Evolution of the DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN subfamily in green plants.

Adapting to unfavorable environments is a necessary step in plant terrestrialization and radiation. The dehydration-responsive element-binding (DREB) protein subfamily plays a pivotal role in plant abiotic stress regulation. However, relationships between the origin and expansion of the DREB subfamily and adaptive evolution of land plants are still being elucidated. Here, we constructed the evolutionary history of the DREB subfamily by compiling APETALA2/ethylene-responsive element-binding protein superfamily genes from 169 representative species of green plants. Through extensive phylogenetic analyses and comparative genomic analysis, our results revealed that the DREB subfamily diverged from the ethylene-responsive factor (ERF) subfamily in the common ancestor of Zygnemophyceae and Embryophyta during the colonization of land by plants, followed by expansions to form three different ancient archetypal genes in Zygnemophyceae species, designated as groups archetype-I, archetype-II/III, and archetype-IV. Four large-scale expansions paralleling the evolution of land plants led to the nine-subgroup divergence of group archetype-II/III in angiosperms, and five whole-genome duplications during Brassicaceae and Poaceae radiation shaped the diversity of subgroup IIb-1. We identified a Poaceae-specific gene in subgroup IIb-1, ERF014, remaining in a Poaceae-specific microsynteny block and co-evolving with a small heat shock protein cluster. Expression analyses demonstrated that heat acclimation may have driven the neofunctionalization of ERF014s in Pooideae by engaging in the conserved heat-responsive module in Poaceae. This study provides insights into lineage-specific expansion and neofunctionalization in the DREB subfamily, together with evolutionary information valuable for future functional studies of plant stress biology.

Harnessing Knowledge from Plant Functional Genomics and Multi-Omics for Genetic Improvement.

Plant biology research has currently entered the post-genomics era with the advances in genomic technologies [...].

Conservation and Divergence of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) Gene Family between Wheat and Rice.

The SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) gene family affects plant architecture, panicle structure, and grain development, representing key genes for crop improvements. The objective of the present study is to utilize the well characterized SPLs' functions in rice to facilitate the functional genomics of TaSPL genes. To achieve these goals, we combined several approaches, including genome-wide analysis of TaSPLs, comparative genomic analysis, expression profiling, and functional study of TaSPL3 in rice. We established the orthologous relationships of 56 TaSPL genes with the corresponding OsSPLs, laying a foundation for the comparison of known SPL functions between wheat and rice. Some TaSPLs exhibited different spatial-temporal expression patterns when compared to their rice orthologs, thus implicating functional divergence. TaSPL2/6/8/10 were identified to respond to different abiotic stresses through the combination of RNA-seq and qPCR expression analysis. Additionally, ectopic expression of TaSPL3 in rice promotes heading dates, affects leaf and stem development, and leads to smaller panicles and decreased yields per panicle. In conclusion, our work provides useful information toward cataloging of the functions of TaSPLs, emphasized the conservation and divergence between TaSPLs and OsSPLs, and identified the important SPL genes for wheat improvement.

TaASR1-D confers abiotic stress resistance by affecting ROS accumulation and ABA signalling in transgenic wheat.

Cultivating new crop cultivars with multiple abiotic stress tolerances is important for crop production. The abscisic acid-stress-ripening (ASR) protein has been shown to confer abiotic stress tolerance in plants. However, the mechanisms of ASR function under stress condition remain largely unclear. In this study, we characterized all ASR family members in common wheat and constitutively overexpressed TaASR1-D in a commercial hexaploid wheat cultivar Zhengmai 9023. The transgenic wheat plants exhibited increased tolerance to multiple abiotic stresses and increased grain yields under salt stress condition. Overexpression of TaASR1-D conferred enhanced antioxidant capacity and ABA sensitivity in transgenic wheat plants. Further, RNA in situ hybridization results showed that TaASR1-D had higher expression levels in the vascular tissues of leaves and the parenchyma cells around the vascular tissues of roots and stems. Yeast one-hybrid and electrophoretic mobility shift assays revealed that TaASR1-D could directly bind the specific cis-elements in the promoters of TaNCED1 and TaGPx1-D. In conclusion, our findings suggest that TaASR1-D can be used to breed new wheat cultivars with increased multiple abiotic stress tolerances, and TaASR1-D enhances abiotic stress tolerances by reinforcing antioxidant capacity and ABA signalling.

