Genome-wide identification of wheat ABC1K gene family and functional dissection of TaABC1K3 and TaABC1K6 involved in drought tolerance.

Activity of BC1 complex kinase (ABC1K) serves as an atypical kinase family involved in plant stress resistance. This study identified 44 ABC1K genes in the wheat genome, which contained three clades (I-III). TaABC1K genes generally had similar structural features, but differences were present in motif and exon compositions from different clade members. More type II functional divergence sites were detected between clade I and clade III and no positive selection site were found in TaABC1K family. The three-dimensional structure prediction by Alphafold2 showed that TaABC1K proteins had more α-helixes with a relatively even distribution, and different clade members had differences in the content of secondary structures. The cis-acting element analysis showed that TaABC1K genes contained abundant cis-acting elements related to plant hormones and environmental stress response in the promoter region, and generally displayed a significantly upregulated expression under drought stress. In particular, both TaABC1K3 and TaABC1K6 genes from clade I was highly induced by drought stress, and their overexpression in yeast and Arabidopsis enhanced drought tolerance by suppressing active oxygen burst and reducing photosynthesis impairment. Meanwhile, TaABC1K3 and TaABC1K6 could, respectively, complement the function of Arabidopsis abc1k3 and abc1k6 mutants and reduce photosynthesis damage caused by drought stress.

Genome-Wide Identification and Characterisation of Wheat MATE Genes Reveals Their Roles in Aluminium Tolerance.

The Multidrug and toxin efflux (MATE) gene family plays crucial roles in plant growth and development and response to adverse stresses. This work investigated the structural and evolutionary characteristics, expression profiling and potential functions involved in aluminium (Al) tolerance from a genome-wide level. In total, 211 wheat MATE genes were identified, which were classified into four subfamilies and unevenly distributed on chromosomes. Duplication analysis showed that fragments and tandem repeats played the main roles in the amplification of TaMATEs, and Type II functional disproportionation had a leading role in the differentiation of TaMATEs. TaMATEs had abundant Al resistance and environmental stress-related elements, and generally had a high expression level in roots and leaves and in response to Al stress. The 3D structure prediction by AlphaFold and molecular docking showed that six TaMATE proteins localised in the plasmalemma could combine with citrate via amino acids in the citrate exuding motif and other sites, and then transport citrate to soil to form citrate aluminium. Meanwhile, citrate aluminium formed in root cells might be transported to leaves by TaMATEs to deposit in vacuoles, thereby alleviating Al toxicity.

Functional conservation and divergence in plant-specific GRF gene family revealed by sequences and expression analysis.

Unique to plants, growth regulatory factors (GRFs) play important roles in plant growth and reproduction. This study investigated the evolutionary and functional characteristics associated with plant growth. Using genome-wide analysis of 15 plant species, 173 members of the GRF family were identified and phylogenetically categorized into six groups. All members contained WRC and QLQ conserved domains, and the family's expansion largely depended on segmental duplication. The promoter region of the GRF gene family mainly contained four types of cis-acting elements (light-responsive elements, development-related elements, hormone-responsive elements, and environmental stress-related elements) that are mainly related to gene expression levels. Functional divergence analysis revealed that changes in amino acid site evolution rate played a major role in the differentiation of the GRF gene family, with ten significant sites identified. Six significant sites were identified for positive selection. Moreover, the four groups of coevolutionary sites identified may play a key role in regulating the transcriptional activation of the GRF protein. Expression profiles revealed that GRF genes were generally highly expressed in young plant tissues and had tissue or organ expression specificity, demonstrating their functional conservation with distinct divergence. The results of these sequence and expression analyses are expected to provide molecular evolutionary and functional references for the plant GRF gene family.

Genome-Wide Analysis of Wheat GATA Transcription Factor Genes Reveals Their Molecular Evolutionary Characteristics and Involvement in Salt and Drought Tolerance.

