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CAMTA3, a Ca 2+ -regulated transcription factor, is a repressor of plant immune responses. A truncated version of CAMTA3; CAMTA3 334 called N-terminal repression module (NRM), and its extended version (CAMTA 447 ), which include the DNA binding domain, were previously reported to complement the camta3/2 mutant phenotype. Here, we generated a series of CAMTA3 truncated versions [the N-terminus (aa 1-517), C-terminus (aa 517-1032), R1 (aa 1-173), R2 (aa 174-345), R3 (aa 346-517), R4 (aa 517-689), R5 (aa 690-861) and R6 (aa 862-1032)], expressed in camta3 mutant and analyzed the phenotypes of the transgenic lines. Interestingly, unlike CAMTA 447 , extending the N-terminal region to 517 aa did not complement the camta3 phenotype, suggesting that the amino acid region from 448-517 (70 aa), which includes a part of the TIG domain suppresses the NRM activity. The C-terminus and other truncated versions (R1-R6) also failed to complement the camta3 mutant . Expressing the full length or NRM of CAMTA3 in camta3 plants suppressed the activation of immune-responsive genes and increased the expression of cold-induced genes. In contrast, the transgenic lines expressing the N- or C-terminus or R1-R6 of CAMTA3 showed expression patterns like those of the camta3 with enhanced expression of the defense genes and suppressed expression of the cold response genes. Furthermore, like camta3 , the transgenic lines expressing the N- or C-terminus, or R1-R6 of CAMTA3 exhibited higher levels of H 2 O 2 and increased resistance to a Pst DC3000 as compared to WT, NRM, or FL-CAMTA3 transgenic plants. Our studies identified a novel regulatory region in CAMTA3 that suppresses the NRM activity., Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01401-w., Competing Interests: Conflict of interestNo conflict of interest is declared by any of the authors., (© Prof. H.S. Srivastava Foundation for Science and Society 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Alternative splicing (AS) is a gene regulatory mechanism modulating gene expression in multiple ways. AS is prevalent in all eukaryotes including plants. AS generates two or more mRNAs from the precursor mRNA (pre-mRNA) to regulate transcriptome complexity and proteome diversity. Advances in next-generation sequencing, omics technology, bioinformatics tools, and computational methods provide new opportunities to quantify and visualize AS-based quantitative trait variation associated with plant growth, development, reproduction, and stress tolerance. Domestication, polyploidization, and environmental perturbation may evolve novel splicing variants associated with agronomically beneficial traits. To date, pre-mRNAs from many genes are spliced into multiple transcripts that cause phenotypic variation for complex traits, both in model plant Arabidopsis and field crops. Cataloguing and exploiting such variation may provide new paths to enhance climate resilience, resource-use efficiency, productivity, and nutritional quality of staple food crops. This review provides insights into AS variation alongside a gene expression analysis to select for novel phenotypic diversity for use in breeding programs. AS contributes to heterosis, enhances plant symbiosis (mycorrhiza and rhizobium), and provides a mechanistic link between the core clock genes and diverse environmental clues.

Calmodulin-binding transcription activators (CAMTAs), a small family of highly conserved transcription factors, function in calcium-mediated signaling pathways. Of the six CAMTAs in Arabidopsis , CAMTA3 regulates diverse biotic and abiotic stress responses. A recent study has shown that CAMTA3 is a guardee of NLRs (Nucleotide-binding, Leucine-rich repeat Receptors) in modulating plant immunity, raising the possibility that CAMTA3 transcriptional activity is dispensable for its function. Here, we show that the DNA-binding activity of CAMTA3 is essential for its role in mediating plant immune responses. Analysis of the DNA-binding (CG-1) domain of CAMTAs in plants and animals showed strong conservation of several amino acids. We mutated six conserved amino acids in the CG-1 domain to investigate their role in CAMTA3 function. Electrophoretic mobility shift assays using these mutants with a promoter of its target gene identified critical amino acid residues necessary for DNA-binding activity. In addition, transient assays showed that these residues are essential for the CAMTA3 function in activating the Rapid Stress Response Element ( RSRE) -driven reporter gene expression. In line with this, transgenic lines expressing the CG-1 mutants of CAMTA3 in the camta3 mutant failed to rescue the mutant phenotype and restore the expression of CAMTA3 downstream target genes. Collectively, our results provide biochemical and genetic evidence that the transcriptional activity of CAMTA3 is indispensable for its function.

Background: Alternative splicing is a widespread regulatory phenomenon that enables a single gene to produce multiple transcripts. Among the different types of alternative splicing, intron retention is one of the least explored despite its high prevalence in both plants and animals. The recent discovery that the majority of splicing is co-transcriptional has led to the finding that chromatin state affects alternative splicing. Therefore, it is plausible that transcription factors can regulate splicing outcomes., Results: We provide evidence for the hypothesis that transcription factors are involved in the regulation of intron retention by studying regions of open chromatin in retained and excised introns. Using deep learning models designed to distinguish between regions of open chromatin in retained introns and non-retained introns, we identified motifs enriched in IR events with significant hits to known human transcription factors. Our model predicts that the majority of transcription factors that affect intron retention come from the zinc finger family. We demonstrate the validity of these predictions using ChIP-seq data for multiple zinc finger transcription factors and find strong over-representation for their peaks in intron retention events., Conclusions: This work opens up opportunities for further studies that elucidate the mechanisms by which transcription factors affect intron retention and other forms of splicing., Availability: Source code available at https://github.com/fahadahaf/chromir., (© 2023. The Author(s).)

Light signals perceived by a group of photoreceptors have profound effects on the physiology, growth, and development of plants. The red/far-red light-absorbing phytochromes (phys) modulate these aspects by intricately regulating gene expression at multiple levels. Here, we report the identification and functional characterization of an RNA-binding splicing factor, SWAP1 (SUPPRESSOR-OF-WHITE-APRICOT/SURP RNA-BINDING DOMAIN-CONTAINING PROTEIN1). Loss-of-function swap1-1 mutant is hyposensitive to red light and exhibits a day length-independent early flowering phenotype. SWAP1 physically interacts with two other splicing factors, (SFPS) SPLICING FACTOR FOR PHYTOCHROME SIGNALING and (RRC1) REDUCED RED LIGHT RESPONSES IN CRY1CRY2 BACKGROUND 1 in a light-independent manner and forms a ternary complex. In addition, SWAP1 physically interacts with photoactivated phyB and colocalizes with nuclear phyB photobodies. Phenotypic analyses show that the swap1sfps , swap1rrc1, and sfpsrrc1 double mutants display hypocotyl lengths similar to that of the respective single mutants under red light, suggesting that they function in the same genetic pathway. The swap1sfps double and swap1sfpsrrc1 triple mutants display pleiotropic phenotypes, including sterility at the adult stage. Deep RNA sequencing (RNA-seq) analyses show that SWAP1 regulates the gene expression and pre-messenger RNA (mRNA) alternative splicing of a large number of genes, including those involved in plant responses to light signaling. A comparative analysis of alternative splicing among single, double, and triple mutants showed that all three splicing factors coordinately regulate the alternative splicing of a subset of genes. Our study uncovered the function of a splicing factor that modulates light-regulated alternative splicing by interacting with photoactivated phyB and other splicing factors.

