular mechanisms underlying variation in dormancy and weight/size in wheat seeds, which is a critical step towards the designing of molecular strategies for enhancing seed yield and quality.

Lignin biosynthesis in wheat (Triticum aestivum L.): its response to waterlogging and association with hormonal levels.

BACKGROUND: Lignin is an important structural component of plant cell wall that confers mechanical strength and tolerance against biotic and abiotic stressors; however it affects the use of biomass such as wheat straw for some industrial applications such as biofuel production. Genetic alteration of lignin quantity and quality has been considered as a viable option to overcome this problem. However, the molecular mechanisms underlying lignin formation in wheat biomass has not been studied. Combining molecular and biochemical approaches, the present study investigated the transcriptional regulation of lignin biosynthesis in two wheat cultivars with varying lodging characteristics and also in response to waterlogging. It also examined the association of lignin level in tissues with that of plant hormones implicated in the control of lignin biosynthesis. RESULTS: Analysis of lignin biosynthesis in the two wheat cultivars revealed a close association of lodging resistance with internode lignin content and expression of 4-coumarate:CoA ligase1 (4CL1), p-coumarate 3-hydroxylase1 (C3H1), cinnamoyl-CoA reductase2 (CCR2), ferulate 5-hydroxylase2 (F5H2) and caffeic acid O-methyltransferase2 (COMT2), which are among the genes highly expressed in wheat tissues, implying the importance of these genes in mediating lignin deposition in wheat stem. Waterlogging of wheat plants reduced internode lignin content, and this effect is accompanied by transcriptional repression of three of the genes characterized as highly expressed in wheat internode including phenylalanine ammonia-lyase6 (PAL6), CCR2 and F5H2, and decreased activity of PAL. Expression of the other genes was, however, induced by waterlogging, suggesting their role in the synthesis of other phenylpropanoid-derived molecules with roles in stress responses. Moreover, difference in internode lignin content between cultivars or change in its level due to waterlogging is associated with the level of cytokinin. CONCLUSION: Lodging resistance, tolerance against biotic and abiotic stresses and feedstock quality of wheat biomass are closely associated with its lignin content. Therefore, the findings of this study provide important insights into the molecular mechanisms underlying lignin formation in wheat, an important step towards the development of molecular tools that can facilitate the breeding of wheat cultivars for optimized lignin content and enhanced feedstock quality without affecting other lignin-related agronomic benefits.

Abscisic acid metabolic genes of wheat (Triticum aestivum L.): identification and insights into their functionality in seed dormancy and dehydration tolerance.

MAIN CONCLUSION: The three homeologues of wheat NCED2 were identified; the wheat NCED2A and CYP707A1B affect seed ABA level and dormancy but not leaf ABA level and transpirational water loss in Arabidopsis. Biosynthesis and catabolism of abscisic acid (ABA) in plants are primarily regulated by 9-cis-epoxycarotenoid dioxygenases (NCEDs) and ABA 8'-hydroxylase (ABA8'OH), respectively. The present study identified the complete coding sequences of a second NCED gene, designated as TaNCED2, and its homeologues (TaNCED2A, TaNCED2B and TaNCED2D) in hexaploid wheat, and characterized its functionality in seed dormancy and leaf dehydration tolerance using the TaNCED2A homeologue. The study also investigated the role of the B genome copy of the cytochrome P450 monooxygenase 707A1 (CYP707A1) gene of hexaploid wheat (TaCYP707A1B), which encodes ABA8'OH, in regulating the two traits as this has not been studied before. Ectopic expression of TaNCED2A and TaCYP707A1B in Arabidopsis resulted in altered seed ABA level and dormancy with no effect on leaf ABA content and transpirational water loss. To gain insights into the physiological roles of TaNCED2 and TaCYP707A1 in wheat, the study examined their spatiotemporal expression patterns and determined the genomic contributions of transcripts to their total expression.

Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis.