GATA transcription factor genes participate in plant growth, development, morphogenesis, and stress response. In this study, we carried out a comprehensive genome-wide analysis of wheat GATA transcription factor genes to reveal their molecular evolutionary characteristics and involvement in salt and drought tolerance. In total, 79 TaGATA genes containing a conserved GATA domain were identified in the wheat genome, which were classified into four subfamilies. Collinear analysis indicated that fragment duplication plays an important role in the amplification of the wheat GATA gene family. Functional disproportionation analysis between subfamilies found that both type I and type II functional divergence simultaneously occurs in wheat GATA genes, which might result in functional differentiation of the TaGATA gene family. Transcriptional expression analysis showed that TaGATA genes generally have a high expression level in leaves and in response to drought and salt stresses. Overexpression of TaGATA62 and TaGATA73 genes significantly enhanced the drought and salt tolerance of yeast and Arabidopsis. Protein-protein docking indicated that TaGATAs can enhance drought and salt tolerance by interacting between the DNA-binding motif of GATA transcription factors and photomorphogenesis-related protein TaCOP9-5A. Our results provided a base for further understanding the molecular evolution and functional characterization of the plant GATA gene family in response to abiotic stresses.

Genome-wide analysis of wheat DNA-binding with one finger (Dof) transcription factor genes: evolutionary characteristics and diverse abiotic stress responses.

BACKGROUND: DNA binding with one finger (Dof) transcription factors play important roles in plant growth and abiotic stress responses. Although genome-wide identification and analysis of the DOF transcription factor family has been reported in other species, no relevant studies have emerged in wheat. The aim of this study was to investigate the evolutionary and functional characteristics associated with plant growth and abiotic stress responses by genome-wide analysis of the wheat Dof transcription factor gene family. RESULTS: Using the recently released wheat genome database (IWGSC RefSeq v1.0), we identified 96 wheat Dof gene family members, which were phylogenetically clustered into five distinct subfamilies. Gene duplication analysis revealed a broad and heterogeneous distribution of TaDofs on the chromosome groups 1 to 7, and obvious tandem duplication genes were present on chromosomes 2 and 3.Members of the same gene subfamily had similar exon-intron structures, while members of different subfamilies had obvious differences. Functional divergence analysis indicated that type-II functional divergence played a major role in the differentiation of the TaDof gene family. Positive selection analysis revealed that the Dof gene family experienced different degrees of positive selection pressure during the process of evolution, and five significant positive selection sites (30A, 31 T, 33A, 102G and 104S) were identified. Additionally, nine groups of coevolving amino acid sites, which may play a key role in maintaining the structural and functional stability of Dof proteins, were identified. The results from the RNA-seq data and qRT-PCR analysis revealed that TaDof genes exhibited obvious expression preference or specificity in different organs and developmental stages, as well as in diverse abiotic stress responses. Most TaDof genes were significantly upregulated by heat, PEG and heavy metal stresses. CONCLUSIONS: The genome-wide analysis and identification of wheat DOF transcription factor family and the discovery of important amino acid sites are expected to provide new insights into the structure, evolution and function of the plant Dof gene family.

Delineation of the Crucial Evolutionary Amino Acid Sites in Trehalose-6-Phosphate Synthase From Higher Plants.

Trehalose-6-phosphate synthase (TPS) is a key enzyme in the biosynthesis of trehalose, with its direct product, trehalose-6-phosphate, playing important roles in regulating whole-plant carbohydrate allocation and utilization. Genes encoding TPS constitute a multigene family in which functional divergence appears to have occurred repeatedly. To identify the crucial evolutionary amino acid sites of TPS in higher plants, a series of bioinformatics tools were applied to investigate the phylogenetic relationships, functional divergence, positive selection, and co-evolution of TPS proteins. First, we identified 150 TPS genes from 13 higher plant species. Phylogenetic analysis placed these TPS proteins into 2 clades: clades A and B, of which clade B could be further divided into 4 subclades (B1-B4). This classification was supported by the intron-exon structures, with more introns present in clade A. Next, detection of the critical functionally divergent amino acid sites resulted in the isolation of a total of 286 sites reflecting nonredundant radical shifts in amino acid properties with a high posterior probability cutoff among subclades. In addition, positively selected sites were identified using a codon substitution model, from which 46 amino acid sites were isolated as exhibiting positive selection at a significant level. Moreover, 18 amino acid sites were highlighted both for functional divergence and positive selection; these may thus potentially represent crucial evolutionary sites in the TPS family. Further co-evolutionary analysis revealed 3 pairs of sites: 11S and 12H, 33S and 34N, and 109G and 110E as demonstrating co-evolution. Finally, the 18 crucial evolutionary amino acid sites were mapped in the 3-dimensional structure. A total of 77 sites harboring functionally and structurally important residues of TPS proteins were found by using the CLIPS-4D online tool; notably, no overlap was observed with the identified crucial evolutionary sites, providing positive evidence supporting their designation. A total of 18 sites were isolated as key amino acids by using multiple bioinformatics tools based on their concomitant functional divergence and positive selection. Almost all these key sites are located in 2 domains of this protein family where they exhibit no overlap with the structurally and functionally conserved sites. These results will provide an improved understanding of the complexity of the TPS gene family and of its function and evolution in higher plants. Moreover, this knowledge may facilitate the exploitation of these sites for protein engineering applications.