Wheat PP2C-a10 regulates seed germination and drought tolerance in transgenic Arabidopsis.

KEY MESSAGE: A wheat protein phosphatase PP2C-a10, which interacted with TaDOG1L1 and TaDOG1L4, promoted seed germination and decreased drought tolerance of transgenic Arabidopsis. Seed dormancy and germination are critical to plant fitness. DELAY OF GERMINATION 1 (DOG1) is a quantitative trait locus for dormancy in Arabidopsis thaliana. Some interactions between DOG1 and the type 2C protein phosphatases (PP2Cs) have been reported in Arabidopsis. However, the research on molecular functions and regulations of DOG1Ls and group A PP2Cs in wheat (Triticum aestivum. L), an important crop plant, is rare. In this study, the whole TaDOG1L family was identified. Expression analysis revealed that TaDOG1L2, TaDOG1L4 and TaDOG1L-N2 specially expressed in wheat grains, while others displayed distinct expression patterns. Yeast two-hybrid analysis of TaDOG1Ls and group A TaPP2Cs revealed interaction patterns differed from those in Arabidopsis, and TaDOG1L1 and TaDOG1L4 interacted with TaPP2C-a10. The qRT-PCR analysis showed that TaPP2C-a10 exhibited the highest transcript level in wheat grains. Further investigation showed that ectopic expression of TaPP2C-a10 in Arabidopsis promoted seed germination and decreased sensitivity to ABA during germination stage. Additionally, TaPP2C-a10 transgenic Arabidopsis exhibited decreased tolerance to drought stress. Finally, the phylogenetic analysis indicated that TaPP2C-a10 gene was conserved in angiosperm during evolutionary process. Overall, our results reveal the role of TaPP2C-a10 in seed germination and abiotic stress response, as well as the functional diversity of TaDOG1L family.

Genomics-Enabled Analysis of Puroindoline b2 Genes Identifies New Alleles in Wheat and Related Triticeae Species.

Kernel hardness is a key trait of wheat seeds, largely controlled by two tightly linked genes Puroindoline a and b (Pina and Pinb). Genes homologous to Pinb, namely Pinb2, have been studied. Whether these genes contribute to kernel hardness and other important seed traits remains inconclusive. Using the high-quality bread wheat reference genome, we show that PINB2 are encoded by three homoeologous loci Pinb2 not syntenic to the Hardness locus, with Pinb2-7A locus containing three tandem copies. PINB2 proteins have several features conserved for the Pin/Pinb2 phylogenetic cluster but lack a structural basis of significant impact on kernel hardness. Pinb2 are seed-specifically expressed with varied expression levels between the homoeologous copies and among wheat varieties. Using the high-quality genome information, we developed new Pinb2 allele specific markers and demonstrated their usefulness by 1) identifying new Pinb2 alleles in Triticeae species; and 2) performing an association analysis of Pinb2 with kernel hardness. The association result suggests that Pinb2 genes may have no substantial contribution to kernel hardness. Our results provide new insights into Pinb2 evolution and expression and the new allele-specific markers are useful to further explore Pinb2's contribution to seed traits in wheat.

Effects of Cold Jet Atmospheric Pressure Plasma on the Structural Characteristics and Immunoreactivity of Celiac-Toxic Peptides and Wheat Storage Proteins.

