Genome-wide analysis of the LAZ1 gene family in Gossypium hirsutum.

BACKGROUND: As the world's leading fiber crop and a major oil-producing crop, cotton fiber yield and fiber quality are affected by environmental stresses, especially heat, drought and salinity. The LAZ1 (Lazarus 1) family genes are responsive to abscisic acid, drought, and salt treatments. Currently, mining and functional analyses of LAZ1 family genes in cotton have not been reported. METHODS AND RESULTS: In this study, 20 GhLAZ1 genes, designated GhLAZ1-1 - GhLAZ1-20, were identified in the genome of Gossypium hirsutum through the construction of an HMM model, and their molecular properties, chromosomal localization, phylogeny, gene structure, evolutionary selection pressure, promoter cis elements and gene expression under salt stress were analyzed. With the exception of GhLAZ1-17 and GhLAZ1-20, the remaining 18 GhLAZ1 genes were unevenly localized on 13 chromosomes in G. hirsutum; evolutionary analysis showed that these genes could be divided into three subfamilies; and evolutionary selection pressure analysis demonstrated that the GhLAZ1 genes were all under purifying selection. Many elements related to light responses, hormone responses, and abiotic stresses were predicted on the GhLAZ1 family gene promoters, and real-time quantitative PCR results showed that GhLAZ1-2, GhLAZ1-8, and GhLAZ1-18 were upregulated significantly in salt-treated cotton leaves. CONCLUSIONS: Our results suggested that GhLAZ1 genes were involved in the salt tolerance mechanism in G. hirsutum and provided a reference for further exploring the function and molecular mechanism of LAZ1 genes.

Genome-wide identification of the geranylgeranyl pyrophosphate synthase (GGPS) gene family involved in chlorophyll synthesis in cotton.

BACKGROUND: Geranylgeranyl pyrophosphate synthase (GGPS) is a structural enzyme of the terpene biosynthesis pathway that is involved in regulating plant photosynthesis, growth and development, but this gene family has not been systematically studied in cotton. RESULTS: In the current research, genome-wide identification was performed, and a total of 75 GGPS family members were found in four cotton species, Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii. The GGPS genes were divided into three subgroups by evolutionary analysis. Subcellular localization prediction showed that they were mainly located in chloroplasts and plastids. The closely related GGPS contains a similar gene structure and conserved motif, but some genes are quite different, resulting in functional differentiation. Chromosome location analysis, collinearity and selection pressure analysis showed that many fragment duplication events occurred in GGPS genes. Three-dimensional structure analysis and conservative sequence analysis showed that the members of the GGPS family contained a large number of α-helices and random crimps, and all contained two aspartic acid-rich domains, DDxxxxD and DDxxD (x is an arbitrary amino acid), suggesting its key role in function. Cis-regulatory element analysis showed that cotton GGPS may be involved in light response, abiotic stress and other processes. A GGPS gene was silenced successfully by virus-induced gene silencing (VIGS), and it was found that the chlorophyll content in cotton leaves decreased significantly, suggesting that the gene plays an important role in plant photosynthesis. CONCLUSIONS: In total, 75 genes were identified in four Gossypium species by a series of bioinformatics analysis. Gene silencing from GGPS members of G. hirsutum revealed that GGPS plays an important regulatory role in photosynthesis. This study provides a theoretical basis for the biological function of GGPS in cotton growth and development.

Combined Metabolomic and Transcriptomic Analysis Reveals Allantoin Enhances Drought Tolerance in Rice.