Functional divergence and adaptive selection of KNOX gene family in plants.

KNOTTED-like homeodomain (KNOX) genes are transcriptional regulators that play an important role in morphogenesis. In the present study, a comparative analysis was performed to investigate the molecular evolution of the characteristics of the KNOX gene family in 10 different plant species. We identified 129 KNOX gene family members, which were categorized into two subfamilies based on multiple sequence alignment and phylogenetic tree reconstruction. Several segmental duplication pairs were found, indicating that different species share a common expansion model. Functional divergence analysis identified the 15 and 52 amino acid sites with significant changes in evolutionary rates and amino acid physicochemical properties as functional divergence sites. Additional selection analysis showed that 14 amino acid sites underwent positive selection during evolution, and two groups of co-evolutionary amino acid sites were identified by Coevolution Analysis using Protein Sequences software. These sites could play critical roles in the molecular evolution of the KNOX gene family in these species. In addition, the expression profiles of KNOX duplicated genes demonstrated functional divergence. Taken together, these results provide novel insights into the structural and functional evolution of the KNOX gene family.

Genome-wide identification and expression analysis of expansin gene family in common wheat (Triticum aestivum L.).

BACKGROUND: Expansin loosens plant cell walls and involves in cell enlargement and various abiotic stresses. Plant expansin superfamily contains four subfamilies: α-expansin (EXPA), ß-expansin (EXPB), expansin-like A (EXLA), and expansin-like B (EXLB). In this work, we performed a comprehensive study on the molecular characterization, phylogenetic relationship and expression profiling of common wheat (Triticum aestivum) expansin gene family using the recently released wheat genome database (IWGSC RefSeq v1.1 with a coverage rate of 94%). RESULTS: Genome-wide analysis identified 241 expansin genes in the wheat genome, which were grouped into three subfamilies (EXPA, EXPB and EXLA) by phylogenetic tree. Molecular structure analysis showed that wheat expansin gene family showed high evolutionary conservation although some differences were present in different subfamilies. Some key amino acid sites that contribute to functional divergence, positive selection, and coevolution were detected. Evolutionary analysis revealed that wheat expansin gene superfamily underwent strong positive selection. The transcriptome map and qRT-PCR analysis found that wheat expansin genes had tissue/organ expression specificity and preference, and generally highly expressed in the roots. The expression levels of some expansin genes were significantly induced by NaCl and polyethylene glycol stresses, which was consistent with the differential distribution of the cis-elements in the promoter region. CONCLUSIONS: Wheat expansin gene family showed high evolutionary conservation and wide range of functional divergence. Different selection constraints may influence the evolution of the three expansin subfamilies. The different expression patterns demonstrated that expansin genes could play important roles in plant growth and abiotic stress responses. This study provides new insights into the structures, evolution and functions of wheat expansin gene family.

Genome-wide identification, molecular evolution, and expression analysis of auxin response factor (ARF) gene family in Brachypodium distachyon L.