The present research reported the effects of structural properties and immunoreactivity of celiac-toxic peptides and wheat storage proteins modified by cold jet atmospheric pressure (CJAP) plasma. It could generate numerous high-energy excited atoms, photons, electrons, and reactive oxygen and nitrogen species, including O3, H2O2, •OH, NO2- and NO3- etc., to modify two model peptides and wheat storage proteins. The Orbitrap HR-LC-MS/MS was utilized to identify and quantify CJAP plasma-modified model peptide products. Backbone cleavage of QQPFP and PQPQLPY at specific proline and glutamine residues, accompanied by hydroxylation at the aromatic ring of phenylalanine and tyrosine residues, contributed to the reduction and modification of celiac-toxic peptides. Apart from fragmentation, oxidation, and agglomeration states were evaluated, including carbonyl formation and the decline of γ-gliadin. The immunoreactivity of gliadin extract declined over time, demonstrating a significant decrease by 51.95% after 60 min of CJAP plasma treatment in vitro. The CJAP plasma could initiate depolymerization of gluten polymer, thereby reducing the amounts of large-sized polymers. In conclusion, CJAP plasma could be employed as a potential technique in the modification and reduction of celiac-toxic peptides and wheat storage proteins.

Genome-wide identification and expression profiling of glutathione transferase gene family under multiple stresses and hormone treatments in wheat (Triticum aestivum L.).

BACKGROUND: Glutathione transferases (GSTs), the ancient, ubiquitous and multi-functional proteins, play significant roles in development, metabolism as well as abiotic and biotic stress responses in plants. Wheat is one of the most important crops, but the functions of GST genes in wheat were less studied. RESULTS: A total of 330 TaGST genes were identified from the wheat genome and named according to the nomenclature of rice and Arabidopsis GST genes. They were classified into eight classes based on the phylogenetic relationship among wheat, rice, and Arabidopsis, and their gene structure and conserved motif were similar in the same phylogenetic class. The 43 and 171 gene pairs were identified as tandem and segmental duplication genes respectively, and the Ka/Ks ratios of tandem and segmental duplication TaGST genes were less than 1 except segmental duplication gene pair TaGSTU24/TaGSTU154. The 59 TaGST genes were identified to have syntenic relationships with 28 OsGST genes. The expression profiling involved in 15 tissues and biotic and abiotic stresses suggested the different expression and response patterns of the TaGST genes. Furthermore, the qRT-PCR data showed that GST could response to abiotic stresses and hormones extensively in wheat. CONCLUSIONS: In this study, a large GST family with 330 members was identified from the wheat genome. Duplication events containing tandem and segmental duplication contributed to the expansion of TaGST family, and duplication genes might undergo extensive purifying selection. The expression profiling and cis-elements in promoter region of 330 TaGST genes implied their roles in growth and development as well as adaption to stressful environments. The qRT-PCR data of 14 TaGST genes revealed that they could respond to different abiotic stresses and hormones, especially salt stress and abscisic acid. In conclusion, this study contributed to the further functional analysis of GST genes family in wheat.

Genome-wide identification and expression profiling of trihelix gene family under abiotic stresses in wheat.

BACKGROUND: The trihelix gene family is a plant-specific transcription factor family that plays important roles in plant growth, development, and responses to abiotic stresses. However, to date, no systemic characterization of the trihelix genes has yet been conducted in wheat and its close relatives. RESULTS: We identified a total of 94 trihelix genes in wheat, as well as 22 trihelix genes in Triticum urartu, 29 in Aegilops tauschii, and 31 in Brachypodium distachyon. We analyzed the chromosomal locations and orthology relations of the identified trihelix genes, and no trihelix gene was found to be located on chromosome 7A, 7B, or 7D of wheat, thereby reflecting the uneven distributions of wheat trihelix genes. Phylogenetic analysis indicated that the 186 identified trihelix proteins in wheat, rice, B. distachyon, and Arabidopsis were clustered into five major clades. The trihelix genes belonging to the same clades usually shared similar motif compositions and exon/intron structural patterns. Five pairs of tandem duplication genes and three pairs of segmental duplication genes were identified in the wheat trihelix gene family, thereby validating the supposition that more intrachromosomal gene duplication events occur in the genome of wheat than in that of other grass species. The tissue-specific expression and differential expression profiling of the identified genes under cold and drought stresses were analyzed by using RNA-seq data. qRT-PCR was also used to confirm the expression profiles of ten selected wheat trihelix genes under multiple abiotic stresses, and we found that these genes mainly responded to salt and cold stresses. CONCLUSIONS: In this study, we identified trihelix genes in wheat and its close relatives and found that gene duplication events are the main driving force for trihelix gene evolution in wheat. Our expression profiling analysis demonstrated that wheat trihelix genes responded to multiple abiotic stresses, especially salt and cold stresses. The results of our study built a basis for further investigation of the functions of wheat trihelix genes and provided candidate genes for stress-resistant wheat breeding programs.