Drought is a misfortune for agriculture and human beings. The annual crop yield reduction caused by drought exceeds the sum of all pathogens. As one of the gatekeepers of China's "granary", rice is the most important to reveal the key drought tolerance factors in rice. Rice seedlings of Nipponbare (Oryza sativa L. ssp. Japonica) were subjected to simulated drought stress, and their root systems were analyzed for the non-targeted metabolome and strand-specific transcriptome. We found that both DEGs and metabolites were enriched in purine metabolism, and allantoin accumulated significantly in roots under drought stress. However, few studies on drought tolerance of exogenous allantoin in rice have been reported. We aimed to further determine whether allantoin can improve the drought tolerance of rice. Under the treatment of exogenous allantoin at different concentrations, the drought resistant metabolites of plants accumulated significantly, including proline and soluble sugar, and reactive oxygen species (ROS) decreased and reached a significant level in 100 µmol L-1. To this end, a follow-up study was identified in 100 µmol L-1 exogenous allantoin and found that exogenous allantoin improved the drought resistance of rice. At the gene level, under allantoin drought treatment, we found that genes of scavenge reactive oxygen species were significantly expressed, including peroxidase (POD), catalase (CATA), ascorbate peroxidase 8 (APX8) and respiratory burst oxidase homolog protein F (RbohF). This indicates that plants treated by allantoin have better ability to scavenge reactive oxygen species to resist drought. Alternative splicing analysis revealed a total of 427 differentially expressed alternative splicing events across 320 genes. The analysis of splicing factors showed that gene alternative splicing could be divided into many different subgroups and play a regulatory role in many aspects. Through further analysis, we restated the key genes and enzymes in the allantoin synthesis and catabolism pathway, and found that the expression of synthetase and hydrolase showed a downward trend. The pathway of uric acid to allantoin is completed by uric acid oxidase (UOX). To find out the key transcription factors that regulate the expression of this gene, we identified two highly related transcription factors OsERF059 and ONAC007 through correlation analysis. They may be the key for allantoin to enhance the drought resistance of rice.

Phylogenetic analysis and stress response of the plant U2 small nuclear ribonucleoprotein B″ gene family.

BACKGROUND: Alternative splicing (AS) is an important channel for gene expression regulation and protein diversification, in addition to a major reason for the considerable differences in the number of genes and proteins in eukaryotes. In plants, U2 small nuclear ribonucleoprotein B″ (U2B″), a component of splicing complex U2 snRNP, plays an important role in AS. Currently, few studies have investigated plant U2B″, and its mechanism remains unclear. RESULT: Phylogenetic analysis, including gene and protein structures, revealed that U2B″ is highly conserved in plants and typically contains two RNA recognition motifs. Subcellular localisation showed that OsU2B″ is located in the nucleus and cytoplasm, indicating that it has broad functions throughout the cell. Elemental analysis of the promoter region showed that it responded to numerous external stimuli, including hormones, stress, and light. Subsequent qPCR experiments examining response to stress (cold, salt, drought, and heavy metal cadmium) corroborated the findings. The prediction results of protein-protein interactions showed that its function is largely through a single pathway, mainly through interaction with snRNP proteins. CONCLUSION: U2B″ is highly conserved in the plant kingdom, functions in the nucleus and cytoplasm, and participates in a wide range of processes in plant growth and development.

The OsCBL8-OsCIPK17 Module Regulates Seedling Growth and Confers Resistance to Heat and Drought in Rice.

The calcium signaling pathway is critical for plant growth, development, and response to external stimuli. The CBL-CIPK pathway has been well characterized as a calcium-signaling pathway. However, in most reports, only a single function for this module has been described. Here, we examined multiple functions of this module. CIPK showed a similar distribution to that of CBL, and OsCBL and OsCIPK families were retained after experiencing whole genome duplication events through the phylogenetic and synteny analysis. This study found that OsCBL8 negatively regulated rice seed germination and seedling growth by interacting with OsCIPK17 with overexpression and gene editing mutant plants as materials combining plant phenotype, physiological indicators and transcriptome sequencing. This process is likely mediated by OsPP2C77, which is a member of the ABA signaling pathway. In addition, OsCBL mediated the targeting of OsNAC77 and OsJAMYB by OsCIPK17, thus conferring resistance to high temperatures and pathogens in rice. Our work reveals a unique signaling pathway, wherein OsCBL8 interacts with OsCIPK17 and provides rice with multiple resistance while also regulating seedling growth.

Genome-wide analysis reveals the spatiotemporal expression patterns of SOS3 genes in the maize B73 genome in response to salt stress.