BACKGROUND: The auxin response factor (ARF) gene family is involved in plant development and hormone regulation. Although the ARF gene family has been studied in some plant species, its structural features, molecular evolution, and expression profiling in Brachypodium distachyon L. are still not clear. RESULTS: Genome-wide analysis identified 19 ARF genes in B. distachyon. A phylogenetic tree constructed with 182 ARF genes from seven plant species revealed three different clades, and the ARF genes from within a clade exhibited structural conservation, although certain divergences occurred in different clades. The branch-site model identified some sites where positive selection may have occurred, and functional divergence analysis found more Type II divergence sites than Type I. In particular, both positive selection and functional divergence may have occurred in 241H, 243G, 244 L, 310 T, 340G and 355 T. Subcellular localization prediction and experimental verification indicated that BdARF proteins were present in the nucleus. Transcript expression analysis revealed that BdARFs were mainly expressed in the leaf and root tips, stems, and developing seeds. Some BdARF genes exhibited significantly upregulated expression under various abiotic stressors. Particularly, BdARF4 and BdARF8 were significantly upregulated in response to abiotic stress factors such as salicylic acid and heavy metals. CONCLUSION: The ARF gene family in B. distachyon was highly conserved. Several important amino acid sites were identified where positive selection and functional divergence occurred, and they may play important roles in functional differentiation. BdARF genes had clear tissue and organ expression preference and were involved in abiotic stress response, suggesting their roles in plant growth and stress resistance.

Molecular Characterization and Expression Profiling of Brachypodium distachyon L. Cystatin Genes Reveal High Evolutionary Conservation and Functional Divergence in Response to Abiotic Stress.

Cystatin is a class of proteins mainly involved in cysteine protease inhibition and plant growth and development, as well as tolerance under various abiotic stresses. In this study, we performed the first comprehensive analysis of the molecular characterization and expression profiling in response to various abiotic stresses of the cystatin gene family in Brachypodium distachyon, a novel model plant for Triticum species with huge genomes. Comprehensive searches of the Brachypodium genome database identified 25 B. distachyon cystatin (BdC) genes that are distributed unevenly on chromosomes; of these, nine and two were involved in tandem and segmental duplication events, respectively. All BdC genes had similar exon/intron structural organization, with three conserved motifs similar to those from other plant species, indicating their high evolutionary conservation. Expression profiling of 10 typical BdC genes revealed ubiquitous expression in different organs at varying expression levels. BdC gene expression in seedling leaves was particularly highly induced by various abiotic stresses, including the plant hormone abscisic acid and various environmental cues (cold, H2O2, CdCl2, salt, and drought). Interestingly, most BdC genes were significantly upregulated under multiple abiotic stresses, including BdC15 under all stresses, BdC7-2 and BdC10 under five stresses, and BdC7-1, BdC2-1, BdC14, and BdC12 under four stresses. The putative metabolic pathways of cytastin genes in response to various abiotic stresses mainly involve the aberrant protein degradation pathway and reactive oxygen species (ROS)-triggered programmed cell death signaling pathways. These observations provide a better understanding of the structural and functional characteristics of the plant cystatin gene family.

The similar and different evolutionary trends of MATE family occurred between rice and Arabidopsis thaliana.

BACKGROUND: Multidrug and toxic compound extrusion (MATE) transporter proteins are present in all organisms. Although the functions of some MATE gene family members have been studied in plants, few studies have investigated the gene expansion patterns, functional divergence, or the effects of positive selection. RESULTS: Forty-five MATE genes from rice and 56 from Arabidopsis were identified and grouped into four subfamilies. MATE family genes have similar exon-intron structures in rice and Arabidopsis; MATE gene structures are conserved in each subfamily but differ among subfamilies. In both species, the MATE gene family has expanded mainly through tandem and segmental duplications. A transcriptome atlas showed considerable differences in expression among the genes, in terms of transcript abundance and expression patterns under normal growth conditions, indicating wide functional divergence in this family. In both rice and Arabidopsis, the MATE genes showed consistent functional divergence trends, with highly significant Type-I divergence in each subfamily, while Type-II divergence mainly occurred in subfamily III. The Type-II coefficients between rice subfamilies I/III, II/III, and IV/III were all significantly greater than zero, while only the Type-II coefficient between Arabidopsis IV/III subfamilies was significantly greater than zero. A site-specific model analysis indicated that MATE genes have relatively conserved evolutionary trends. A branch-site model suggested that the extent of positive selection on each subfamily of rice and Arabidopsis was different: subfamily II of Arabidopsis showed higher positive selection than other subfamilies, whereas in rice, positive selection was highest in subfamily III. In addition, the analyses identified 18 rice sites and 7 Arabidopsis sites that were responsible for positive selection and for Type-I and Type-II functional divergence; there were no common sites between rice and Arabidopsis. Five coevolving amino acid sites were identified in rice and three in Arabidopsis; these sites might have important roles in maintaining local structural stability and protein functional domains. CONCLUSIONS: We demonstrate that the MATE gene family expanded through tandem and segmental duplication in both rice and Arabidopsis. Overall, the results of our analyses contribute to improved understanding of the molecular evolution and functions of the MATE gene family in plants.