Co-expression of high-molecular-weight glutenin subunit 1Ax1 and Puroindoline a (Pina) genes in transgenic durum wheat (Triticum turgidum ssp. durum) improves milling and pasting quality.

BACKGROUND: Durum wheat is considered not suitable for making many food products that bread wheat can. This limitation is largely due to: (i) lack of grain-hardness controlling genes (Puroindoline a and b) and consequently extremely-hard kernel; (ii) lack of high- and low-molecular-weight glutenin subunit loci (Glu-D1 and Glu-D3) that contribute to gluten strength. To improve food processing quality of durum wheat, we stacked transgenic Pina and HMW-glutenin subunit 1Ax1 in durum wheat and developed lines with medium-hard kernel texture. RESULTS: Here, we demonstrated that co-expression of Pina + 1Ax1 in durum wheat did not affect the milling performance that was enhanced by Pina expression. While stacking of Pina + 1Ax1 led to increased flour yield, finer flour particles and decreased starch damage compared to the control lines. Interestingly, Pina and 1Ax1 co-expression showed synergistic effects on the pasting attribute peak viscosity. Moreover, Pina and 1Ax1 co-expression suggests that PINA impacts gluten aggregation via interaction with gluten protein matrix. CONCLUSIONS: The results herein may fill the gap of grain hardness between extremely-hard durum wheat and the soft kernel durum wheat, the latter of which has been developed recently. Our results may also serve as a proof of concept that stacking Puroindolines and other genes contributing to wheat end-use quality from the A and/or D genomes could improve the above-mentioned bottleneck traits of durum wheat and help to expand its culinary uses.

Genome-wide identification and analysis of WD40 proteins in wheat (Triticum aestivum L.).

BACKGROUND: WD40 domains are abundant in eukaryotes, and they are essential subunits of large multiprotein complexes, which serve as scaffolds. WD40 proteins participate in various cellular processes, such as histone modification, transcription regulation, and signal transduction. WD40 proteins are regarded as crucial regulators of plant development processes. However, the systematic identification and analysis of WD40 proteins have yet to be reported in wheat. RESULTS: In this study, a total of 743 WD40 proteins were identified in wheat, and they were grouped into 5 clusters and 11 subfamilies. Their gene structures, chromosomal locations, and evolutionary relationships were analyzed. Among them, 39 and 46 pairs of TaWD40s were distinguished as tandem duplication and segmental duplication genes. The 123 OsWD40s were identified to exhibit synteny with TaWD40s. TaWD40s showed the specific characteristics at the reproductive developmental stage, and numerous TaWD40s were involved in responses to stresses, including cold, heat, drought, and powdery mildew infection pathogen, based on the result of RNA-seq data analysis. The expression profiles of some TaWD40s in wheat seed development were confirmed through qRT-PCR technique. CONCLUSION: In this study, 743 TaWD40s were identified from the wheat genome. As the main driving force of evolution, duplication events were observed, and homologous recombination was another driving force of evolution. The expression profiles of TaWD40s revealed their importance for the growth and development of wheat and their response to biotic and abiotic stresses. Our study also provided important information for further functional characterization of some WD40 proteins in wheat.

Correction to: Genome-wide identification and analysis of WD40 proteins in wheat (Triticum aestivum L.).

Following the publication of this article [1], the authors reported the following errors.

Expression of TaGF14b, a 14-3-3 adaptor protein gene from wheat, enhances drought and salt tolerance in transgenic tobacco.