Understanding gene expression and regulation requires insights into RNA transcription, processing, modification, and translation. However, the relationship between the epitranscriptome and the proteome under drought stress remains undetermined in poplar (Populus trichocarpa). In this study, we used Nanopore direct RNA sequencing and tandem mass tag-based proteomic analysis to examine epitranscriptomic and proteomic regulation induced by drought treatment in stem-differentiating xylem (SDX). Our results revealed a decreased full-length read ratio under drought treatment and, especially, a decreased association between transcriptome and proteome changes in response to drought. Epitranscriptome analysis of cellulose- and lignin-related genes revealed an increased N6-Methyladenosine (m6A) ratio, which was accompanied by decreased RNA abundance and translation, under drought stress. Interestingly, usage of the distal poly(A) site increased during drought stress. Finally, we found that transcripts of highly expressed genes tend to have shorter poly(A) tail length (PAL), and drought stress increased the percentage of transcripts with long PAL. These findings provide insights into the interplay among m6A, polyadenylation, PAL, and translation under drought stress in P. trichocarpa SDX., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis., Results: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage., Conclusions: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species., (© 2022. The Author(s).)

Abiotic stresses profoundly affect plant growth and development and limit crop productivity. Pre-mRNA splicing is a major form of gene regulation that helps plants cope with various stresses. Serine/arginine (SR)-rich splicing factors play a key role in pre-mRNA splicing to regulate different biological processes under stress conditions. Alternative splicing (AS) of SR transcripts and other transcripts of stress-responsive genes generates multiple splice isoforms that contribute to protein diversity, modulate gene expression, and affect plant stress tolerance. Here, we investigated the function of the plant-specific SR protein RS33 in regulating pre-mRNA splicing and abiotic stress responses in rice. The loss-of-function mutant rs33 showed increased sensitivity to salt and low-temperature stresses. Genome-wide analyses of gene expression and splicing in wild-type and rs33 seedlings subjected to these stresses identified multiple splice isoforms of stress-responsive genes whose AS are regulated by RS33. The number of RS33-regulated genes was much higher under low-temperature stress than under salt stress. Our results suggest that the plant-specific splicing factor RS33 plays a crucial role during plant responses to abiotic stresses.

Background: Despite recent progress in basecalling of Oxford nanopore DNA sequencing data, its wide adoption is still being hampered by its relatively low accuracy compared to short read technologies. Furthermore, very little of the recent research was focused on basecalling of RNA data, which has different characteristics than its DNA counterpart., Results: We fill this gap by benchmarking a fully convolutional deep learning basecalling architecture with improved performance compared to Oxford nanopore's RNA basecallers., Availability: The source code for our basecaller is available at: https://github.com/biodlab/RODAN ., (© 2022. The Author(s).)

The natural auxin indole-3-acetic acid (IAA) is a key regulator of many aspects of plant growth and development. Synthetic auxin herbicides such as 2,4-D mimic the effects of IAA by inducing strong auxinic-signaling responses in plants. To determine the mechanism of 2,4-D resistance in a Sisymbrium orientale (Indian hedge mustard) weed population, we performed a transcriptome analysis of 2,4-D-resistant (R) and -susceptible (S) genotypes that revealed an in-frame 27-nucleotide deletion removing nine amino acids in the degron tail (DT) of the auxin coreceptor Aux/IAA2 ( SoIAA2 ). The deletion allele cosegregated with 2,4-D resistance in recombinant inbred lines. Further, this deletion was also detected in several 2,4-D-resistant field populations of this species. Arabidopsis transgenic lines expressing the SoIAA2 mutant allele were resistant to 2,4-D and dicamba. The IAA2-DT deletion reduced binding to TIR1 in vitro with both natural and synthetic auxins, causing reduced association and increased dissociation rates. This mechanism of synthetic auxin herbicide resistance assigns an in planta function to the DT region of this Aux/IAA coreceptor for its role in synthetic auxin binding kinetics and reveals a potential biotechnological approach to produce synthetic auxin-resistant crops using gene-editing., Competing Interests: Competing interest statement: A pending patent application related to the use of the IAA2 mutation to generate herbicide-resistant plants has been filed by Colorado State University Foundation and University of Adelaide for inventors T.A.G., M.R.A.F., A.K., and C.P. and application number WO2020185907A1., (Copyright © 2022 the Author(s). Published by PNAS.)