Programmed cell death (PCD) in multicellular organisms is a vital process in growth, development, and stress responses that contributes to the formation of tissues and organs. Although numerous studies have defined the molecular participants in apoptotic and PCD cascades, successful identification of early master regulators that target specific cells to live or die is limited. Using Zea mays somatic embryogenesis as a model system, we report that the expressions of two plant hemoglobin (Hb) genes (ZmHb1 and ZmHb2) regulate the cell survival/death decision that influences somatic embryogenesis through their cell-specific localization patterns. Suppression of either of the two ZmHbs is sufficient to induce PCD through a pathway initiated by elevated NO and Zn2+ levels and mediated by production of reactive oxygen species. The effect of the death program on the fate of the developing embryos is dependent on the localization patterns of the two ZmHbs. During somatic embryogenesis, ZmHb2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, whereas ZmHb1 transcripts extend to several embryonic domains. Suppression of ZmHb2 induces PCD in the anchoring cells, allowing the embryos to develop further, whereas suppression of ZmHb1 results in massive PCD, leading to abortion. We conclude that regulation of the expression of these ZmHbs has the capability to determine the developmental fate of the embryogenic tissue during somatic embryogenesis through their effect on PCD. This unique regulation might have implications for development and differentiation in other species.

Modulation in the ratio of abscisic acid to gibberellin level determines genetic variation of seed dormancy in barley (Hordeum vulgare L.).

Abscisic acid (ABA) and gibberellin (GA) are major regulators of seed dormancy, an adaptive trait closely associated with preharvest sprouting. This study examined transcriptional regulation of ABA and GA metabolism genes and modulation of ABA and GA levels in seeds of barley genotypes exhibiting a range of dormancy phenotype. We observed a very strong negative correlation between genetic variation in seed germination and embryonic ABA level (r = 0.85), which is regulated by transcriptional modulation of HvNCED1 and/or HvCYP707A genes. A strong positive correlation was evident between variation in seed germination and GA level (r = 0.64), mediated via transcriptional regulation of GA biosynthesis genes, HvGA20ox2 and/or HvGA3oxs, and GA catabolism genes, HvGA2ox3 and/or HvGA3ox6. Modulation of the ABA and GA levels in the genotypes led to the prevalence of ABA to GA level ratio that exhibited a very strong negative correlation (r = 0.84) with seed germination, highlighting the importance of a shift in ABA/GA ratio in determining genetic variation of dormancy in barley seeds. Our results overall show that transcriptional regulation of specific ABA and GA metabolism genes underlies genetic variation in ABA/GA ratio and seed dormancy, reflecting the potential use of these genes as molecular tools for enhancing preharvest sprouting resistance in barley.

Identification and characterization of the three homeologues of a new sucrose transporter in hexaploid wheat (Triticum aestivum L.).

BACKGROUND: Sucrose transporters (SUTs) play important roles in regulating the translocation of assimilates from source to sink tissues. Identification and characterization of new SUTs in economically important crops such as wheat provide insights into their role in determining seed yield. To date, however, only one SUT of wheat has been reported and functionally characterized. The present study reports the isolation and characterization of a new SUT, designated as TaSUT2, and its homeologues (TaSUT2A, TaSUT2B and TaSUT2D) in hexaploid wheat (Triticum aestivum L.). RESULTS: TaSUT2A and TaSUT2B genes each encode a protein with 506 amino acids, whereas TaSUT2D encodes a protein of 508 amino acids. The molecular mass of these proteins is predicted to be ~ 54 kDA. Topological analysis of the amino acid sequences of the three homeologues revealed that they contain 12 transmembrane spanning helices, which are described as distinct characteristic features of glycoside-pentoside-hexuronide cation symporter family that includes all known plant SUTs, and a histidine residue that appears to be localized at and associated conformationally with the sucrose binding site. Yeast SUSY7/ura3 strain cells transformed with TaSUT2A, TaSUT2B and TaSUT2D were able to uptake sucrose and grow on a medium containing sucrose as a sole source of carbon; however, our subcellular localization study with plant cells revealed that TaSUT2 is localized to the tonoplast. The expression of TaSUT2 was detected in the source, including flag leaf blade, flag leaf sheath, peduncle, glumes, palea and lemma, and sink (seed) tissues. The relative contributions of the three genomes of wheat to the total expression of TaSUT2 appear to differ with tissues and developmental stages. At the cellular level, TaSUT2 is expressed mainly in the vein of developing seeds and subepidermal mesophyll cells of the leaf blade. CONCLUSION: This study demonstrated that TaSUT2 is a new wheat SUT protein. Given that TaSUT2 is localized to the tonoplast and sucrose is temporarily stored in the vacuoles of both source and sink tissues, our data imply that TaSUT2 is involved in the intracellular partitioning of sucrose, particularly between the vacuole and cytoplasm.