MAIN CONCLUSION: TaGF14b enhances tolerance to multiple stresses through ABA signaling pathway by altering physiological and biochemical processes, including ROS-scavenging system, stomatal closure, compatible osmolytes, and stress-related gene expressions in tobaccos. The 14-3-3 proteins are involved in plant growth, development, and in responding to abiotic stresses. However, the precise functions of 14-3-3s in responding to drought and salt stresses remained unclear, especially in wheat. In this study, a 14-3-3 gene from wheat, designated TaGF14b, was cloned and characterized. TaGF14b was upregulated by polyethylene glycol 6000, sodium chloride, hydrogen peroxide, and abscisic acid (ABA) treatments. Ectopic expression of TaGF14b in tobacco conferred enhanced tolerance to drought and salt stresses. Transgenic tobaccos had longer root, better growth status, and higher relative water content, survival rate, photosynthetic rate, and water use efficiency than control plants under drought and salt stresses. The contribution of TaGF14b to drought and salt tolerance relies on the regulations of ABA biosynthesis and ABA signaling, as well as stomatal closure and stress-related gene expressions. Moreover, TaGF14b expression could significantly enhance the reactive oxygen species (ROS) scavenging system to ameliorate oxidative damage to cells. In addition, TaGF14b increased tolerance to osmotic stress evoked by drought and salinity through modifying water conservation and compatible osmolytes in plants. In conclusion, TaGF14b enhances tolerance to multiple abiotic stresses through the ABA signaling pathway in transgenic tobaccos by altering physiological and biochemical processes.

Ectopic expression of BdCIPK31 confers enhanced low-temperature tolerance in transgenic tobacco plants.

Calcineurin B-like protein (CBL), the Ca2+ sensor, and its interacting protein kinases (CIPKs) play essential roles in plants' response to stress. However, few studies have focused on the functions of CIPKs in low-temperature response. In the present study, BdCIPK31, a cold-responsive CIPK in Brachypodium distachyon, was found to participate in low-temperature response. Ectopic expression of BdCIPK31 conferred cold tolerance in transgenic tobaccos. Further analyses indicated that expression of BdCIPK31 improved ROS detoxication and omsoprotectant biosynthesis in transgenic plants under low-temperature treatment, suggesting that the BdCIPK31 functions positively in plant adaption to the cold-induced oxidative and osmotic stresses. Moreover, BdCIPK31 could upregulate the expressions of some representative stress-related genes under cold stress. In conclusion, these findings suggest that BdCIPK31 functions positively in plant cold response.

Identification of the ASR gene family from Brachypodium distachyon and functional characterization of BdASR1 in response to drought stress.

KEY MESSAGE: A genome-wide investigation identified five B. distachyon ASR genes. BdASR1 may be a transcription factor that confers drought resistance by activating antioxidant systems involving ROS-scavenging enzymes and non-enzymatic antioxidants. Abscisic acid-, stress-, and ripening-induced (ASR) proteins belong to a family of plant-specific, small, and hydrophilic proteins with important roles in responses to abiotic stresses. Although several ASR genes involved in drought tolerance have been characterized in various plant species, the mechanisms regulating ASR activities are still uncharacterized. Additionally, no research on Brachypodium distachyon ASR proteins have been completed. In this study, five B. distachyon BdASR genes were identified through genome-wide analyses. Phylogenetic analyses revealed that BdASR genes originated from tandem and whole genome duplications. Expression analyses revealed the BdASR genes responded to various abiotic stresses, including cold, drought, and salinity, as well as signaling molecules such as abscisic acid, ethylene, and H2O2. BdASR1, which localizes to the nucleus and is transcriptionally active, was functionally characterized. BdASR1 overexpression considerably enhanced drought tolerance in transgenic tobacco plants, which was accompanied by increased superoxide dismutase, catalase, and peroxidase activities, as well as an increased abundance of antioxidants such as ascorbate, tocopherols, and glutathione. BdASR1 may function as a transcription factor that provides drought stress resistance by inducing the production of reactive oxygen species-scavenging enzymes and non-enzymatic antioxidants.

Investigation of the ASR family in foxtail millet and the role of ASR1 in drought/oxidative stress tolerance.

