Plant serine/arginine-rich proteins: versatile players in RNA processing.

MAIN CONCLUSION: Serine/arginine-rich (SR) proteins participate in RNA processing by interacting with precursor mRNAs or other splicing factors to maintain plant growth and stress responses. Alternative splicing is an important mechanism involved in mRNA processing and regulation of gene expression at the posttranscriptional level, which is the main reason for the diversity of genes and proteins. The process of alternative splicing requires the participation of many specific splicing factors. The SR protein family is a splicing factor in eukaryotes. The vast majority of SR proteins' existence is an essential survival factor. Through its RS domain and other unique domains, SR proteins can interact with specific sequences of precursor mRNA or other splicing factors and cooperate to complete the correct selection of splicing sites or promote the formation of spliceosomes. They play essential roles in the composition and alternative splicing of precursor mRNAs, providing pivotal functions to maintain growth and stress responses in animals and plants. Although SR proteins have been identified in plants for three decades, their evolutionary trajectory, molecular function, and regulatory network remain largely unknown compared to their animal counterparts. This article reviews the current understanding of this gene family in eukaryotes and proposes potential key research priorities for future functional studies.

Global Analysis of Dark- and Heat-Regulated Alternative Splicing in Arabidopsis.

Alternative splicing (AS) is one of the major post-transcriptional regulation mechanisms that contributes to plant responses to various environmental perturbations. Darkness and heat are two common abiotic factors affecting plant growth, yet the involvement and regulation of AS in the plant responses to these signals remain insufficiently examined. In this study, we subjected Arabidopsis seedlings to 6 h of darkness or heat stress and analyzed their transcriptome through short-read RNA sequencing. We revealed that both treatments altered the transcription and AS of a subset of genes yet with different mechanisms. Dark-regulated AS events were found enriched in photosynthesis and light signaling pathways, while heat-regulated AS events were enriched in responses to abiotic stresses but not in heat-responsive genes, which responded primarily through transcriptional regulation. The AS of splicing-related genes (SRGs) was susceptible to both treatments; while dark treatment mostly regulated the AS of these genes, heat had a strong effect on both their transcription and AS. PCR analysis showed that the AS of the Serine/Arginine-rich family gene SR30 was reversely regulated by dark and heat, and heat induced the upregulation of multiple minor SR30 isoforms with intron retention. Our results suggest that AS participates in plant responses to these two abiotic signals and reveal the regulation of splicing regulators during these processes.

Phylogeny and conservation of plant U2A/U2A', a core splicing component in U2 spliceosomal complex.

MAIN CONCLUSION: This study systematically identifies 112 U2A genes from 80 plant species by combinatory bioinformatics analysis, which is important for understanding their phylogenetic history, expression profiles and for predicting specific functions. In eukaryotes, a pre-mRNA can generate multiple transcripts by removing certain introns and joining corresponding exons, thus greatly expanding the transcriptome and proteome diversity. The spliceosome is a mega-Dalton ribonucleoprotein (RNP) complex that is essential for the process of splicing. In spliceosome components, the U2 small nuclear ribonucleoprotein (U2 snRNP) forms the pre-spliceosome by association with the branch site. An essential component that promotes U2 snRNP assembly, named U2A, has been extensively identified in humans, yeast and nematodes. However, studies examining U2A genes in plants are scarce. In this study, we performed a comprehensive analysis and identified a total of 112 U2A genes from 80 plant species representing dicots, monocots, mosses and algae. Comparisons of the gene structures, protein domains, and expression patterns of 112 U2A genes indicated that the conserved functions were likely retained by plant U2A genes and important for responses to internal and external stimuli. In addition, analysis of alternative transcripts and splice sites of U2A genes indicated that the fifth intron contained a conserved alternative splicing event that might be important for its molecular function. Our work provides a general understanding of this splicing factor family in terms of genes and proteins, and it will serve as a fundamental resource that will contribute to further mechanistic characterization in plants.

Full-Length Transcript-Based Proteogenomics of Rice Improves Its Genome and Proteome Annotation.