Integrated analysis of seed proteome and mRNA oxidation reveals distinct post-transcriptional features regulating dormancy in wheat (Triticum aestivum L.).

Wheat seeds can be released from a dormant state by after-ripening; however, the underlying molecular mechanisms are still mostly unknown. We previously identified transcriptional programmes involved in the regulation of after-ripening-mediated seed dormancy decay in wheat (Triticum aestivum L.). Here, we show that seed dormancy maintenance and its release by dry after-ripening in wheat is associated with oxidative modification of distinct seed-stored mRNAs that mainly correspond to oxidative phosphorylation, ribosome biogenesis, nutrient reservoir and α-amylase inhibitor activities, suggesting the significance of post-transcriptional repression of these biological processes in regulating seed dormancy. We further show that after-ripening induced seed dormancy release in wheat is mediated by differential expression of specific proteins in both dry and hydrated states, including those involved in proteolysis, cellular signalling, translation and energy metabolism. Among the genes corresponding to these proteins, the expression of those encoding α-amylase/trypsin inhibitor and starch synthase appears to be regulated by mRNA oxidation. Co-expression analysis of the probesets differentially expressed and oxidized during dry after-ripening along with those corresponding to proteins differentially regulated between dormant and after-ripened seeds produced three co-expressed gene clusters containing more candidate genes potentially involved in the regulation of seed dormancy in wheat. Two of the three clusters are enriched with elements that are either abscisic acid (ABA) responsive or recognized by ABA-regulated transcription factors, indicating the association between wheat seed dormancy and ABA sensitivity.

Transcriptomic data of imbibed wheat (Triticum aestivum L.) seeds developed at different temperatures.

The present data profile a large scale transcriptome changes associated with variations in seed dormancy level induced by seed development temperature in hexaploid wheat. Seed dormancy is an important trait that inhibits seed germination under optimal conditions and therefore has important implication in preventing the incidence of preharvest sprouting, which refers to the germination of seeds on the mother plant prior to harvest, in wheat. Since preharvest sprouting, which causes a significant reduction in seed yield and quality in wheat, is closely associated with low level of seed dormancy manifested in modern wheat cultivars, it is important to develop wheat cultivars with optimal level of dormancy to enhance wheat yield and quality. Thus, elucidation of the molecular mechanisms that regulate seed dormancy is critical for the development of preharvest sprouting resistant wheat cultivars. The data we are presenting here were generated from total RNA samples extracted from imbibed seeds of a dormant wheat (Triticum aestivum L.) genotype that were developed at different temperatures using the Affymetrix GeneChip Wheat Genome Array. The raw and normalized formats of these data are deposited in the NCBI's gene expression data repository, Gene Expression Ominbus (GEO) with accession number GSE153527.

Temporal and spatial transcriptional regulation of phytohormone metabolism during seed development in barley (Hordeum vulgare L.).