BACKGROUND: Salt damage is an important abiotic stress that affects the growth and yield of maize worldwide. As an important member of the salt overly sensitive (SOS) signal transduction pathway, the SOS3 gene family participates in the transmission of stress signals and plays a vital role in improving the salt tolerance of plants. RESULTS: In this study, we identified 59 SOS3 genes in the maize B73 genome using bioinformatics methods and genome-wide analyses. SOS3 proteins were divided into 5 different subfamilies according to the phylogenetic relationships. A close relationship between the phylogenetic classification and intron mode was observed, with most SOS3 genes in the same group sharing common motifs and similar exon-intron structures in the corresponding genes. These genes were unequally distributed on five chromosomes of B73. A total of six SOS3 genes were identified as repeated genes, and 12 pairs of genes were proven to be segmentally duplicated genes, indicating that gene duplication may play an important role in the expansion of the SOS3 gene family. The expression analysis of 10 genes that were randomly selected from different subgroups suggested that all 10 genes were significantly differentially expressed within 48 h after salt treatment, of which eight SOS3 genes showed a significant decline while Zm00001d025938 and Zm00001d049665 did not. By observing the subcellular localization results, we found that most genes were expressed in chloroplasts while some genes were expressed in the cell membrane and nucleus. CONCLUSIONS: Our study provides valuable information for elucidating the evolutionary relationship and functional characteristics of the SOS3 gene family and lays the foundation for further study of the SOS3 gene family in the maize B73 genome.

Combined metabolomic and transcriptomic analysis reveals key components of OsCIPK17 overexpression improves drought tolerance in rice.

Oryza Sativa is one of the most important food crops in China, which is easily affected by drought during its growth and development. As a member of the calcium signaling pathway, CBL-interacting protein kinase (CIPK) plays an important role in plant growth and development as well as environmental stress. However, there is no report on the function and mechanism of OsCIPK17 in rice drought resistance. We combined transcriptional and metabonomic analysis to clarify the specific mechanism of OsCIPK17 in response to rice drought tolerance. The results showed that OsCIPK17 improved drought resistance of rice by regulating deep roots under drought stress; Response to drought by regulating the energy metabolism pathway and controlling the accumulation of citric acid in the tricarboxylic acid (TCA) cycle; Our exogenous experiments also proved that OsCIPK17 responds to citric acid, and this process involves the auxin metabolism pathway; Exogenous citric acid can improve the drought resistance of overexpression plants. Our research reveals that OsCIPK17 positively regulates rice drought resistance and participates in the accumulation of citric acid in the TCA cycle, providing new insights for rice drought resistance.

Phylogenetic Analysis of the Plant U2 snRNP Auxiliary Factor Large Subunit A Gene Family in Response to Developmental Cues and Environmental Stimuli.

In all organisms, splicing occurs through the formation of spliceosome complexes, and splicing auxiliary factors are essential during splicing. U2AF65 is a crucial splicing cofactor, and the two typical RNA-recognition motifs at its center recognize and bind the polypyrimidine sequence located between the intron branch site and the 3'-splice site. U2AF65A is a member of the U2AF65 gene family, with pivotal roles in diseases in mammals, specifically humans; however, few studies have investigated plant U2AF65A, and its specific functions are poorly understood. Therefore, in the present study, we systematically identified U2AF65A in plant species from algae to angiosperms. Based on 113 putative U2AF65A sequences from 33 plant species, phylogenetic analyses were performed, followed by basic bioinformatics, including the comparisons of gene structure, protein domains, promoter motifs, and gene expression levels. In addition, using rice as the model crop, we demonstrated that the OsU2AF65A protein is localized to the nucleus and cytoplasm, and it is involved in responses to various stresses, such as drought, high salinity, low temperature, and heavy metal exposure (e.g., cadmium). Using Arabidopsis thaliana and rice mutants, we demonstrated that U2AF65A is involved in the accumulation of plant biomass, growth of hypocotyl upon thermal stimulation, and reduction of tolerance of high temperature stress. These findings offer an overview of the U2AF65 gene family and its stress response functions, serving as the reference for further comprehensive functional studies of the essential specific splicing cofactor U2AF65A in the plant kingdom.

