Bei Gao

Characterization of reference genes for RT-qPCR in the desert moss Syntrichia caninervis in response to abiotic stress and desiccation/rehydration

Syntrichia caninervis is the dominant bryophyte of the biological soil crusts found in the Gurbantunggut desert. The extreme desert environment is characterized by prolonged drought, temperature extremes, high radiation and frequent cycles of hydration and dehydration. S. caninervis is an ideal organism for the identification and characterization of genes related to abiotic stress tolerance. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) expression analysis is a powerful analytical technique that requires the use of stable reference genes. Using available S. caninervis transcriptome data, we selected 15 candidate reference genes and analyzed their relative expression stabilities in S. caninervis gametophores exposed to a range of abiotic stresses or a hydration-desiccation-rehydration cycle. The programs geNorm, NormFinder, and RefFinder were used to assess and rank the expression stability of the 15 candidate genes. The stability ranking results of reference genes under each specific experimental condition showed high consistency using different algorithms. For abiotic stress treatments, the combination of two genes (α-TUB2 and CDPK) were sufficient for accurate normalization. For the hydration-desiccation-rehydration process, the combination of two genes (α-TUB1 and CDPK) were sufficient for accurate normalization. 18S was among the least stable genes in all of the experimental sets and was unsuitable as reference gene in S. caninervis. This is the first systematic investigation and comparison of reference gene selection for RT-qPCR work in S. caninervis. This research will facilitate gene expression studies in S. caninervis, related moss species from the Syntrichia complex and other mosses.

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.

Desiccation tolerance in bryophytes: The dehydration and rehydration transcriptomes in the desiccation-tolerant bryophyte Bryum argenteum

The desiccation tolerant bryophyte Bryum argenteum is an important component of desert biological soil crusts (BSCs) and is emerging as a model system for studying vegetative desiccation tolerance. Here we present and analyze the hydration-dehydration-rehydration transcriptomes in B. argenteum to establish a desiccation-tolerance transcriptomic atlas. B. argenteum gametophores representing five different hydration stages (hydrated (H0), dehydrated for 2 h (D2), 24 h (D24), then rehydrated for 2 h (R2) and 48 h (R48)), were sampled for transcriptome analyses. Illumina high throughput RNA-Seq technology was employed and generated more than 488.46 million reads. An in-house de novo transcriptome assembly optimization pipeline based on Trinity assembler was developed to obtain a reference Hydration-Dehydration-Rehydration (H-D-R) transcriptome comprising of 76,206 transcripts, with an N50 of 2,016 bp and average length of 1,222 bp. Comprehensive transcription factor (TF) annotation discovered 978 TFs in 62 families, among which 404 TFs within 40 families were differentially expressed upon dehydration-rehydration. Pfam term enrichment analysis revealed 172 protein families/domains were significantly associated with the H-D-R cycle and confirmed early rehydration (i.e. the R2 stage) as exhibiting the maximum stress-induced changes in gene expression.

Alternative splicing and translation play important roles in parallel with transcriptional regulation during rice hypoxic germination

Post-transcriptional mechanisms, including alternative splicing (AS) and alternative translation initiation (ATI), have been used to explain the protein diversity involved in plant developmental processes and stress responses. Rice germination under hypoxia conditions is a classical model system for the study of low oxygen stress. It is known that there is transcriptional regulation during rice hypoxic germination, but the potential roles of AS and ATI in this process are not well understood. In this study, a proteogenomic approach was used to integrate the data from RNA sequencing, qualitative and quantitative proteomics to discover new players or pathways in the response to hypoxia stress. The improved analytical pipeline of proteogenomics led to the identification of 10,253 intron-containing genes, 1,729 of which were not present in the current annotation. Approximately 1,741 differentially expressed AS (DAS) events from 811 genes were identified in hypoxia-treated seeds in comparison to controls. Over 95% of these were not present in the list of differentially expressed genes (DEG). In particular, regulatory pathways such as spliceosome, ribosome, ER 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 traditional transcriptional regulation. Massive AS changes were identified, including the preference usage of certain non-conventional splice sites and enrichment of splicing factors in the DAS datasets. In addition, using self-constructed protein libraries by 6-frame translation, thousands of novel proteins/peptides contributed by ATI were identified. In summary, these results provide deeper insights towards understanding the underlying mechanisms of AS and ATI during rice hypoxic germination.

Genome-wide identification and functional analysis of the splicing component SYF2/NTC31/p29 across different plant species

Main conclusionThis study systematically identifies plant SYF2/NTC31/p29 genes from 62 plant species by a combinatory bioinformatics approach, revealing the importance of this gene family in phylogenetics, duplication, transcriptional, and post-transcriptional regulation.Alternative splicing is a post-transcriptional regulatory mechanism, which is critical for plant development and stress responses. The entire process is strictly attenuated by a complex of splicing-related proteins, designated splicing factors. Human p29, also referred to as synthetic lethal with cdc forty 2 (SYF2) or the NineTeen complex 31 (NTC31), is a core protein found in the NTC complex of humans and yeast. This splicing factor participates in a variety of biological processes, including DNA damage repair, control of the cell cycle, splicing, and tumorigenesis. However, its function in plants has been seldom reported. Thus, we have systematically identified 89 putative plant SYF2s from 62 plant species among the deposited entries in the Phytozome database. The phylogenetic relationships and evolutionary history among these plant SYF2s were carefully examined. The results revealed that plant SYF2s exhibited distinct patterns regarding their gene structure, promoter sequences, and expression levels, suggesting their functional diversity in response to developmental cues or stress treatments. Although local duplication events, such as tandem duplication and retrotransposition, were found among several plant species, most of the plant species contained only one copy of SYF2, suggesting the existence of additional mechanisms to confer duplication resistance. Further investigation using the model dicot and monocot representatives Arabidopsis and rice SYF2s indicated that the splicing pattern and resulting protein isoforms might play an alternative role in the functional diversity.