Molecular Characterization of the 14-3-3 Gene Family in Brachypodium distachyon L. Reveals High Evolutionary Conservation and Diverse Responses to Abiotic Stresses.

The 14-3-3 gene family identified in all eukaryotic organisms is involved in a wide range of biological processes, particularly in resistance to various abiotic stresses. Here, we performed the first comprehensive study on the molecular characterization, phylogenetics, and responses to various abiotic stresses of the 14-3-3 gene family in Brachypodium distachyon L. A total of seven 14-3-3 genes from B. distachyon and 120 from five main lineages among 12 species were identified, which were divided into five well-conserved subfamilies. The molecular structure analysis showed that the plant 14-3-3 gene family is highly evolutionarily conserved, although certain divergence had occurred in different subfamilies. The duplication event investigation revealed that segmental duplication seemed to be the predominant form by which the 14-3-3 gene family had expanded. Moreover, seven critical amino acids were detected, which may contribute to functional divergence. Expression profiling analysis showed that BdGF14 genes were abundantly expressed in the roots, but showed low expression in the meristems. All seven BdGF14 genes showed significant expression changes under various abiotic stresses, including heavy metal, phytohormone, osmotic, and temperature stresses, which might play important roles in responses to multiple abiotic stresses mainly through participating in ABA-dependent signaling and reactive oxygen species-mediated MAPK cascade signaling pathways. In particular, BdGF14 genes generally showed upregulated expression in response to multiple stresses of high temperature, heavy metal, abscisic acid (ABA), and salicylic acid (SA), but downregulated expression under H2O2, NaCl, and polyethylene glycol (PEG) stresses. Meanwhile, dynamic transcriptional expression analysis of BdGF14 genes under longer treatments with heavy metals (Cd(2+), Cr(3+), Cu(2+), and Zn(2+)) and phytohormone (ABA) and recovery revealed two main expression trends in both roots and leaves: up-down and up-down-up expression from stress treatments to recovery. This study provides new insights into the structures and functions of plant 14-3-3 genes.

Molecular characterization and evolutionary origins of farinin genes in Brachypodium distachyon L.

Farinins are one of the oldest members of the gluten family in wheat and Aegilops species, and they influence dough properties. Here, we performed the first detailed molecular genetic study on farinin genes in Brachypodium distachyon L., the model species for Triticum aestivum. A total of 51 b-type farinin genes were cloned and characterized, including 27 functional and 24 non-functional pseudogenes from 14 different B. distachyon accessions. All genes were highly similar to those previously reported from wheat and Aegilops species. The identification of deduced amino acid sequences showed that b-type farinins across Triticeae genomes could be classified as b1-, b2-, b3-, and b4-type farinins; however, B. distachyon had only b3- and b4-type farinins. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that farinin genes are transcribed into mRNA in B. distachyon at much lower levels than in Triticeae, despite the presence of cis-acting elements in promoter regions. Phylogenetic analysis suggested that Brachypodium farinins may have closer relationships with common wheat and further confirmed four different types of b-type farinins in Triticeae and Brachypodium genomes, corresponding to b1, b2, b3 (group 1), and b4 (group 2). A putative evolutionary origin model of farinin genes in Brachypodium, Triticum, and the related species suggests that all b-type farinins diverged from their common ancestor ~3.2 million years ago (MYA). The b3 and b4 types could be considered older in the farinin family. The results explain the loss of b1- and b2-type farinin alleles in Brachypodium.