Among polyploid species with complex genomic architecture, variations in the regulation of alternative splicing (AS) provide opportunities for transcriptional and proteomic plasticity and the potential for generating trait diversities. However, the evolution of AS and its influence on grain development in diploid grass and valuable polyploid wheat crops are poorly understood. To address this knowledge gap, we developed a pipeline for the analysis of alternatively spliced transcript isoforms, which takes the high sequence similarity among polyploid wheat subgenomes into account. Through analysis of synteny and detection of collinearity of homoeologous subgenomes, conserved and specific AS events across five wheat and grass species were identified. A global analysis of the regulation of AS in diploid grass and polyploid wheat grains revealed diversity in AS events not only between the endosperm, pericarp and embryo overdevelopment, but also between subgenomes. Analysis of AS in homoeologous triads of polyploid wheats revealed evolutionary divergence between gene-level and transcript-level regulation of embryogenesis. Evolutionary age analysis indicated that the generation of novel transcript isoforms has occurred in young genes at a more rapid rate than in ancient genes. These findings, together with the development of comprehensive AS resources for wheat and grass species, advance understanding of the evolution of regulatory features of AS during embryogenesis and grain development in wheat., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Circular RNAs (circRNAs) are a recently discovered type of non-coding RNA derived from pre-mRNAs. R-loops consist of a DNA:RNA hybrid and the associated single-stranded DNA. In Arabidopsis thaliana, circRNA:DNA R-loops regulate alternative splicing (AS) of SEPALLATA3 (SEP3). However, the occurrence and functions of circRNAs and R-loops in Populus trichocarpa are largely unexplored. Here, we performed circRNA-enriched sequencing in the stem-differentiating xylem (SDX) of P. trichocarpa and identified 2,742 distinct circRNAs, including circ-CESA4, circ-IRX7, and circ-GUX1, which are generated from genes involved in cellulose, and hemicellulose biosynthesis, respectively. To investigate the roles of circRNAs in modulating alternative splicing (AS), we detected 7,836 AS events using PacBio Iso-Seq and identified 634 circRNAs that overlapped with 699 AS events. Furthermore, using DNA:RNA hybrid immunoprecipitation followed by sequencing (DRIP-seq), we identified 8,932 R-loop peaks that overlapped with 181 circRNAs and 672 AS events. Notably, several SDX-related circRNAs overlapped with R-loop peaks, pointing to their possible roles in modulating AS in SDX. Indeed, overexpressing circ-IRX7 increased the levels of R-loop structures and decreased the frequency of intron retention in linear IRX7 transcripts. This study provides a valuable R-loop atlas resource and uncovers the interplay between circRNAs and AS in SDX of P. trichocarpa., (© 2021 Institute of Botany, Chinese Academy of Sciences.)

Populus trichocarpa ( P. trichocarpa ) is a model tree for the investigation of wood formation. In recent years, researchers have generated a large number of high-throughput sequencing data in P. trichocarpa . However, no comprehensive database that provides multi-omics associations for the investigation of secondary growth in response to diverse stresses has been reported. Therefore, we developed a public repository that presents comprehensive measurements of gene expression and post-transcriptional regulation by integrating 144 RNA-Seq, 33 ChIP-seq, and six single-molecule real-time (SMRT) isoform sequencing (Iso-seq) libraries prepared from tissues subjected to different stresses. All the samples from different studies were analyzed to obtain gene expression, co-expression network, and differentially expressed genes (DEG) using unified parameters, which allowed comparison of results from different studies and treatments. In addition to gene expression, we also identified and deposited pre-processed data about alternative splicing (AS), alternative polyadenylation (APA) and alternative transcription initiation (ATI). The post-transcriptional regulation, differential expression, and co-expression network datasets were integrated into a new P. trichocarpa Stem Differentiating Xylem (PSDX) database (http://forestry.fafu.edu.cn/db/SDX), which further highlights gene families of RNA-binding proteins and stress-related genes. The PSDX also provides tools for data query, visualization, a genome browser, and the BLAST option for sequence-based query. Much of the data is also available for bulk download. The availability of PSDX contributes to the research related to the secondary growth in response to stresses in P. trichocarpa , which will provide new insights that can be useful for the improvement of stress tolerance in woody plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Liu, Dai, Yang, Luo, Gao, Liu, Wei, Wang, Xu, Reddy, Jaiswal, Li, Liu and Gu.)

The SF3B complex, a multiprotein component of the U2 snRNP of the spliceosome, plays a crucial role in recognizing branch point sequence and facilitates spliceosome assembly and activation. Several chemicals that bind SF3B1 and PHF5A subunits of the SF3B complex inhibit splicing. We recently generated a splicing inhibitor-resistant SF3B1 mutant named SF3B1 GEX1A RESISTANT 4 (SGR4) using CRISPR-mediated directed evolution, whereas splicing inhibitor-resistant mutant of PHF5A (Overexpression-PHF5A GEX1A Resistance, OGR) was generated by expressing an engineered version PHF5A-Y36C. Global analysis of splicing in wild type and these two mutants revealed the role of SF3B1 and PHF5A in splicing regulation. This analysis uncovered a set of genes whose intron retention is regulated by both proteins. Further analysis of these retained introns revealed that they are shorter, have a higher GC content, and contain shorter and weaker polypyrimidine tracts. Furthermore, splicing inhibition increased seedlings sensitivity to salt stress, consistent with emerging roles of splicing regulation in stress responses. In summary, we uncovered the functions of two members of the plant branch point recognition complex. The novel strategies described here should be broadly applicable in elucidating functions of splicing regulators, especially in studying the functions of redundant paralogs in plants.

Improvements in yield and quality of rice are crucial for global food security. However, global rice production is substantially hindered by various biotic and abiotic stresses. Making further improvements in rice yield is a major challenge to the rice research community, which can be accomplished through developing abiotic stress-resilient rice varieties and engineering durable agrochemical-independent pathogen resistance in high-yielding elite rice varieties. This, in turn, needs increased understanding of the mechanisms by which stresses affect rice growth and development. Alternative splicing (AS), a post-transcriptional gene regulatory mechanism, allows rapid changes in the transcriptome and can generate novel regulatory mechanisms to confer plasticity to plant growth and development. Mounting evidence indicates that AS has a prominent role in regulating rice growth and development under stress conditions. Several regulatory and structural genes and splicing factors of rice undergo different types of stress-induced AS events, and the functional significance of some of them in stress tolerance has been defined. Both rice and its pathogens use this complex regulatory mechanism to devise strategies against each other. This review covers the current understanding and evidence for the involvement of AS in biotic and abiotic stress-responsive genes, and its relevance to rice growth and development. Furthermore, we discuss implications of AS for the virulence of different rice pathogens and highlight the areas of further research and potential future avenues to develop climate-smart and disease-resistant rice varieties.

RNAs transmit information from DNA to encode proteins that perform all cellular processes and regulate gene expression in multiple ways. From the time of synthesis to degradation, RNA molecules are associated with proteins called RNA-binding proteins (RBPs). The RBPs play diverse roles in many aspects of gene expression including pre-mRNA processing and post-transcriptional and translational regulation. In the last decade, the application of modern techniques to identify RNA-protein interactions with individual proteins, RNAs, and the whole transcriptome has led to the discovery of a hidden landscape of these interactions in plants. Global approaches such as RNA interactome capture (RIC) to identify proteins that bind protein-coding transcripts have led to the identification of close to 2000 putative RBPs in plants. Interestingly, many of these were found to be metabolic enzymes with no known canonical RNA-binding domains. Here, we review the methods used to analyze RNA-protein interactions in plants thus far and highlight the understanding of plant RNA-protein interactions these techniques have provided us. We also review some recent protein-centric, RNA-centric, and global approaches developed with non-plant systems and discuss their potential application to plants. We also provide an overview of results from classical studies of RNA-protein interaction in plants and discuss the significance of the increasingly evident ubiquity of RNA-protein interactions for the study of gene regulation and RNA biology in plants.