Bismuth Vanadium Oxide Can Promote Growth and Activity in Arabidopsis thaliana.

The excellent properties of nanomaterials have been confirmed in many fields, but their effects on plants are still unclear. In this study, different concentrations of bismuth vanadate (BV) were added to the growth medium to analyze the growth of seedlings, including taproots, lateral roots, leaf stomata, root activity, and superoxide anion O2 .- generation. Gene expression levels related to root growth were determined by quantitative PCR in Arabidopsis thaliana. The results showed that BV promoted the growth of taproots and the development of lateral roots, enhanced the length of the extension zone in roots, increased the number and size of leaf stomata and root activity, reduced the accumulation of ROS in seedlings, and changed the expression levels of genes related to polyamines or hormones. At the same time, we investigated the antibacterial activity of BV against a variety of common pathogens causing crop diseases. The results showed that BV could effectively inhibit the growth of Fusarium wilt of cotton and rice sheath blight. These results provide a new prospect for the development of nanomaterial-assisted plants, which is expected to become one of the ways to solve the problem of controlling and promoting the development of plants. At the same time, it also provides a reference for the study of the effect of BV on plants.

Functional Analysis of OsCIPK17 in Rice Grain Filling.

We used mutant cipk17 and Nipponbare in field experiments to analyze agronomic traits, photosynthetic parameters, transcriptome, and gene expression. The results demonstrated cytoplasmic localization of OsCIPK17, while GUS allogeneic (A. thaliana) tissue-staining and quantitative analysis showed the gene was expressed in many organs, including flower buds; furthermore, it was involved in root, stem, and leaf growth. Compared to Nipponbare plants, grain filling rate and final grain weight decreased in plants of the knockout mutant owing to a delay in attainment of maximum grain filling rate. Photosystem II (PSII) efficiency was also reduced. Enrichment analysis showed that the functions of differentially expressed genes (DEGs) focused on nucleoside-, nucleotide-, and lipid-binding, as well as hydrolase, transferase, and phosphorylase activities. Signaling pathways mainly included starch and sucrose metabolism, as well as photosynthesis. Additionally, some DEGs were verified by fluorescence analysis. The results showed that knockout of OsCIPK17 affected photosynthesis and starch-, sucrose-, and amino acid metabolism-related gene expression; furthermore, the mutation reduced PSII utilization efficiency, it blocked the synthesis and metabolism of starch and sucrose, and affected the formation and transport of assimilates, thereby reducing final grain weight.

Spermidine Enhanced Free Polyamine Levels and Expression of Polyamine Biosynthesis Enzyme Gene in Rice Spikelets under Heat Tolerance before Heading.

High temperatures (HT) before heading strongly inhibit the development of spikelets in rice. Spermidine (Spd) can improve rice's resistance to HT stress; however, the mechanism underlying this effect has not been elucidated. This study investigated several parameters, including yield, superoxide anion (O2.-), protective enzyme activities, and polyamine content, in a heat-sensitive genotype, Shuanggui 1. The yield and yield components decreased dramatically when subjected to HT stress, while this reduction could be partially recovered by exogenous Spd. Spd also slowed the generation rate of O2.- and increased protective enzyme, superoxide dismutase (SOD) and catalase (CAT) activities both under normal and high temperatures, which suggested that Spd may participate in the antioxidant system. Furthermore, genes involved in polyamine synthesis were analyzed. The results show that HT before heading significantly increased the expression of arginine decarboxylase OsADC1, Spd synthase OsSPDS1 and OsSPDS3 and had little effect on the expression of the S-adenosylmethionine decarboxylase OsSAMDC2 and ornithine decarboxylase OsODC1. In addition, exogenous Spd considerably reduced the expression of OsSAMDC2, OsSPDS1 and OsSPDS3 under HT but not the expression of OsADC1. The above mentioned results indicate that the exogenous Spd could help young rice spikelets to resist HT stress by reducing the expression of OsSAMDC2, OsSPDS1 and OsSPDS3, resulting in higher levels of endogenous Spd and Spm, which were also positively correlated with yield. In conclusion, the adverse effect of HT stress on young spikelets seems to be alleviated by increasing the amounts of Spd and Spm, which provides guidance for adaptation to heat stress during rice production.