Molecular Characterization and Expression Profiling of NAC Transcription Factors in Brachypodium distachyon L.

NAC (NAM, ATAF1/2, CUC2) transcription factors are involved in regulating plant developmental processes and response to environmental stresses. Brachypodium distachyon is an emerging model system for cereals, temperate grasses and biofuel crops. In this study, a comprehensive investigation of the molecular characterizations, phylogenetics and expression profiles under various abiotic stresses of the NAC gene family in Brachypodium distachyon was performed. In total, 118 BNAC genes in B. distachyon were identified, of which 22 (18.64%) were tandemly duplicated and segmentally duplicated, respectively. The Bayesian phylogenetic inference using Markov Chain Monte Carlo (MCMC) algorithms showed that they were divided into two clades and fourteen subfamilies, supported by similar motif compositions within one subfamily. Some critical amino acids detected using DIVERGE v3.0 might contribute to functional divergence among subfamilies. The different exon-intron organizations among subfamilies revealed structural differentiation. Promoter sequence predictions showed that the BNAC genes were involved in various developmental processes and diverse stress responses. Three NAC domain-encoding genes (BNAC012, BNAC078 and BNAC108), orthologous of NAC1, were targeted by five miRNA164 (Bdi-miR164a-c, e, f), suggesting that they might function in lateral organ enlargement, floral development and the responses to abiotic stress. Eleven (~9.32%) BNAC proteins containing α-helical transmembrane motifs were identified. 23 representative BNAC genes were analyzed by quantitative real-time PCR, showing different expression patterns under various abiotic stresses, of which 18, 17 and 11 genes were up-regulated significantly under drought, H2O2 and salt stresses, respectively. Only four and two genes were up-regulated under cold and cadmium stresses, respectively. Dynamic transcriptional expression analysis revealed that six genes showed constitutive expression and period-specific expression. The current results provide novel insights into the structure and function of the plant NAC gene family.

The divergence and positive selection of the plant-specific BURP-containing protein family.

BURP domain-containing proteins belong to a plant-specific protein family and have diverse roles in plant development and stress responses. However, our understanding about the genetic divergence patterns and evolutionary rates of these proteins remain inadequate. In this study, 15 plant genomes were explored to elucidate the genetic origins, divergence, and functions of these proteins. One hundred and twenty-five BURP protein-encoding genes were identified from four main plant lineages, including 13 higher plant species. The absence of BURP family genes in unicellular and multicellular algae suggests that this family (1) appeared when plants shifted from relatively stable aquatic environments to land, where conditions are more variable and stressful, and (2) is critical in the adaptation of plants to adverse environments. Promoter analysis revealed that several responsive elements to plant hormones and external environment stresses are concentrated in the promoter region of BURP protein-encoding genes. This finding confirms that these genes influence plant stress responses. Several segmentally and tandem-duplicated gene pairs were identified from eight plant species. Thus, in general, BURP domain-containing genes have been subject to strong positive selection, even though these genes have conformed to different expansion models in different species. Our study also detected certain critical amino acid sites that may have contributed to functional divergence among groups or subgroups. Unexpectedly, all of the critical amino acid residues of functional divergence and positive selection were exclusively located in the C-terminal region of the BURP domain. In conclusion, our results contribute novel insights into the genetic divergence patterns and evolutionary rates of BURP proteins.

Unusual tandem expansion and positive selection in subgroups of the plant GRAS transcription factor superfamily.