Alternative splicing (AS) is a major gene regulatory mechanism in plants. Recent evidence supports co-transcriptional splicing in plants, hence the chromatin state can impact AS. However, how dynamic changes in the chromatin state such as nucleosome occupancy influence the cold-induced AS remains poorly understood. Here, we generated transcriptome (RNA-Seq) and nucleosome positioning (MNase-Seq) data for Arabidopsis thaliana to understand how nucleosome positioning modulates cold-induced AS. Our results show that characteristic nucleosome occupancy levels are strongly associated with the type and abundance of various AS events under normal and cold temperature conditions in Arabidopsis. Intriguingly, exitrons, alternatively spliced internal regions of protein-coding exons, exhibit distinctive nucleosome positioning pattern compared to other alternatively spliced regions. Likewise, nucleosome patterns differ between exitrons and retained introns, pointing to their distinct regulation. Collectively, our data show that characteristic changes in nucleosome positioning modulate AS in plants in response to cold., (© 2020 The Authors New Phytologist © 2020 New Phytologist Trust.)

There are no comprehensive methods to identify N 6 -methyladenosine (m 6 A) at single-base resolution for every single transcript, which is necessary for the estimation of m 6 A abundance. We develop a new pipeline called Nanom6A for the identification and quantification of m 6 A modification at single-base resolution using Nanopore direct RNA sequencing based on an XGBoost model. We validate our method using methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and m 6 A-sensitive RNA-endoribonuclease-facilitated sequencing (m6A-REF-seq), confirming high accuracy. Using this method, we provide a transcriptome-wide quantification of m 6 A modification in stem-differentiating xylem and reveal that different alternative polyadenylation (APA) usage shows a different ratio of m 6 A.

Next-generation sequencing (NGS) technologies - Illumina RNA-seq, Pacific Biosciences isoform sequencing (PacBio Iso-seq), and Oxford Nanopore direct RNA sequencing (DRS) - have revealed the complexity of plant transcriptomes and their regulation at the co-/post-transcriptional level. Global analysis of mature mRNAs, transcripts from nuclear run-on assays, and nascent chromatin-bound mRNAs using short as well as full-length and single-molecule DRS reads have uncovered potential roles of different forms of RNA polymerase II during the transcription process, and the extent of co-transcriptional pre-mRNA splicing and polyadenylation. These tools have also allowed mapping of transcriptome-wide start sites in cap-containing RNAs, poly(A) site choice, poly(A) tail length, and RNA base modifications. The emerging theme from recent studies is that reprogramming of gene expression in response to developmental cues and stresses at the co-/post-transcriptional level likely plays a crucial role in eliciting appropriate responses for optimal growth and plant survival under adverse conditions. Although the mechanisms by which developmental cues and different stresses regulate co-/post-transcriptional splicing are largely unknown, a few recent studies indicate that the external cues target spliceosomal and splicing regulatory proteins to modulate alternative splicing. In this review, we provide an overview of recent discoveries on the dynamics and complexities of plant transcriptomes, mechanistic insights into splicing regulation, and discuss critical gaps in co-/post-transcriptional research that need to be addressed using diverse genomic and biochemical approaches., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

N 6 -methyladenosine (m 6 A) is a prevalent modification in messenger RNAs and circular RNAs that play important roles in regulating various aspects of RNA metabolism. However, the occurrence of the m 6 A modification in plant circular RNAs has not been reported. A widely used method to identify m 6 A modifications relies on m 6 A-specific antibodies followed by next-generation sequencing of precipitated RNAs (MeRIP-Seq). However, one limitation of MeRIP-Seq is that it does not provide the precise location of m 6 A at single-nucleotide resolution. Although more recent sequencing techniques such as Nanopore-based direct RNA sequencing (DRS) can overcome such limitations, the technology does not allow sequencing of circular RNAs, as these molecules lack a poly(A) tail. Here, we developed a novel method to detect the precise location of m 6 A modifications in circular RNAs using Nanopore DRS. We first enriched our samples for circular RNAs, which we then fragmented and sequenced on the Nanopore platform with a customized protocol. Using this method, we identified 470 unique circular RNAs from DRS reads based on the back-spliced junction region. Among exonic circular RNAs, about 10% contained m 6 A sites, which mainly occurred around acceptor and donor splice sites. This study demonstrates the utility of our antibody-independent method in identifying total and methylated circular RNAs using Nanopore DRS. This method has the additional advantage of providing the exact location of m 6 A sites at single-base resolution in circular RNAs or linear transcripts from non-coding RNA without poly(A) tails., (© 2020 Institute of Botany, Chinese Academy of Sciences.)

Alternative splicing (AS) of pre-mRNAs increases transcriptome and proteome diversity, regulates gene expression through multiple mechanisms, and plays important roles in plant development and stress responses. However, the prevalence of genome-wide plant AS changes during infection and the mechanisms by which pathogens modulate AS remain poorly understood. Here, we examined the global AS changes in tomato leaves infected with Phytophthora infestans, the infamous Irish famine pathogen. We show that more than 2000 genes exhibiting significant changes in AS are not differentially expressed, indicating that AS is a distinct layer of transcriptome reprogramming during plant-pathogen interactions. Furthermore, our results show that P. infestans subverts host immunity by repressing the AS of positive regulators of plant immunity and promoting the AS of susceptibility factors. To study the underlying mechanism, we established a luminescence-based AS reporter system in Nicotiana benthamiana to screen pathogen effectors modulating plant AS. We identified nine splicing regulatory effectors (SREs) from 87 P. infestans effectors. Further studies revealed that SRE3 physically binds U1-70K to manipulate the plant AS machinery and subsequently modulates AS-mediated plant immunity. Our study not only unveils genome-wide plant AS reprogramming during infection but also establishes a novel AS screening tool to identify SREs from a wide range of plant pathogens, providing opportunities to understand the splicing regulatory mechanisms through which pathogens subvert plant immunity., (Copyright © 2020 The Author. Published by Elsevier Inc. All rights reserved.)