Comprehensive transcriptome analysis of faba bean in response to vernalization.

MAIN CONCLUSION: This study unravels the transcriptional response of a highly productive faba bean cultivar under vernalization treatment. Faba bean (Vicia faba L.) is a member of the Leguminosae family and an important food crop worldwide providing valuable nutrients for humans. However, genome-wide studies and comprehensive sequencing resources of faba bean remain limited. Vernalization is crucial for enhanced yields in a number of winter-sown crops. However, the effects of vernalization on faba bean remain unknown. In this study, we generated a high-quality transcriptome assembly and functional annotation source for vernalized faba bean (Vicia faba L.) cv. Tongxian-2, a domesticated cultivar from southern China. A total of 369.9 million clean Illumina paired-end RNA-Seq reads were generated, and the transcriptome was assembled into 68,683 unigene sequences, with an average length of 1018 bp and an N50 of 1652 bp. Comprehensive functional annotation provided putative functional descriptions for more than 70% of the faba bean transcripts. We annotated a total of 1560 faba bean transcripts encoding transcription factors (TFs) belonging to 55 distinct TF families. The bHLH (168 transcripts), ERF (123 transcripts) and WRKY (105 transcripts) contained the largest number of TFs in response to vernalization. Genome-wide transcript changes comparing vernalized and unvernalized seedlings were investigated using bioinformatics approaches, which revealed a strong repression of photosynthesis and carbon metabolism, while genes participating in 'response to stress' were significantly induced. We also specifically identified vernalization-induced twenty-two 'pollen-pistil interaction' genes. A detailed functional annotation and expression profile analyses unveiled a number of protein kinases, which were specifically induced in vernalized seedlings. We also identified a total of 6852 simple sequence repeats (SSRs) in 6552 transcripts, representing a valuable genomic molecular marker resource for faba bean. In summary, this study provides new insights into the vernalization process in this economically valuable crop. The transcriptome data obtained provides us with a valuable candidate gene resource for future functional and molecular breeding studies. These data will contribute to the genome annotation for ensuing genome projects.

Rhizosheath formation and involvement in foxtail millet (Setaria italica) root growth under drought stress.

The rhizosheath, a layer of soil particles that adheres firmly to the root surface by a combination of root hairs and mucilage, may improve tolerance to drought stress. Setaria italica (L.) P. Beauv. (foxtail millet), a member of the Poaceae family, is an important food and fodder crop in arid regions and forms a larger rhizosheath under drought conditions. Rhizosheath formation under drought conditions has been studied, but the regulation of root hair growth and rhizosheath size in response to soil moisture remains unclear. To address this question, in this study we monitored root hair growth and rhizosheath development in response to a gradual decline in soil moisture. Here, we determined that a soil moisture level of 10%-14% (w/w) stimulated greater rhizosheath production compared to other soil moisture levels. Root hair density and length also increased at this soil moisture level, which was validated by measurement of the expression of root hair-related genes. These findings contribute to our understanding of rhizosheath formation in response to soil water stress.

Alternative splicing and translation play important roles in hypoxic germination in rice.

