Increased DNA methylation of the splicing regulator SR45 suppresses seed abortion in litchi.

The gene regulatory networks that govern seed development are complex, yet very little is known about the genes and processes that are controlled by DNA methylation. Here, we performed single-base resolution DNA methylome analysis and found that CHH methylation increased significantly throughout seed development in litchi. Based on the association analysis of differentially methylated regions and weighted gene co-expression network analysis (WGCNA), 46 genes were identified as essential DNA methylation-regulated candidate genes involved in litchi seed development, including LcSR45, a homolog of the serine/arginine-rich (SR) splicing regulator SR45. LcSR45 is predominately expressed in the funicle, embryo, and seed integument, and displayed increased CHH methylation in the promoter during seed development. Notably, silencing of LcSR45 in a seed-aborted litchi cultivar significantly improved normal seed development, whereas the ectopic expression of LcSR45 in Arabidopsis caused seed abortion. Furthermore, LcSR45-dependent alternative splicing events were found to regulate genes involved in seed development. Together, our findings demonstrate that LcSR45 is hypermethylated, and plays a detrimental role in litchi seed development, indicating a global increase in DNA methylation at this stage.

Shedding Light on Iron Nutrition: Exploring Intersections of transcription factor cascades in light and iron deficiency signaling.

In the dynamic environment of plants, the interplay between light-dependent growth and iron nutrition is a recurring challenge. Plants respond to low iron levels by adjusting growth and physiology through enhanced iron acquisition from the rhizosphere and internal iron pool reallocation. Iron deficiency response assays and gene co-expression networks aid in documenting physiological reactions and unraveling gene regulatory cascades, offering insight into the interplay between hormonal and external signaling pathways. However, research directly exploring the significance of light in iron nutrition remains limited. This review provides an overview on iron deficiency regulation and its cross-connection with distinct light signals, focusing on transcription factor cascades and long-distance signaling. The circadian clock and retrograde signaling influence iron uptake and allocation. The light-activated shoot-to-root mobile transcription factor ELONGATED HYPOCOTYL5 (HY5) affects iron homeostasis responses in roots. Blue light triggers the formation of biomolecular condensates containing iron deficiency-induced protein complexes. The potential of exploiting the connection between light and iron signaling remains underutilized. With climate change and soil alkalinity on the rise, there is a need to develop crops with improved nutrient use efficiency and modified light dependencies. More research is needed to understand and leverage the interplay between light signaling and iron nutrition.

The Arabidopsis SR45 splicing factor bridges the splicing machinery and the exon-exon junction complex.

The Arabidopsis splicing factor serine/arginine-rich 45 (SR45) contributes to several biological processes. The sr45-1 loss-of-function mutant exhibits delayed root development, late flowering, unusual numbers of floral organs, shorter siliques with decreased seed sets, narrower leaves and petals, and altered metal distribution. SR45 bears a unique RNA recognition motif (RRM) flanked by one serine/arginine-rich (RS) domain on both sides. Here, we studied the function of each SR45 domains by examining their involvement in: (i) the spatial distribution of SR45; (ii) the establishment of a protein-protein interaction network including spliceosomal and exon-exon junction complex (EJC) components; and (iii) the RNA binding specificity. We report that the endogenous SR45 promoter is active during vegetative and reproductive growth, and that the SR45 protein localizes in the nucleus. We demonstrate that the C-terminal arginine/serine-rich domain is a determinant of nuclear localization. We show that the SR45 RRM domain specifically binds purine-rich RNA motifs via three residues (H101, H141, and Y143), and is also involved in protein-protein interactions. We further show that SR45 bridges both mRNA splicing and surveillance machineries as a partner of EJC core components and peripheral factors, which requires phosphoresidues probably phosphorylated by kinases from both the CLK and SRPK families. Our findings provide insights into the contribution of each SR45 domain to both spliceosome and EJC assemblies.

Altered metal distribution in the sr45-1 Arabidopsis mutant causes developmental defects.

The plant serine/arginine-rich (SR) splicing factor SR45 plays important roles in several biological processes, such as splicing, DNA methylation, innate immunity, glucose regulation, and abscisic acid signaling. A homozygous Arabidopsis sr45-1 null mutant is viable, but exhibits diverse phenotypic alterations, including delayed root development, late flowering, shorter siliques with fewer seeds, narrower leaves and petals, and unusual numbers of floral organs. Here, we report that the sr45-1 mutant presents an unexpected constitutive iron deficiency phenotype characterized by altered metal distribution in the plant. RNA-Sequencing highlighted severe perturbations in metal homeostasis, the phenylpropanoid pathway, oxidative stress responses, and reproductive development. Ionomic quantification and histochemical staining revealed strong iron accumulation in the sr45-1 root tissues accompanied by iron starvation in aerial parts. Mis-splicing of several key iron homeostasis genes, including BTS, bHLH104, PYE, FRD3, and ZIF1, was observed in sr45-1 roots. We showed that some sr45-1 developmental abnormalities can be complemented by exogenous iron supply. Our findings provide new insight into the molecular mechanisms governing the phenotypes of the sr45-1 mutant.