Alternative polyadenylation (APA) regulates diverse developmental and physiological processes through its effects on gene expression, mRNA stability, translatability, and transport. Sorghum is a major cereal crop in the world and, despite its importance, not much is known about the role of post-transcriptional regulation in mediating responses to abiotic stresses in Sorghum. A genome-wide APA analysis unveiled widespread occurrence of APA in Sorghum in response to drought, heat, and salt stress. Abiotic stress treatments incited changes in poly(A) site choice in a large number of genes. Interestingly, abiotic stresses led to the re-directing of transcriptional output into non-productive pathways defined by the class of poly(A) site utilized. This result revealed APA to be part of a larger global response of Sorghum to abiotic stresses that involves the re-direction of transcriptional output into non-productive transcriptional and translational pathways. Large numbers of stress-inducible poly(A) sites could not be linked with known, annotated genes, suggestive of the existence of numerous unidentified genes whose expression is strongly regulated by abiotic stresses. Furthermore, we uncovered a novel stress-specific cis-element in intronic poly(A) sites used in drought- and heat-stressed plants that might play an important role in non-canonical poly(A) site choice in response to abiotic stresses., (© 2020 The Authors The Plant Journal © 2020 John Wiley & Sons Ltd.)

Soil salinity, a prevalent abiotic stress, causes enormous losses in global crop yields annually. Previous studies have shown that salt stress-induced reprogramming of gene expression contributes to the survival of plants under this stress. However, mechanisms regulating gene expression in response to salt stress at the posttranscriptional level are not well understood. In this study, we show that salt stress increases the level of Signal Responsive 1 (SR1) mRNA, a member of signal-responsive Ca2+/calmodulin-regulated transcription factors, by enhancing its stability. We present multiple lines of evidence indicating that reactive oxygen species generated by NADPH oxidase activity mediate salt-induced SR1 transcript stability. Using mutants impaired in either nonsense-mediated decay, XRN4 or mRNA decapping pathways, we show that neither the nonsense-mediated mRNA decay pathway, XRN4 nor the decapping of SR1 mRNA is required for its decay. We analyzed the salt-induced accumulation of eight truncated versions of the SR1 coding region (∼3 kb) in the sr1 mutant background. This analysis identified a 500-nt region at the 3' end of the SR1 coding region to be required for the salt-induced stability of SR1 mRNA. Potential mechanisms by which this region confers SR1 transcript stability in response to salt are discussed., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Drought is a major limiting factor of crop yields. In response to drought, plants reprogram their gene expression, which ultimately regulates a multitude of biochemical and physiological processes. The timing of this reprogramming and the nature of the drought-regulated genes in different genotypes are thought to confer differential tolerance to drought stress. Sorghum is a highly drought-tolerant crop and has been increasingly used as a model cereal to identify genes that confer tolerance. Also, there is considerable natural variation in resistance to drought in different sorghum genotypes. Here, we evaluated drought resistance in four genotypes to polyethylene glycol (PEG)-induced drought stress at the seedling stage and performed transcriptome analysis in seedlings of sorghum genotypes that are either drought-resistant or drought-sensitive to identify drought-regulated changes in gene expression that are unique to drought-resistant genotypes of sorghum. Our analysis revealed that about 180 genes are differentially regulated in response to drought stress only in drought-resistant genotypes and most of these (over 70%) are up-regulated in response to drought. Among these, about 70 genes are novel with no known function and the remaining are transcription factors, signaling and stress-related proteins implicated in drought tolerance in other crops. This study revealed a set of drought-regulated genes, including many genes encoding uncharacterized proteins that are associated with drought tolerance at the seedling stage., Competing Interests: The authors declare no conflicts interests. The founding sponsors had no role in the design of the study, in the collection, analysis or interpretation of the data, in writing of the manuscript, and in the decision to publish the results.

Alternative splicing (AS) of pre-mRNAs contributes to transcriptome diversity and enables plants to generate different protein isoforms from a single gene and/or fine-tune gene expression during different development stages and environmental changes. Although AS is pervasive, the genetic basis for differential isoform usage in plants is still emerging. In this study, we performed genome-wide analysis in 666 geographically distributed diverse ecotypes of Arabidopsis thaliana to identify genomic regions [splicing quantitative trait loci (sQTLs)] that may regulate differential AS. These ecotypes belong to different microclimatic conditions and are part of the relict and non-relict populations. Although sQTLs were spread across the genome, we observed enrichment for trans -sQTL ( trans -sQTLs hotspots) on chromosome one. Furthermore, we identified several sQTL (911) that co-localized with trait-linked single nucleotide polymorphisms (SNP) identified in the Arabidopsis genome-wide association studies (AraGWAS). Many sQTLs were enriched among circadian clock, flowering, and stress-responsive genes, suggesting a role for differential isoform usage in regulating these important processes in diverse ecotypes of Arabidopsis. In conclusion, the current study provides a deep insight into SNPs affecting isoform ratios/genes and facilitates a better mechanistic understanding of trait-associated SNPs in GWAS studies. To the best of our knowledge, this is the first report of sQTL analysis in a large set of Arabidopsis ecotypes and can be used as a reference to perform sQTL analysis in the Brassicaceae family. Since whole genome and transcriptome datasets are available for these diverse ecotypes, it could serve as a powerful resource for the biological interpretation of trait-associated loci, splice isoform ratios, and their phenotypic consequences to help produce more resilient and high yield crop varieties., (Copyright © 2019 Khokhar, Hassan, Reddy, Chaudhary, Jabre, Byrne and Syed.)