BACKGROUND: GRAS proteins belong to a plant transcription factor family that is involved with multifarious roles in plants. Although previous studies of this protein family have been reported for Arabidopsis, rice, Chinese cabbage and other species, investigation of expansion patterns and evolutionary rate on the basis of comparative genomics in different species remains inadequate. RESULTS: A total of 289 GRAS genes were identified in Arabidopsis, B. distachyon, rice, soybean, S. moellendorffii, and P. patens and were grouped into seven subfamilies, supported by the similarity of their exon-intron patterns and structural motifs. All of tandem duplicated genes were found in group II except one cluster of rice, indicating that tandem duplication greatly promoted the expansion of group II. Furthermore, segment duplications were mainly found in the soybean genome, whereas no single expansion pattern dominated in other plant species indicating that GRAS genes from these five species might be subject to a more complex evolutionary mechanism. Interestingly, branch-site model analyses of positive selection showed that a number of sites were positively selected under foreground branches I and V. These results strongly indicated that these groups were experiencing higher positive selection pressure. Meanwhile, the site-specific model revealed that the GRAS genes were under strong positive selection in P. patens. DIVERGE v2.0 was used to detect critical amino acid sites, and the results showed that the shifted evolutionary rate was mainly attributed to the functional divergence between the GRAS genes in the two groups. In addition, the results also demonstrated the expression divergence of the GRAS duplicated genes in the evolution. In short, the results above provide a solid foundation for further functional dissection of the GRAS gene superfamily. CONCLUSIONS: In this work, differential expression, evolutionary rate, and expansion patterns of the GRAS gene family in the six species were predicted. Especially, tandem duplication events played an important role in expansion of group II. Together, these results contribute to further functional analysis and the molecular evolution of the GRAS gene superfamily.

Molecular cloning, phylogenetic analysis, and expression profiling of endoplasmic reticulum molecular chaperone BiP genes from bread wheat (Triticum aestivum L.).

BACKGROUND: The endoplasmic reticulum chaperone binding protein (BiP) is an important functional protein, which is involved in protein synthesis, folding assembly, and secretion. In order to study the role of BiP in the process of wheat seed development, we cloned three BiP homologous cDNA sequences in bread wheat (Triticum aestivum), completed by rapid amplification of cDNA ends (RACE), and examined the expression of wheat BiP in wheat tissues, particularly the relationship between BiP expression and the subunit types of HMW-GS using near-isogenic lines (NILs) of HMW-GS silencing, and under abiotic stress. RESULTS: Sequence analysis demonstrated that all BiPs contained three highly conserved domains present in plants, animals, and microorganisms, indicating their evolutionary conservation among different biological species. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that TaBiP (Triticum aestivum BiP) expression was not organ-specific, but was predominantly localized to seed endosperm. Furthermore, immunolocalization confirmed that TaBiP was primarily located within the protein bodies (PBs) in wheat endosperm. Three TaBiP genes exhibited significantly down-regulated expression following high molecular weight-glutenin subunit (HMW-GS) silencing. Drought stress induced significantly up-regulated expression of TaBiPs in wheat roots, leaves, and developing grains. CONCLUSIONS: The high conservation of BiP sequences suggests that BiP plays the same role, or has common mechanisms, in the folding and assembly of nascent polypeptides and protein synthesis across species. The expression of TaBiPs in different wheat tissue and under abiotic stress indicated that TaBiP is most abundant in tissues with high secretory activity and with high proportions of cells undergoing division, and that the expression level of BiP is associated with the subunit types of HMW-GS and synthesis. The expression of TaBiPs is developmentally regulated during seed development and early seedling growth, and under various abiotic stresses.

Screening and identification of soybean seed-specific genes by using integrated bioinformatics of digital differential display, microarray, and RNA-seq data.

Soybean is one of the most economically important crops in the world. Soybean seeds have abundant protein and lipid content and very high economic value. In this study, a total of 184 seed-specific genes were obtained using online microarray databases, DDD, and RNA-seq data. The reported seed-specific genes in soybean and the 184 seed-specific genes analyzed in this paper were compared. Of the screened genes, 26 were common to both previous reports and the current screening. Meanwhile, 90 of the 184 genes have homologous counterparts in Arabidopsis, among which 24 have seed-specific expression, as indicated by microarray data for Arabidopsis. Furthermore, promoter analysis showed that almost all seed-specific genes contain at least one seed specific-related element. Seed-specific element Skn-1 motif exists in most, if not all, of the seed-specific genes screened. Five genes were randomly selected from 184 soybean seed specific gene pool and their expressions were quantified using quantitative real time polymerase chain reaction (qRT-PCR) to further confirm the specificity of the screened genes. The results indicated that all five genes showed seed-specific expression. Moreover, the identification of genes with seed-specific expression screened in this study provides information valuable to the in-depth study of soybean.