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

BackgroundThe 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.MethodsTo 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.ResultsThe 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.ConclusionThe 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.

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

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.

Desiccation tolerance in bryophytes: the rehydration proteomes of Bryum argenteum provide insights into the resuscitation mechanism

Bryum argenteum Hedw. is a desiccation tolerant bryophyte and belongs to one of the most important components of the biological soil crusts (BSCs) found in the deserts of Central Asia. Limited information is available on rehydration-responsive proteins in desiccation tolerant plants. As a complement to our previous research analyzing the rehydration transcriptome, we present a parallel quantitative proteomic effort to study rehydration-responsive proteins. Bryophyte gametophores were desiccated (Dry) and rehydrated for 2 h (R2) and 24 h (R24). Proteins from Dry, R2 and R24 gametophores were labeled by isobaric tags for relative and absolute quantitation (iTRAQ) to determine the relative abundance of rehydration-responsive proteins. A total of 5503 non-redundant protein sequences were identified and 4772 (86.7%) protein sequences were annotated using Gene Ontology (GO) terms and Pfam classifications. Upon rehydration 239 proteins were elevated and 461 proteins were reduced as compared to the desiccated protein sample. Differentially up-regulated proteins were classified into a number of categories including reactive oxygen species scavenging enzymes, detoxifying enzymes, Late Embryogenesis Abundant (LEA) proteins, heat shock proteins, proteasome components and proteases, and photosynthesis and translation related proteins. Furthermore, the results of the correlation between transcriptome and proteome revealed the discordant changes in the expression between protein and mRNA.

Evolution by duplication: paleopolyploidy events in plants reconstructed by deciphering the evolutionary history of VOZ transcription factors

BackgroundFacilitated by the rapid progress of sequencing technology, comparative genomic studies in plants have unveiled recurrent whole genome duplication (i.e. polyploidization) events throughout plant evolution. The evolutionary past of plant genes should be analyzed in a background of recurrent polyploidy events in distinctive plant lineages. The Vascular Plant One Zinc-finger (VOZ) gene family encode transcription factors associated with a number of important traits including control of flowering time and photoperiodic pathways, but the evolutionary trajectory of this gene family remains uncharacterized.ResultsIn this study, we deciphered the evolutionary history of the VOZ gene family by analyses of 107 VOZ genes in 46 plant genomes using integrated methods: phylogenic reconstruction, Ks-based age estimation and genomic synteny comparisons. By scrutinizing the VOZ gene family phylogeny the core eudicot γ event was well circumscribed, and relics of the precommelinid τ duplication event were detected by incorporating genes from oil palm and banana. The more recent T and ρ polyploidy events, closely coincident with the species diversification in Solanaceae and Poaceae, respectively, were also identified. Other important polyploidy events captured included the “salicoid” event in poplar and willow, the “early legume” and “soybean specific” events in soybean, as well as the recent polyploidy event in Physcomitrella patens. Although a small transcription factor gene family, the evolutionary history of VOZ genes provided an outstanding record of polyploidy events in plants. The evolutionary past of VOZ gene family demonstrated a close correlation with critical plant polyploidy events which generated species diversification and provided answer to Darwin’s “abominable mystery”.ConclusionsWe deciphered the evolutionary history of VOZ transcription factor family in plants and ancestral polyploidy events in plants were recapitulated simultaneously. This analysis allowed for the generation of an idealized plant gene tree demonstrating distinctive retention and fractionation patterns following polyploidy events.

Regulation of Gene Expression in the Remobilization of Carbon Reserves in Rice Stems During Grain Filling

Carbon reserves in rice straw (stem and sheath) before flowering contribute to a significant portion of grain filling. However, the molecular mechanism of carbon reserve remobilization from straw to grains remains unclear. In this study, super rice LYP9 and conventional rice 9311 showed different carbon reserve remobilization behaviors. The transcriptomic profiles of straws of LYP9 and 9311 were analyzed at three stages of grain filling. Among the differentially expressed genes (DGs), 5,733 genes were uniquely up- or down-regulated at 30 days after anthesis (DAA) between LYP9 and 9311 in comparison with 681 at 10 DAA and 495 at 20 DAA, suggesting that the gene expression profile of LYP9 was very different from that of 9311 at the late stage of grain filling. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Ontology (GO) classification of DGs both showed that the carbohydrate catabolic pathway, plant hormone signal transduction and photosynthesis pathway were enriched in DGs, suggesting their roles in carbon reserve remobilization, which explains to a certain extent the difference in non-structural carbohydrate content, photosynthesis and ABA content between the two cultivars during grain filling. Further comparative analysis and confirmation by quantitative real-time PCR and enzyme assays suggest that genes involved in trehalose synthesis (trehalose-phosphate phosphatase and trehalose 6-phosphate synthase/phosphatase), starch degradation (β-amylase) and sucrose synthesis (sucrose-phosphate synthase and sucrose synthase) were important for carbon reserve remobilization, whereas ABA content was determined by the counteraction of NCED1 and ABA8ox1 genes. The higher expression level of all these genes and ABA content in 9311 resulted in better efficiency of carbon reserve remobilization in 9311 than in LYP9.