Plant hormones play important roles in seed development; however, transcriptional regulation of their metabolism and levels of the respective bioactive forms during barley seed development is poorly understood. To this end, this study performed a comprehensive analysis of changes in the expression patterns phytohormone metabolism genes and levels of the respective bioactive forms in the embryo and endosperm tissues. Our study showed the presence of elevated levels of abscisic acid (ABA), bioactive forms of gibberellins (GAs), jasmonate (JA) and cytokinins (CKs), auxin and salicylic acid (SA) in the endosperm and embryo tissues at early stage of seed filling (SF). The levels of all hormones in both tissues, except that of ABA, decreased to low levels during SF. In contrast, embryonic ABA level increased during SF and peaked at physiological maturity (PM) while the endospermic ABA was maintained at a similar level observed during SF. Although its level decreased high amount of ABA was still present in the embryo during post-PM. We detected low levels of ABA in the endosperm and all the other hormones in both tissues during post-PM phase except the relatively higher levels of jasmonoyl-isoleucine and SA detected at late stage of post-PM. Our data also showed that spatiotemporal changes in the levels of plant hormones during barley seed development are mediated by the expression of specific genes involved in their respective metabolic pathways. These results indicate that seed development in barley is mediated by spatiotemporal modulation in the metabolism and levels of plant hormones.

Heterologous functional analysis and expression patterns of gibberellin 2-oxidase genes of barley (Hordeum vulgare L.).

The level of bioactive gibberellins (GAs) in plants is regulated partly by their inactivation, mainly by the action of GA 2-oxidases (GA2oxs). This study identified three new GA2ox genes in barley: HvGA2ox1, HvGA2ox3 and HvGA2ox6. Analysis of their nucleotide and putative amino acid sequences revealed that they share high sequence identity with other plant GA2oxs and their corresponding proteins. Phylogenetic analysis revealed the HvGA2ox1, HvGA2ox3 and HvGA2ox6 belong to GA2ox structural classes II, I, and III, respectively. Feeding the HvGA2ox1 and HvGA2ox3 recombinant proteins with the C19-GAs, GA1 and GA20, resulted in the production of GA8 and GA29, respectively, with no product detected when they were fed with the C20-GA, GA12. Whereas the HvGA2ox6 recombinant protein was able to convert GA12 to GA110, and no product was detected when it was fed with GA1 or GA20. HvGA2ox1 and HvGA2ox3 were highly expressed in internodes and the endosperm of maturing seeds while HvGA2ox6 was predominantly expressed in the embryos. Salinity stress upregulated the expression of all three genes in seedling tissues. Our results indicate that HvGA2ox1, HvGA2ox3 and HvGA2ox6 encode functional GA2oxs that can regulate GA levels, and therefore growth and development of a barley plant, and its interaction with environment.

Transcriptional programs regulating seed dormancy and its release by after-ripening in common wheat (Triticum aestivum L.).

Seed dormancy is an important agronomic trait in wheat (Trticum aestivum). Seeds can be released from a physiologically dormant state by after-ripening. To understand the molecular mechanisms underlying the role of after-ripening in conferring developmental switches from dormancy to germination in wheat seeds, we performed comparative transcriptomic analyses between dormant (D) and after-ripened (AR) seeds in both dry and imbibed states. Transcriptional activation of genes represented by a core of 22 and 435 probesets was evident in the dry and imbibed states of D seeds, respectively. Furthermore, two-way ANOVA analysis identified 36 probesets as specifically regulated by dormancy. These data suggest that biological functions associated with these genes are involved in the maintenance of seed dormancy. Expression of genes encoding protein synthesis/activity inhibitors was significantly repressed during after-ripening, leading to dormancy decay. Imbibing AR seeds led to transcriptional activation of distinct biological processes, including those related to DNA replication, nitrogen metabolism, cytoplasmic membrane-bound vesicle, jasmonate biosynthesis and cell wall modification. These after-ripening-mediated transcriptional programs appear to be regulated by epigenetic mechanisms. Clustering of our microarray data produced 16 gene clusters; dormancy-specific probesets and abscisic acid (ABA)-responsive elements were significantly overrepresented in two clusters, indicating the linkage of dormancy in wheat with that of seed sensitivity to ABA. The role of ABA signalling in regulating wheat seed dormancy was further supported by the down-regulation of ABA response-related probesets in AR seeds and absence of differential expression of ABA metabolic genes between D and AR seeds.