Motivation: MicroRNA (miRNA) and alternative splicing (AS)-mediated post-transcriptional regulation has been extensively studied in most eukaryotes. However, the interplay between AS and miRNAs has not been explored in plants. To our knowledge, the overall profile of miRNA target sites in circular RNAs (circRNA) generated by alternative back splicing has never been reported previously. To address the challenge, we identified miRNA target sites located in alternatively spliced regions of the linear and circular splice isoforms using the up-to-date single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) and Illumina sequencing data in eleven plant species., Results: In total, we identified 399 401 and 114 574 AS events from linear and circular RNAs, respectively. Among them, there were 64 781 and 41 146 miRNA target sites located in linear and circular AS region, respectively. In addition, we found 38 913 circRNAs to be overlapping with 45 648 AS events of its own parent isoforms, suggesting circRNA regulation of AS of linear RNAs by forming R-loop with the genomic locus. Here, we present a comprehensive database of miRNA targets in alternatively spliced linear and circRNAs (ASmiR) and a web server for deposition and identification of miRNA target sites located in the alternatively spliced region of linear and circular RNAs. This database is accompanied by an easy-to-use web query interface for meaningful downstream analysis. Plant research community can submit user-defined datasets to the web service to search AS regions harboring small RNA target sites. In conclusion, this study provides an unprecedented resource to understand regulatory relationships between miRNAs and AS in both gymnosperms and angiosperms., Availability and Implementation: The readily accessible database and web-based tools are available at http://forestry.fafu.edu.cn/bioinfor/db/ASmiR., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Calcium (Ca 2+ ) signals are decoded by the Ca 2+ -sensor protein calmodulin (CaM) and are transduced to Ca 2+ /CaM-binding transcription factors to directly regulate gene expression necessary for acclimation responses in plants. The molecular mechanisms of Ca 2+ /CaM signal transduction processes and their functional significance remains enigmatic. Here we report a novel Ca 2+ /CaM signal transduction mechanism that allosterically regulates DNA-binding activity of GT2-LIKE 1 (GTL1), a transrepressor of STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1), to repress stomatal development in response to water stress. We demonstrated that Ca 2+ /CaM interaction with the 2 nd helix of the GTL1 N-terminal trihelix DNA-binding domain (GTL1N) destabilizes a hydrophobic core of GTL1N and allosterically inhibits 3 rd helix docking to the SDD1 promoter, leading to osmotic stress-induced Ca 2+ /CaM-dependent activation (de-repression) of SDD1 expression. This resulted in GTL1-dependent repression of stomatal development in response to water-deficit stress. Together, our results demonstrate that a Ca 2+ /CaM-regulated transcriptional switch on a trihelix transrepressor directly transduces osmotic stress to repress stomatal development to improve plant water-use efficiency as an acclimation response.

Key Message: Functions of most splice isoforms that are generated by alternative splicing are unknown. We show that two splice variants that encode proteins differing in only eight amino acids have distinct functions in a stress response. Serine/arginine-rich (SR) and SR-like proteins, a conserved family of RNA binding proteins across eukaryotes, play important roles in pre-mRNA splicing and other post-transcriptional processes. Pre-mRNAs of SR and SR-like proteins undergo extensive alternative splicing in response to diverse stresses and produce multiple splice isoforms. However, the functions of most splice isoforms remain elusive. Alternative splicing of pre-mRNA of Arabidopsis SR45, which encodes an SR-like splicing regulator, generates two isoforms (long-SR45.1 and short-SR45.2). The proteins encoded by these two isoforms differ in eight amino acids. Here, we investigated the role of SR45 and its splice variants in salt stress tolerance. The loss of SR45 resulted in enhanced sensitivity to salt stress and changes in expression and splicing of genes involved in regulating salt stress response. Interestingly, only the long isoform (SR45.1) rescued the salt-sensitive phenotype as well as the altered gene expression and splicing patterns in the mutant. These results suggest that SR45 positively regulates salt tolerance. Furthermore, only the long isoform is required for SR45-mediated salt tolerance.

Plants, unlike animals, exhibit a very high degree of plasticity in their growth and development and employ diverse strategies to cope with the variations during diurnal cycles and stressful conditions. Plants and animals, despite their remarkable morphological and physiological differences, share many basic cellular processes and regulatory mechanisms. Alternative splicing (AS) is one such gene regulatory mechanism that modulates gene expression in multiple ways. It is now well established that AS is prevalent in all multicellular eukaryotes including plants and humans. Emerging evidence indicates that in plants, as in animals, transcription and splicing are coupled. Here, we reviewed recent evidence in support of co-transcriptional splicing in plants and highlighted similarities and differences between plants and humans. An unsettled question in the field of AS is the extent to which splice isoforms contribute to protein diversity. To take a critical look at this question, we presented a comprehensive summary of the current status of research in this area in both plants and humans, discussed limitations with the currently used approaches and suggested improvements to current methods and alternative approaches. We end with a discussion on the potential role of epigenetic modifications and chromatin state in splicing memory in plants primed with stresses.

Serine/arginine-rich (SR) proteins are conserved RNA-binding proteins that play major roles in RNA metabolism. They function as molecular adaptors, facilitate spliceosome assembly and modulate constitutive and alternative splicing of pre-mRNAs. Pre-mRNAs encoding SR proteins and many other proteins involved in stress responses are extensively alternatively spliced in response to diverse stresses. Hence, it is proposed that stress-induced changes in splice isoforms contribute to the adaptation of plants to stress responses. However, functions of most SR genes and their splice isoforms in stress responses are not known. Lack of easy and robust tools hindered the progress in this area. Emerging technologies such as CRISPR/Cas9 will facilitate studies of SR function by enabling the generation of single and multiple knock-out mutants of SR subfamily members. Moreover, CRISPR/Cas13 allows targeted manipulation of splice isoforms from SR and other genes in a constitutive or tissue-specific manner to evaluate functions of individual splice variants. Identification of the in vivo targets of SR proteins and their splice variants using the recently developed TRIBE (Targets of RNA-binding proteins Identified By Editing) and other methods will help unravel their mode of action and splicing regulatory elements under various conditions. These new approaches are expected to provide significant new insights into the roles of SRs and splice isoforms in plants adaptation to diverse stresses., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Alternative splicing (AS) generates multiple transcripts from the same gene, however, AS contribution to proteome complexity remains elusive in plants. AS is prevalent under stress conditions in plants, but it is counterintuitive why plants would invest in protein synthesis under declining energy supply. We propose that plants employ AS not only to potentially increasing proteomic complexity, but also to buffer against the stress-responsive transcriptome to reduce the metabolic cost of translating all AS transcripts. To maximise efficiency under stress, plants may make fewer proteins with disordered domains via AS to diversify substrate specificity and maintain sufficient regulatory capacity. Furthermore, we suggest that chromatin state-dependent AS engenders short/long-term stress memory to mediate reproducible transcriptional response in the future., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Plants display exquisite control over gene expression to elicit appropriate responses under normal and stress conditions. Alternative splicing (AS) of pre-mRNAs, a process that generates two or more transcripts from multi-exon genes, adds another layer of regulation to fine-tune condition-specific gene expression in animals and plants. However, exactly how plants control splice isoform ratios and the timing of this regulation in response to environmental signals remains elusive. In mammals, recent evidence indicate that epigenetic and epitranscriptome changes, such as DNA methylation, chromatin modifications and RNA methylation, regulate RNA polymerase II processivity, co-transcriptional splicing, and stability and translation efficiency of splice isoforms. In plants, the role of epigenetic modifications in regulating transcription rate and mRNA abundance under stress is beginning to emerge. However, the mechanisms by which epigenetic and epitranscriptomic modifications regulate AS and translation efficiency require further research. Dynamic changes in the chromatin landscape in response to stress may provide a scaffold around which gene expression, AS and translation are orchestrated. Finally, we discuss CRISPR/Cas-based strategies for engineering chromatin architecture to manipulate AS patterns (or splice isoforms levels) to obtain insight into the epigenetic regulation of AS., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nanopore sequencing from Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) are revolutionizing the way transcriptomes are analyzed. These methods offer many advantages over most widely used high-throughput short-read RNA sequencing (RNA-Seq) approaches and allow a comprehensive analysis of transcriptomes in identifying full-length splice isoforms and several other post-transcriptional events. In addition, direct RNA-Seq provides valuable information about RNA modifications, which are lost during the PCR amplification step in other methods. Here, we present a comprehensive summary of important applications of these technologies in plants, including identification of complex alternative splicing (AS), full-length splice variants, fusion transcripts, and alternative polyadenylation (APA) events. Furthermore, we discuss the impact of the newly developed nanopore direct RNA-Seq in advancing epitranscriptome research in plants. Additionally, we summarize computational tools for identifying and quantifying full-length isoforms and other co/post-transcriptional events and discussed some of the limitations with these methods. Sequencing of transcriptomes using these new single-molecule long-read methods will unravel many aspects of transcriptome complexity in unprecedented ways as compared to previous short-read sequencing approaches. Analysis of plant transcriptomes with these new powerful methods that require minimum sample processing is likely to become the norm and is expected to uncover novel co/post-transcriptional gene regulatory mechanisms that control biological outcomes during plant development and in response to various stresses.