Rice (Oryza sativa) molecular breeding has gained considerable attention in recent years, but inaccurate genome annotation hampers its progress and functional studies of the rice genome. In this study, we applied single-molecule long-read RNA sequencing (lrRNA_seq)-based proteogenomics to reveal the complexity of the rice transcriptome and its coding abilities. Surprisingly, approximately 60% of loci identified by lrRNA_seq are associated with natural antisense transcripts (NATs). The high-density genomic arrangement of NAT genes suggests their potential roles in the multifaceted control of gene expression. In addition, a large number of fusion and intergenic transcripts have been observed. Furthermore, 906,456 transcript isoforms were identified, and 72.9% of the genes can generate splicing isoforms. A total of 706,075 posttranscriptional events were subsequently categorized into 10 subtypes, demonstrating the interdependence of posttranscriptional mechanisms that contribute to transcriptome diversity. Parallel short-read RNA sequencing indicated that lrRNA_seq has a superior capacity for the identification of longer transcripts. In addition, over 190,000 unique peptides belonging to 9,706 proteoforms/protein groups were identified, expanding the diversity of the rice proteome. Our findings indicate that the genome organization, transcriptome diversity, and coding potential of the rice transcriptome are far more complex than previously anticipated.

Comprehensive transcriptome and proteome analyses reveal a novel sodium chloride responsive gene network in maize seed tissues during germination.

Germination is a plant developmental process by which radicle of mature seeds start to penetrate surrounding barriers for seedling establishment and multiple environmental factors have been shown to affect it. Little is known how high salinity affects seed germination of C4 plant, Zea mays. Preliminary germination assay suggested that isolated embryo alone was able to germinate under 200 mM NaCl treatment, whereas the intact seeds were highly repressed. We hypothesized that maize endosperm may function in perception and transduction of salt signal to surrounding tissues such as embryo, showing a completely different response to that in Arabidopsis. Since salt response involves ABA, we analysed in vivo ABA distribution and quantity and the result demonstrated that ABA level in isolated embryo under NaCl treatment failed to increase in comparison with the water control, suggesting that the elevation of ABA level is an endosperm dependent process. Subsequently, by using advanced profiling techniques such as RNA sequencing and SWATH-MS-based quantitative proteomics, we found substantial differences in post-transcriptional and translational changes between salt-treated embryo and endosperm. In summary, our results indicate that these regulatory mechanisms, such as alternative splicing, are likely to mediate early responses to salt stress during maize seed germination.

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.

SWATH-MS-facilitated proteomic profiling of fruit skin between Fuji apple and a red skin bud sport mutant.

BACKGROUND: Apple is one of the most popular fruit crops world-wide and its skin color is an important quality consideration essential for commercial value. However, the strategy on genetic breeding for red skin apple and the genetic basis of skin color differentiation is very limited and still largely unknown. RESULTS: Here, we reported a bud sport mutant of Fuji apple with red skin color and enhanced anthocyanins accumulation. Quantitative SWATH-MS (sequential window acquisition of all theoretical spectra-mass spectrometry) proteomics investigations revealed proteome changes in the apple red skin bud mutation and a total of 451 differentially expressed proteins were identified in apple skin. The mutant showed significantly increased expression levels of photosynthesis-related proteins, stress-related proteins as well as anthocyanins biosynthesis pathway. On the other hand, substantial downregulation of mitogen-activated protein kinase 4 (MAPK4) and mevalonate kinase (MVK) were detected, indicating a promising role for the red skin color development in the mutant. Furthermore, we also hypothesize that a post-transcriptional regulation of the skin color formation occurs in the mutant through the advanced SWATH-MS analysis. CONCLUSION: Our work provides important information on the application of proteomic methods for analysing proteomes changes in Fuji apple and highlights a clade of regulatory proteins potentially contributing for the molecular breeding of fruit skin color.

Comparative metabolite profiling of two switchgrass ecotypes reveals differences in drought stress responses and rhizosheath weight.