Post-transcriptional mechanisms (PTMs), including alternative splicing (AS) and alternative translation initiation (ATI), may explain the diversity of proteins involved in plant development and stress responses. Transcriptional regulation is important during the hypoxic germination of rice seeds, but the potential roles of PTMs in this process have not been characterized. We used a combination of proteomics and RNA sequencing to discover how AS and ATI contribute to plant responses to hypoxia. In total, 10 253 intron-containing genes were identified. Of these, ~1741 differentially expressed AS (DAS) events from 811 genes were identified in hypoxia-treated seeds compared with controls. Over 95% of these were not present in the list of differentially expressed genes. In particular, regulatory pathways such as the spliceosome, ribosome, endoplasmic reticulum protein processing and export, proteasome, phagosome, oxidative phosphorylation, and mRNA surveillance showed substantial AS changes under hypoxia, suggesting that AS responses are largely independent of transcriptional regulation. Considerable AS changes were identified, including the preferential usage of some non-conventional splice sites and enrichment of splicing factors in the DAS data sets. Taken together, these results not only demonstrate that AS and ATI function during hypoxic germination but they have also allowed the identification of numerous novel proteins/peptides produced via ATI.

Comparative performance of the BGISEQ-500 and Illumina HiSeq4000 sequencing platforms for transcriptome analysis in plants.

BACKGROUND: The next-generation sequencing (NGS) technology has greatly facilitated genomic and transcriptomic studies, contributing significantly in expanding the current knowledge on genome and transcriptome. However, the continually evolving variety of sequencing platforms, protocols and analytical pipelines has led the research community to focus on cross-platform evaluation and standardization. As a NGS pioneer in China, the Beijing Genomics Institute (BGI) has announced its own NGS platform designated as BGISEQ-500, since 2016. The capability of this platform in large-scale DNA sequencing and small RNA analysis has been already evaluated. However, the comparative performance of BGISEQ-500 platform in transcriptome analysis remains yet to be elucidated. The Illumina series, a leading sequencing platform in China's sequencing market, would be a preferable reference to evaluate new platforms. METHODS: To this end, we describe a cross-platform comparative study between BGISEQ-500 and Illumina HiSeq4000 for analysis of Arabidopsis thaliana WT (Col 0) transcriptome. The key parameters in RNA sequencing and transcriptomic data processing were assessed in biological replicate experiments, using aforesaid platforms. RESULTS: The results from the two platforms BGISEQ-500 and Illumina HiSeq4000 shared high concordance in both inter- (correlation, 0.88-0.93) and intra-platform (correlation, 0.95-0.98) comparison for gene quantification, identification of differentially expressed genes and alternative splicing events. However, the two platforms yielded highly variable interpretation results for single nucleotide polymorphism and insertion-deletion analysis. CONCLUSION: The present case study provides a comprehensive reference dataset to validate the capability of BGISEQ-500 enabling it to be established as a competitive and reliable platform in plant transcriptome analysis.

Natural variation in the promoter of rice calcineurin B-like protein10 (OsCBL10) affects flooding tolerance during seed germination among rice subspecies.

Rice (Oryza sativa L.) has two ecotypes, upland and lowland rice, that have been observed to show different tolerance levels under flooding stress. In this study, two rice cultivars, upland (Up221, flooding-intolerant) and lowland (Low88, flooding-tolerant), were initially used to study their molecular mechanisms in response to flooding germination. We observed that variations in the OsCBL10 promoter sequences in these two cultivars might contribute to this divergence in flooding tolerance. Further analysis using another eight rice cultivars revealed that the OsCBL10 promoter could be classified as either a flooding-tolerant type (T-type) or a flooding-intolerant type (I-type). The OsCBL10 T-type promoter only existed in japonica lowland cultivars, whereas the OsCBL10 I-type promoter existed in japonica upland, indica upland and indica lowland cultivars. Flooding-tolerant rice cultivars containing the OsCBL10 T-type promoter have shown lower Ca2+ flow and higher α-amylase activities in comparison to those in flooding-intolerant cultivars. Furthermore, the OsCBL10 overexpression lines were sensitive to both flooding and hypoxic treatments during rice germination with enhanced Ca2+ flow in comparison to wild-type. Subsequent findings also indicate that OsCBL10 may affect OsCIPK15 protein abundance and its downstream pathways. In summary, our results suggest that the adaptation to flooding stress during rice germination is associated with two different OsCBL10 promoters, which in turn affect OsCBL10 expression in different cultivars and negatively affect OsCIPK15 protein accumulation and its downstream cascade.

iTRAQ-based quantitative proteomic analysis in vernalization-treated faba bean (Vicia faba L.).

