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.

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.

Optimization of ultrasonic-assisted enzymatic hydrolysis for the extraction of luteolin and apigenin from celery.

The extraction of flavonoids is of increasing interest because of their various pharmacological effects. This study is the first attempt for the ultrasonic-assisted enzymatic hydrolysis (USAEH) applied in the extraction of 2 bioactive flavonoid compounds in celery--luteolin and apigenin. The quantitative yields of luteolin and apigenin were determined by high-performance liquid chromatography (HPLC). To achieve high yields of extracted compounds, the procedure was optimized with regard to the relative parameters involved. The optimal conditions for enzymatic hydrolysis using pectinase treatment were a reaction time of 30 min and a concentration of 0.4 mg/mL at pH 3 for luteolin and pH 5.5 for apigenin. The optimal ultrasonic parameters were an exposure period of 30 min at a temperature of 25 °C using a power source of 80 W. Under these optimal conditions, the yields of luteolin and apigenin were increased to 42.5 and 25.3 mg/g, respectively, which represented a 26.1-fold and a 32.2-fold increase in the yields of these 2 compounds, respectively, compared with the control model of aqueous extraction without enzyme or ultrasonic treatment.