Soil salinity, a significant problem in agriculture, severely limits the productivity of crop plants. Plants respond to and cope with salt stress by reprogramming gene expression via multiple signaling pathways that converge on transcription factors. To develop strategies to generate salt-tolerant crops, it is necessary to identify transcription factors that modulate salt stress responses in plants. In this study, we investigated the role of VOZ (VASCULAR PLANT ONE-ZINC FINGER PROTEIN) transcription factors (VOZs) in salt stress response. Transcriptome analysis in WT (wild-type), voz1-1 , voz2-1 double mutant and a VOZ2 complemented line revealed that many stress-responsive genes are regulated by VOZs. Enrichment analysis for gene ontology terms in misregulated genes in voz double mutant confirmed previously identified roles of VOZs and suggested a new role for them in salt stress. To confirm VOZs role in salt stress, we analyzed seed germination and seedling growth of WT, voz1 , voz2-1 , voz2-2 single mutants, voz1-1 voz2-1 double mutant and a complemented line under different concentrations of NaCl. Only the double mutant exhibited hypersensitivity to salt stress as compared to WT, single mutants, and a complemented line. Expression analysis showed that hypersensitivity of the double mutant was accompanied by reduced expression of salt-inducible genes. These results suggest that VOZ transcription factors act as positive regulators of several salt-responsive genes and that the two VOZs are functionally redundant in salt stress.

Abiotic and biotic stresses limit crop productivity. Exposure to a non-lethal stress, referred to as priming, can allow plants to survive subsequent and otherwise lethal conditions; the priming effect persists even after a prolonged stress-free period. However, the molecular mechanisms underlying priming are not fully understood. Here, we investigated the molecular basis of heat-shock memory and the role of priming in Arabidopsis thaliana. Comprehensive analysis of transcriptome-wide changes in gene expression and alternative splicing in primed and non-primed plants revealed that alternative splicing functions as a novel component of heat-shock memory. We show that priming of plants with a non-lethal heat stress results in de-repression of splicing after a second exposure to heat stress. By contrast, non-primed plants showed significant repression of splicing. These observations link 'splicing memory' to the ability of plants to survive subsequent and otherwise lethal heat stress. This newly discovered priming-induced splicing memory may represent a general feature of heat-stress responses in plants and other organisms as many of the key components are conserved among eukaryotes. Furthermore, this finding could facilitate the development of novel approaches to improve plant survival under extreme heat stress.

Abiotic stresses affect plant physiology, development, growth, and alter pre-mRNA splicing. Western poplar is a model woody tree and a potential bioenergy feedstock. To investigate the extent of stress-regulated alternative splicing (AS), we conducted an in-depth survey of leaf, root, and stem xylem transcriptomes under drought, salt, or temperature stress. Analysis of approximately one billion of genome-aligned RNA-Seq reads from tissue- or stress-specific libraries revealed over fifteen millions of novel splice junctions. Transcript models supported by both RNA-Seq and single molecule isoform sequencing (Iso-Seq) data revealed a broad array of novel stress- and/or tissue-specific isoforms. Analysis of Iso-Seq data also resulted in the discovery of 15,087 novel transcribed regions of which 164 show AS. Our findings demonstrate that abiotic stresses profoundly perturb transcript isoform profiles and trigger widespread intron retention (IR) events. Stress treatments often increased or decreased retention of specific introns - a phenomenon described here as differential intron retention (DIR). Many differentially retained introns were regulated in a stress- and/or tissue-specific manner. A subset of transcripts harboring super stress-responsive DIR events showed persisting fluctuations in the degree of IR across all treatments and tissue types. To investigate coordinated dynamics of intron-containing transcripts in the study we quantified absolute copy number of isoforms of two conserved transcription factors (TFs) using Droplet Digital PCR. This case study suggests that stress treatments can be associated with coordinated switches in relative ratios between fully spliced and intron-retaining isoforms and may play a role in adjusting transcriptome to abiotic stresses.

Background: Pre-mRNA splicing is an essential post-transcriptional process in all eukaryotes. In vitro splicing systems using nuclear or cytoplasmic extracts from mammalian cells, yeast, and Drosophila have provided a wealth of mechanistic insights into assembly and composition of the spliceosome, splicing regulatory proteins and mechanisms of pre-mRNA splicing in non-plant systems. The lack of an in vitro splicing system prepared from plant cells has been a major limitation in splicing research in plants., Results: Here we report an in vitro splicing assay system using plant nuclear extract. Several lines of evidence indicate that nuclear extract derived from Arabidopsis seedlings can convert pre-mRNA substrate ( LHCB3 ) into a spliced product. These include: (1) generation of an RNA product that corresponds to the size of expected mRNA, (2) a junction-mapping assay using S1 nuclease revealed that the two exons are spliced together, (3) the reaction conditions are similar to those found with non-plant extracts and (4) finally mutations in conserved donor and acceptor sites abolished the production of the spliced product., Conclusions: This first report on the plant in vitro splicing assay opens new avenues to investigate plant spliceosome assembly and composition, and splicing regulatory mechanisms specific to plants.