MAIN CONCLUSION: Rhizosheath comprises soil that adheres firmly to roots. In this study, two ecotypes of switchgrass with different rhizosheath sizes after drought stress were analyzed which showed metabolic differences under drought conditions. The rhizosheath comprises soil that adheres firmly to roots by a combination of root hairs and mucilage and may aid in root growth under soil drying. The aim of this work is to reveal the potential metabolites involved in rhizosheath formation under drought stress conditions. Panicum virgatum L. (switchgrass), which belongs to the Poaceae family, is an important biofuel and fodder crop in drought areas. Five switchgrass ecotypes (cv. Alamo, cv. Blackwake, cv. Summer, cv. Cave-in-Rock and cv. Kanlow) have a broad range of rhizosheath weight under drought conditions. For two selected ecotypes with contrast rhizosheath weight (cv. Alamo and cv. Kanlow), root hair length and density, lateral root number, root morphological parameters were measured, and real-time qRT-PCR was performed. Gas chromatography mass spectrophotometry (GC-MS) was used to determine the primary metabolites in the shoots and roots of selected ecotypes under drought stress conditions. The change trends of root hair length and density, lateral root number and related gene expression were consistent with rhizosheath weight in Alamo and Kanlow under drought and watered conditions. For root morphological parameters, Alamo grew deeper than Kanlow, while Kanlow exhibited higher values for other parameters. In this study, the levels of amino acids, sugars and organic acids were significantly changed in response to drought stress in two switchgrass ecotypes. Several metabolites including amino acids (arginine, isoleucine, methionine and cysteine) and sugars (kestose, raffinose, fructose, fucose, sorbose and xylose) in the large soil-sheathed roots of Alamo and Kanlow were significantly increased compared to small or no soil-sheathed roots of Alamo and Kanlow. Difference in rhizosheath size is reflected in the plant internal metabolites under drought stress conditions. Additionally, our results highlight the importance of using metabolite profiling and provide a better understanding of rhizosheath formation at the cellular level.

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

MAIN CONCLUSION: This 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.

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.

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.

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 (ß-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.

Abscisic acid regulates early seed development in Arabidopsis by ABI5-mediated transcription of SHORT HYPOCOTYL UNDER BLUE1.

Seed development includes an early stage of endosperm proliferation and a late stage of embryo growth at the expense of the endosperm in Arabidopsis thaliana. Abscisic acid (ABA) has known functions during late seed development, but its roles in early seed development remain elusive. In this study, we report that ABA-deficient mutants produced seeds with increased size, mass, and embryo cell number but delayed endosperm cellularization. ABSCISIC ACID DEFICIENT2 (ABA2) encodes a unique short-chain dehydrogenase/reductase that functions in ABA biosynthesis, and its expression pattern overlaps that of SHORT HYPOCOTYL UNDER BLUE1 (SHB1) during seed development. SHB1 RNA accumulation was significantly upregulated in the aba2-1 mutant and was downregulated by the application of exogenous ABA. Furthermore, RNA accumulation of the basic/region leucine zipper transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5), involved in ABA signaling, was decreased in aba2-1. Consistent with this, seed size was also increased in abi5. We further show that ABI5 directly binds to two discrete regions in the SHB1 promoter. Our results suggest that ABA negatively regulates SHB1 expression, at least in part, through the action of its downstream signaling component ABI5. Our findings provide insights into the molecular mechanisms by which ABA regulates early seed development.

Morphological and molecular analyses reveal two new species of Microcera (Nectriaceae, Hypocreales) associated with scale insects on walnut in China.

The fungal genus Microcera consists of species mostly occurring as parasites of scale insects, but are also commonly isolated from soil or lichens. In the present study, we surveyed the diversity and assess the taxonomy of entomopathogenic fungi in Sichuan Province, China. Two new species of Microcera, viz. M.chrysomphaludis and M.pseudaulacaspidis, were isolated from scale insects colonising walnut (Juglansregia). Maximum Likelihood and Bayesian Inference analyses of ITS, LSU, tef1-α, rpb1, rpb2, acl1, act, tub2, cmdA and his3 sequence data provide evidence for the validity of the two species and their placement in Nectriaceae (Hypocreales). Microcerapseudaulacaspidis primarily differs from similar species by having more septate and smaller cylindrical macroconidia, as well as DNA sequence data. Meanwhile, Microcerachrysomphaludis has elliptical, one-septate ascospores with acute ends and cylindrical, slightly curved with 4-6 septate macroconidia up to 78 µm long. Morphological descriptions with illustrations of the novel species and DNA-based phylogeny generated from analyses of multigene dataset are also provided to better understand species relationships.

In Situ Observation of Abscisic Acid Distribution in Major Crop Species by Immunofluorescence Labeling.