Delineation of plant caleosin residues critical for functional divergence, positive selection and coevolution.

BACKGROUND: The caleosin genes encode proteins with a single conserved EF hand calcium-binding domain and comprise small gene families found in a wide range of plant species. These proteins may be involved in many cellular and biological processes coupled closely to the synthesis, degradation, or stability of oil bodies. Although previous studies of this protein family have been reported for Arabidopsis and other species, understanding of the evolution of the caleosin gene family in plants remains inadequate. RESULTS: In this study, comparative genomic analysis was performed to investigate the phylogenetic relationships, evolutionary history, functional divergence, positive selection, and coevolution of caleosins. First, 84 caleosin genes were identified from five main lineages that included 15 species. Phylogenetic analysis placed these caleosins into five distinct subfamilies (sub I-V), including two subfamilies that have not been previously identified. Among these subfamilies, sub II coincided with the distinct P-caleosin isoform recently identified in the pollen oil bodies of lily; caleosin genes from the same lineage tended to be clustered together in the phylogenetic tree. A special motif was determined to be related with the classification of caleosins, which may have resulted from a deletion in sub I and sub III occurring after the evolutionary divergence of monocot and dicot species. Additionally, several segmentally and tandem-duplicated gene pairs were identified from seven species, and further analysis revealed that caleosins of different species did not share a common expansion model. The ages of each pair of duplications were calculated, and most were consistent with the time of genome-wide duplication events in each species. Functional divergence analysis showed that changes in functional constraints have occurred between subfamilies I/IV, II/IV, and II/V, and some critical amino acid sites were identified during the functional divergence. Additional analyses revealed that caleosins were under positive selection during evolution, and seven candidate amino acid sites (70R, 74G, 88 L, 89G, 100 K, 106A, 107S) for positive selection were identified. Interestingly, the critical amino acid residues of functional divergence and positive selection were mainly located in C-terminal domain. Finally, three groups of coevolved amino acid sites were identified. Among these coevolved sites, seven from group 2 were located in the Ca2+-binding region of crucial importance. CONCLUSION: In this study, the evolutionary and expansion patterns of the caleosin gene family were predicted, and a series of amino acid sites relevant to their functional divergence, adaptive evolution, and coevolution were identified. These findings provide data to facilitate further functional analysis of caleosin gene families in the plant lineage.

Molecular characterization and expression profiling of the protein disulfide isomerase gene family in Brachypodium distachyon L.

Protein disulfide isomerases (PDI) are involved in catalyzing protein disulfide bonding and isomerization in the endoplasmic reticulum and functions as a chaperone to inhibit the aggregation of misfolded proteins. Brachypodium distachyon is a widely used model plant for temperate grass species such as wheat and barley. In this work, we report the first molecular characterization, phylogenies, and expression profiles of PDI and PDI-like (PDIL) genes in B. distachyon in different tissues under various abiotic stresses. Eleven PDI and PDIL genes in the B. distachyon genome by in silico identification were evenly distributed across all five chromosomes. The plant PDI family has three conserved motifs that are involved in catalyzing protein disulfide bonding and isomerization, but a different exon/intron structural organization showed a high degree of structural differentiation. Two pairs of genes (BdPDIL4-1 and BdPDIL4-2; BdPDIL7-1 and BdPDIL7-2) contained segmental duplications, indicating each pair originated from one progenitor. Promoter analysis showed that Brachypodium PDI family members contained important cis-acting regulatory elements involved in seed storage protein synthesis and diverse stress response. All Brachypodium PDI genes investigated were ubiquitously expressed in different organs, but differentiation in expression levels among different genes and organs was clear. BdPDIL1-1 and BdPDIL5-1 were expressed abundantly in developing grains, suggesting that they have important roles in synthesis and accumulation of seed storage proteins. Diverse treatments (drought, salt, ABA, and H2O2) induced up- and down-regulated expression of Brachypodium PDI genes in seedling leaves. Interestingly, BdPDIL1-1 displayed significantly up-regulated expression following all abiotic stress treatments, indicating that it could be involved in multiple stress responses. Our results provide new insights into the structural and functional characteristics of the plant PDI gene family.