KEY MESSAGE: Six foxtail millet ASR genes were regulated by various stress-related signals. Overexpression of ASR1 increased drought and oxidative tolerance by controlling ROS homeostasis and regulating oxidation-related genes in tobacco plants. Abscisic acid stress ripening (ASR) proteins with ABA/WDS domains constituted a class of plant-specific transcription factors, playing important roles in plant development, growth and abiotic stress responses. However, only a few ASRs genes have been characterized in crop plants and none was reported so far in foxtail millet (Setaria italic), an important drought-tolerant crop and model bioenergy grain crop. In the present study, we identified six foxtail millet ASR genes. Gene structure, protein alignments and phylogenetic relationships were analyzed. Transcript expression patterns of ASR genes revealed that ASRs might play important roles in stress-related signaling and abiotic stress responses in diverse tissues in foxtail millet. Subcellular localization assays showed that SiASR1 localized in the nucleus. Overexpression of SiASR1 in tobacco remarkably increased tolerance to drought and oxidative stresses, as determined through developmental and physiological analyses of germination rate, root growth, survival rate, relative water content, ion leakage, chlorophyll content and antioxidant enzyme activities. Furthermore, expression of SiASR1 modulated the transcript levels of oxidation-related genes, including NtSOD, NtAPX, NtCAT, NtRbohA and NtRbohB, under drought and oxidative stress conditions. These results provide a foundation for evolutionary and functional characterization of the ASR gene family in foxtail millet.

Molecular basis of purinergic signal metabolism by ectonucleotide pyrophosphatase/phosphodiesterases 4 and 1 and implications in stroke.

NPP4 is a type I extracellular membrane protein on brain vascular endothelium inducing platelet aggregation via the hydrolysis of Ap3A, whereas NPP1 is a type II extracellular membrane protein principally present on the surface of chondrocytes that regulates tissue mineralization. To understand the metabolism of purinergic signals resulting in the physiologic activities of the two enzymes, we report the high resolution crystal structure of human NPP4 and explore the molecular basis of its substrate specificity with NPP1. Both enzymes cleave Ap3A, but only NPP1 can hydrolyze ATP. Comparative structural analysis reveals a tripartite lysine claw in NPP1 that stabilizes the terminal phosphate of ATP, whereas the corresponding region of NPP4 contains features that hinder this binding orientation, thereby inhibiting ATP hydrolysis. Furthermore, we show that NPP1 is unable to induce platelet aggregation at physiologic concentrations reported in human blood, but it could stimulate platelet aggregation if localized at low nanomolar concentrations on vascular endothelium. The combined studies expand our understanding of NPP1 and NPP4 substrate specificity and range and provide a rational mechanism by which polymorphisms in NPP1 confer stroke resistance.

TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis.

BACKGROUND: NAC (NAM, ATAF, and CUC) transcription factors play important roles in plant biological processes, including phytohormone homeostasis, plant development, and in responses to various environmental stresses. METHODS: TaNAC29 was introduced into Arabidopsis using the Agrobacterium tumefaciens-mediated floral dipping method. TaNAC29-overexpression plants were subjected to salt and drought stresses for examining gene functions. To investigate tolerant mechanisms involved in the salt and drought responses, expression of related marker genes analyses were conducted, and related physiological indices were also measured. Expressions of genes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: A novel NAC transcription factor gene, designated TaNAC29, was isolated from bread wheat (Triticum aestivum). Sequence alignment suggested that TaNAC29 might be located on chromosome 2BS. TaNAC29 was localized to the nucleus in wheat protoplasts, and proved to have transcriptional activation activities in yeast. TaNAC29 was expressed at a higher level in the leaves, and expression levels were much higher in senescent leaves, indicating that TaNAC29 might be involved in the senescence process. TaNAC29 transcripts were increased following treatments with salt, PEG6000, H2O2, and abscisic acid (ABA). To examine TaNAC29 function, transgenic Arabidopsis plants overexpressing TaNAC29 were generated. Germination and root length assays of transgenic plants demonstrated that TaNAC29 overexpression plants had enhanced tolerances to high salinity and dehydration, and exhibited an ABA-hypersensitive response. When grown in the greenhouse, TaNAC29-overexpression plants showed the same tolerance response to salt and drought stresses at both the vegetative and reproductive period, and had delayed bolting and flowering in the reproductive period. Moreover, TaNAC29 overexpression plants accumulated lesser malondialdehyde (MDA), H2O2, while had higher superoxide dismutase (SOD) and catalase (CAT) activities under high salinity and/or dehydration stress. CONCLUSIONS: Our results demonstrate that TaNAC29 plays important roles in the senescence process and response to salt and drought stresses. ABA signal pathway and antioxidant enzyme systems are involved in TaNAC29-mediated stress tolerance mechanisms.