Background: Intron retention (IR) is the most prevalent form of alternative splicing in plants. IR, like other forms of alternative splicing, has an important role in increasing gene product diversity and regulating transcript functionality. Splicing is known to occur co-transcriptionally and is influenced by the speed of transcription which in turn, is affected by chromatin structure. It follows that chromatin structure may have an important role in the regulation of splicing, and there is preliminary evidence in metazoans to suggest that this is indeed the case; however, nothing is known about the role of chromatin structure in regulating IR in plants. DNase I-seq is a useful experimental tool for genome-wide interrogation of chromatin accessibility, providing information on regions of chromatin with very high likelihood of cleavage by the enzyme DNase I, known as DNase I Hypersensitive Sites (DHSs). While it is well-established that promoter regions are highly accessible and are over-represented with DHSs, not much is known about DHSs in the bodies of genes, and their relationship to splicing in general, and IR in particular., Results: In this study we use publicly available DNase I-seq data in arabidopsis and rice to investigate the relationship between IR and chromatin structure. We find that IR events are highly enriched in DHSs in both species. This implies that chromatin is more open in retained introns, which is consistent with a kinetic model of the process whereby higher speeds of transcription in those regions give less time for the spliceosomal machinery to recognize and splice out those introns co-transcriptionally. The more open chromatin in IR can also be the result of regulation mediated by DNA-binding proteins. To test this, we performed an exhaustive search for footprints left by DNA-binding proteins that are associated with IR. We identified several hundred short sequence elements that exhibit footprints in their DNase I-seq coverage, the telltale sign for binding events of a regulatory protein, protecting its binding site from cleavage by DNase I. A highly significant fraction of those sequence elements are conserved between arabidopsis and rice, a strong indication of their functional importance., Conclusions: In this study we have established an association between IR and chromatin accessibility, and presented a mechanistic hypothesis that explains the observed association from the perspective of the co-transcriptional nature of splicing. Furthermore, we identified conserved sequence elements for DNA-binding proteins that affect splicing.

Abiotic and biotic stresses cause significant yield losses in all crops. Acquisition of stress tolerance in plants requires rapid reprogramming of gene expression. SR1/CAMTA3, a member of signal responsive transcription factors (TFs), functions both as a positive and a negative regulator of biotic stress responses and as a positive regulator of cold stress-induced gene expression. Using high throughput RNA-seq, we identified ~3000 SR1-regulated genes. Promoters of about 60% of the differentially expressed genes have a known DNA binding site for SR1, suggesting that they are likely direct targets. Gene ontology analysis of SR1-regulated genes confirmed previously known functions of SR1 and uncovered a potential role for this TF in salt stress. Our results showed that SR1 mutant is more tolerant to salt stress than the wild type and complemented line. Improved tolerance of sr1 seedlings to salt is accompanied with the induction of salt-responsive genes. Furthermore, ChIP-PCR results showed that SR1 binds to promoters of several salt-responsive genes. These results suggest that SR1 acts as a negative regulator of salt tolerance by directly repressing the expression of salt-responsive genes. Overall, this study identified SR1-regulated genes globally and uncovered a previously uncharacterized role for SR1 in salt stress response.

The Arabidopsis FLAGELLIN SENSITIVE2 (FLS2) protein is a leucine-rich repeat receptor-like kinase (LRR-RLK) that plays important roles in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). The binding of bacterial flagellin, one of the PAMPs, to the extracellular domain of FLS2 leads to activation of signaling cascades resulting in activation or repression of a specific set of genes involved in plant defense. The mechanisms at the cell membrane that lead to the activation of this signalling pathway are, however, not fully understood. Recently, we have shown that after ligand-treatment the mobility of FLS2 in the cell membrane is reduced and that the activation of FLS2 does not involve its constitutive or ligand-dependent homodimerization. Our data together with recently published reports suggest that FLS2 activation involves its association with other proteins, including BRI1-associated kinase 1 (BAK1), another LRR-RLK, and localization to less mobile areas, probably lipid rafts, in a ligand-dependent manner to initiate PTI.

Pectate lyases catalyse the eliminative cleavage of de-esterified homogalacturonan in pectin, a major component of the primary cell walls in higher plants. In the completed genome of Arabidopsis, there are 26 genes (AtPLLs) that encode pectate lyase-like proteins. Here, we analysed the expression pattern of all AtPLLs in different organs, at different stages of seedling development and in response to various hormones and stresses. The expression of PLLs varied considerably in different organs, with no expression of some PLLs in vegetative organs. Interestingly, all PLL genes are expressed in flowers. Several PLLs are expressed highly in pollen, suggesting a role for these in pollen development and/or function. Analysis of expression of all PLL genes in seedlings treated with hormones, abiotic stresses and elicitors of defense responses revealed significant changes in the expression of some PLLs without affecting the other PLLs. The stability of transcripts of PLLs varied considerably among different genes. Our results indicate a complex regulation of expression of PLLs and involvement of PLLs in some of the hormonal and stress responses.

KCBP (kinesin-like calmodulin [CaM]-binding proteins), a member of the carboxy-terminal kinesin-like proteins (KLPs), is unique among KLPs in having a CaM-binding domain (CBD). CaM-binding KLPs have been identified from flowering plants and the sea urchin. To determine if CaM-binding KLP is present in phylogenetically divergent protists, we probed Cyanophora paradoxa protein extract with affinity-purified KCBP antibody. The KCBP antibody detected a polypeptide with a molecular mass of about 133 kDa in the crude extract. In a CaM-Sepharose column-purified fraction, the same band was detected with both KCBP antibody and biotinylated CaM. In a PCR reaction using degenerate primers corresponding to two conserved regions in the motor domain of kinesin, a 500-bp fragment (CpKLP1) was amplified from a cDNA library. The predicted amino acid sequence of CpKLP1 showed significant sequence similarity with KCBPs. In phylogenetic analysis, CpKLP1 fell into the KCBP group within the carboxy-terminal subfamily. These biochemical data, sequence, and phylogenetic analysis strongly suggest the presence of a calmodulin-binding KLP in C. paradoxa and that it is related to Ca 2 +/calmodulin regulated KLPs from plants. This is the first report on identification of any motor protein in C. paradoxa. Furthermore, our data suggest that CaM-binding KLPs may have evolved long before the divergence of plants and animals.