Immunofluorescent approach uses fluorophore-conjugated antibodies to target molecules of interest for the observation of their distribution and microenvironment at cellular or tissue level. In connection with modern fluorescence microscopy, immunofluorescence has high sensitivity and specificity, resulting in a wide range of applications in animal studies. However, the protocols of immunofluorescence are seldom reported. To this end, we describe an optimized protocol for the detection of plant hormone abscisic acid (ABA) in monocot crop species, including maize and rice. By using immunofluorescence technique provided from this protocol, investigation of ABA distribution will continue to deeper our insights in crop developments and stress responses.

Morphology and phylogeny of ascomycetes associated with walnut trees (Juglans regia) in Sichuan province, China.

In Sichuan province, walnuts, consisting of Juglans regia, Juglans sigillata, and the hybrid J. regia × J. sigillata, are commercially important edible nuts, and J. regia is the most widespread plant. To date, the diversity and distribution of fungi inhabiting on Juglans have not received enough attention, although there have been studies focusing on pathogens from fruit and stem. In order to update the checklist of fungi associated with Sichuan walnuts, a survey on fungi associated with the three Juglans species from 15 representative regions in Sichuan was conducted. In this article, ten fungi distributed in two classes of Ascomycota (Dothideomycetes and Sordariomycetes) were described based on morpho-molecular analyses, and two novel species, Neofusicoccum sichuanense and Sphaerulina juglandina, a known species of Ophiognomonia leptostyla, and seven new hosts or geographical records of Cladosporium tenuissimum, Diatrypella vulgaris, Helminthosporium juglandinum, Helminthosporium velutinum, Loculosulcatispora hongheensis, Periconia byssoides, and Rhytidhysteron subrufulum were included. Morphological descriptions and illustrations of these fungi are provided.

Morpho-Molecular Characterization of Microfungi Associated with Phyllostachys (Poaceae) in Sichuan, China.

In the present study, we surveyed the ascomycetes from bamboo of Phyllostachys across Sichuan Province, China. A biphasic approach based on morphological characteristics and multigene phylogeny confirmed seven species, including one new genus, two new species, and five new host record species. A novel genus Paralloneottiosporina is introduced to accommodate Pa. sichuanensis that was collected from leaves of Phyllostachys violascens. Moreover, the newly introduced species Bifusisporella sichuanensis was isolated from leaves of P. edulis, and five species were newly recorded on bamboos, four species belonging to Apiospora, viz. Ap. yunnana, Ap. neosubglobosa, Ap. jiangxiensis, and Ap. hydei, and the last species, Seriascoma yunnanense, isolated from dead culms of P. heterocycla. Morphologically similar and phylogenetically related taxa were compared. Comprehensive descriptions, color photo plates of micromorphology are provided.

SWATH-MS-Based Proteomics: Strategies and Applications in Plants.

Most applied proteomic approaches require labeling steps. Recent technological advances provide an alternative label-free proteomics approach: SWATH-MS. This powerful tool is now widely used in animal studies but has drawn far less attention in plants. Here we summarize how this promising technology can be applied to facilitate functional analysis in plant research.

Insight into the Systematics of Novel Entomopathogenic Fungi Associated with Armored Scale Insect, Kuwanaspis howardi (Hemiptera: Diaspididae) in China.

This study led to the discovery of three entomopathogenic fungi associated with Kuwanaspis howardi, a scale insect on Phyllostachys heteroclada (fishscale bamboo) and Pleioblastus amarus (bitter bamboo) in China. Two of these species belong to Podonectria: P. kuwanaspidis X.L. Xu & C.L. Yang sp. nov. and P. novae-zelandiae Dingley. The new species P. kuwanaspidis has wider and thicker setae, longer and wider asci, longer ascospores, and more septa as compared with similar Podonectria species. The morphs of extant species P. novae-zelandiae is confirmed based on sexual and asexual morphologies. Maximum likelihood and Bayesian inference analyses of ITS, LSU, SSU, tef1-α, and rpb2 sequence data provide further evidence for the validity of the two species and their placement in Podonectriaceae (Pleosporales). The second new species, Microcera kuwanaspidis X.L. Xu & C.L. Yang sp. nov., is established based on DNA sequence data from ITS, LSU, SSU, tef1-α, rpb1, rpb2, acl1, act, cmdA, and his3 gene regions, and it is characterized by morphological differences in septum numbers and single conidial mass.