Identification and comprehensive analyses of the CBL and CIPK gene families in wheat (Triticum aestivum L.).

BACKGROUND: Calcineurin B-like (CBL) proteins belong to a unique group of calcium sensors in plant that decode the Ca(2+) signature by interacting with CBL-interacting protein kinases (CIPKs). Although CBL-CIPK complexes have been shown to play important roles in the responses to various stresses in plants, little is known about their functions in wheat. RESULTS: A total of seven TaCBL and 20 TaCIPK genes were amplified from bread wheat, Triticum aestivum cv. Chinese Spring. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and in silico expression analyses showed that TaCBL and TaCIPK genes were expressed at different levels in different tissues, or maintained at nearly constant expression levels during the whole life cycle of the wheat plant. Some TaCBL and TaCIPK genes showed up- or down-regulated expressions during seed germination. Preferential interactions between TaCBLs and TaCIPKs were observed in yeast two-hybrid and bimolecular fluorescence complementation experiments. Analyses of a deletion series of TaCIPK proteins with amino acid variations at the C-terminus provided new insights into the specificity of the interactions between TaCIPKs and TaCBLs, and indicated that the TaCBL-TaCIPK signaling pathway is very complex in wheat because of its hexaploid genome. The expressions of many TaCBLs and TaCIPKs were responsive to abiotic stresses (salt, cold, and simulated drought) and abscisic acid treatment. Transgenic Arabidopsis plants overexpressing TaCIPK24 exhibited improved salt tolerance through increased Na(+) efflux and an enhanced reactive oxygen species scavenging capacity. CONCLUSIONS: These results contribute to our understanding of the functions of CBL-CIPK complexes and provide the basis for selecting appropriate genes for in-depth functional studies of CBL-CIPK in wheat.

The lycopene ß-cyclase plays a significant role in provitamin A biosynthesis in wheat endosperm.

BACKGROUND: Lycopene ß-cyclase (LCYB) is a key enzyme catalyzing the biosynthesis of ß-carotene, the main source of provitamin A. However, there is no documented research about this key cyclase gene's function and relationship with ß-carotene content in wheat. Therefore, the objectives of this study were to clone TaLCYB and characterize its function and relationship with ß-carotene biosynthesis in wheat grains. We also aimed to obtain more information about the endogenous carotenoid biosynthetic pathway and thus provide experimental support for carotenoid metabolic engineering in wheat. RESULTS: In the present study, a lycopene ß-cyclase gene, designated TaLCYB, was cloned from the hexaploid wheat cultivar Chinese Spring. The cyclization activity of the encoded protein was demonstrated by heterologous complementation analysis. The TaLCYB gene was expressed differentially in different tissues of wheat. Although TaLCYB had a higher expression level in the later stages of grain development, the ß-carotene content still showed a decreasing tendency. The expression of TaLCYB in leaves was dramatically induced by strong light and the ß-carotene content variation corresponded with changes of TaLCYB expression. A post-transcriptional gene silencing strategy was used to down-regulate the expression of TaLCYB in transgenic wheat, resulting in a decrease in the content of ß-carotene and lutein, accompanied by the accumulation of lycopene to partly compensate for the total carotenoid content. In addition, changes in TaLCYB expression also affected the expression of several endogenous carotenogenic genes to varying degrees. CONCLUSION: Our results suggest that TaLCYB is a genuine lycopene cyclase gene and plays a crucial role in ß-carotene biosynthesis in wheat. Our attempt to silence it not only contributes to elucidating the mechanism of carotenoid accumulation in wheat but may also help in breeding wheat varieties with high provitamin A content through RNA interference (RNAi) to block specific carotenogenic genes in the wheat endosperm.