Vernalization is classically defined as the induction of flowering process by exposure of the plants to a prolonged cold condition. Normally, it is considered as a precondition of flowering. Vicia faba, commonly known as faba bean, belongs to family Fabaceae. It is one of the plant species that has been cultivated in the earliest human settlements. In this study, an iTRAQ-LC-MS/MS-based quantitative proteomic analysis has been conducted to compare the vernalized faba bean seedlings and its corresponding control. In total, 91 proteins from various functional categories were observed to be differentially accumulated in vernalized faba bean seedlings. Subsequent gene ontology analysis indicated that several biological processes or metabolic pathways including photosynthesis and phytic acid metabolism were differentially respond to vernalization in comparison to the control sample. Further investigation revealed that a family of proteins nominated as glycine-rich RNA-binding factor was accumulated in vernalized seedlings, indicating an extra layer of regulation by alternative splicing on transcript abundance in response to vernalization. These findings raise a possibility that these candidate proteins could be important to represent the responsive network under vernalization process. Therefore, we propose that the regulation of vernalization in faba bean not only occurs at the transcriptional level as previously reported, but also at the post-transcriptional level.

Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings.

In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues; however, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short-read RNA sequencing, single molecule long-read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron-containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR); however, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non-conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment.

A Phylogenetically Informed Comparison of GH1 Hydrolases between Arabidopsis and Rice Response to Stressors.

Glycoside hydrolases Family 1 (GH1) comprises enzymes that can hydrolyze ß-O-glycosidic bond from a carbohydrate moiety. The plant GH1 hydrolases participate in a number of developmental processes and stress responses, including cell wall modification, plant hormone activation or deactivation and herbivore resistance. A large number of members has been observed in this family, suggesting their potential redundant functions in various biological processes. In this study, we have used 304 sequences of plant GH1 hydrolases to study the evolution of this gene family in plant lineage. Gene duplication was found to be a common phenomenon in this gene family. Although many members of GH1 hydrolases showed a high degree of similarity in Arabidopsis and rice, they showed substantial tissue specificity and differential responses to various stress treatments. This differential regulation implies each enzyme may play a distinct role in plants. Furthermore, some of salt-responsive Arabidopsis GH1 hydrolases were selected to test their genetic involvement in salt responses. The knockout mutants of AtBGLU1 and AtBGLU19 were observed to be less-sensitive during NaCl treatment in comparison to the wild type seedlings, indicating their participation in salt stress response. In summary, Arabidopsis and rice GH1 glycoside hydrolases showed distinct features in their evolutionary path, transcriptional regulation and genetic functions.

SWATH-MS Quantitative Analysis of Proteins in the Rice Inferior and Superior Spikelets during Grain Filling.

Modern rice cultivars have large panicle but their yield potential is often not fully achieved due to poor grain-filling of late-flowering inferior spikelets (IS). Our earlier work suggested a broad transcriptional reprogramming during grain filling and showed a difference in gene expression between IS and earlier-flowering superior spikelets (SS). However, the links between the abundances of transcripts and their corresponding proteins are unclear. In this study, a SWATH-MS (sequential window acquisition of all theoretical spectra-mass spectrometry) -based quantitative proteomic analysis has been applied to investigate SS and IS proteomes. A total of 304 proteins of widely differing functionality were observed to be differentially expressed between IS and SS. Detailed gene ontology analysis indicated that several biological processes including photosynthesis, protein metabolism, and energy metabolism are differentially regulated. Further correlation analysis revealed that abundances of most of the differentially expressed proteins are not correlated to the respective transcript levels, indicating that an extra layer of gene regulation which may exist during rice grain filling. Our findings raised an intriguing possibility that these candidate proteins may be crucial in determining the poor grain-filling of IS. Therefore, we hypothesize that the regulation of proteome changes not only occurs at the transcriptional, but also at the post-transcriptional level, during grain filling in rice.