OsFKBP20-1b interacts with the splicing factor OsSR45 and participates in the environmental stress response at the post-transcriptional level in rice.

Sessile plants have evolved distinct mechanisms to respond and adapt to adverse environmental conditions through diverse mechanisms including RNA processing. While the role of RNA processing in the stress response is well understood for Arabidopsis thaliana, limited information is available for rice (Oryza sativa). Here, we show that OsFKBP20-1b, belonging to the immunophilin family, interacts with the splicing factor OsSR45 in both nuclear speckles and cytoplasmic foci, and plays an essential role in post-transcriptional regulation of abiotic stress response. The expression of OsFKBP20-1b was highly upregulated under various abiotic stresses. Moreover genetic analysis revealed that OsFKBP20-1b positively affected transcription and pre-mRNA splicing of stress-responsive genes under abiotic stress conditions. In osfkbp20-1b loss-of-function mutants, the expression of stress-responsive genes was downregulated, while that of their splicing variants was increased. Conversely, in plants overexpressing OsFKBP20-1b, the expression of the same stress-responsive genes was strikingly upregulated under abiotic stress. In vivo experiments demonstrated that OsFKBP20-1b directly maintains protein stability of OsSR45 splicing factor. Furthermore, we found that the plant-specific OsFKBP20-1b gene has uniquely evolved as a paralogue only in some Poaceae species. Together, our findings suggest that OsFKBP20-1b-mediated RNA processing contributes to stress adaptation in rice.

The wheat Sr22, Sr33, Sr35 and Sr45 genes confer resistance against stem rust in barley.

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley production in Africa and Europe. In contrast to wheat with 60 designated stem rust (Sr) resistance genes, barley's genetic variation for stem rust resistance is very narrow with only ten resistance genes genetically identified. Of these, only one complex locus consisting of three genes is effective against TTKSK, a widely virulent Pgt race of the Ug99 tribe which emerged in Uganda in 1999 and has since spread to much of East Africa and parts of the Middle East. The objective of this study was to assess the functionality, in barley, of cloned wheat Sr genes effective against race TTKSK. Sr22, Sr33, Sr35 and Sr45 were transformed into barley cv. Golden Promise using Agrobacterium-mediated transformation. All four genes were found to confer effective stem rust resistance. The barley transgenics remained susceptible to the barley leaf rust pathogen Puccinia hordei, indicating that the resistance conferred by these wheat Sr genes was specific for Pgt. Furthermore, these transgenic plants did not display significant adverse agronomic effects in the absence of disease. Cloned Sr genes from wheat are therefore a potential source of resistance against wheat stem rust in barley.

Dual roles of the serine/arginine-rich splicing factor SR45a in promoting and interacting with nuclear cap-binding complex to modulate the salt-stress response in Arabidopsis.

Alternative splicing (AS) is emerging as a critical co-transcriptional regulation for plants in response to environmental stresses. Although multiple splicing factors have been linked to the salt-sensitive signaling network, the molecular mechanism remains unclear. We discovered that a conserved serine/arginine-rich (SR)-like protein, SR45a, as a component of the spliceosome, was involved in post-transcriptional regulation of salinity tolerance in Arabidopsis thaliana. Furthermore, SR45a was required for the AS and messenger RNA (mRNA) maturation of several salt-tolerance genes. Two alternatively spliced variants of SR45a were induced by salt stress, full-length SR45a-1a and the truncated isoform SR45a-1b, respectively. Lines with overexpression of SR45a-1a and SR45a-1b exhibited hypersensitive to salt stress. Our data indicated that SR45a directly interacted with the cap-binding complex (CBC) subunit cap-binding protein 20 (CBP20) which mediated salt-stress responses. Instead of binding to other spliceosome components, SR45a-1b promoted the association of SR45a-1a with CBP20, therefore mediating salt-stress signal transduction pathways. Additionally, the mutations in SR45a and CBP20 led to different salt-stress phenotypes. Together, these results provide the evidence that SR45a-CBP20 acts as a regulatory complex to regulate the plant response to salt stress, through a regulatory mechanism to fine-tune the splicing factors, especially in stressful conditions.

The Arabidopsis splicing regulator SR45 confers salt tolerance in a splice isoform-dependent manner.

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.

Transcriptome analyses reveal SR45 to be a neutral splicing regulator and a suppressor of innate immunity in Arabidopsis thaliana.

BACKGROUND: Regulation of pre-mRNA splicing diversifies protein products and affects many biological processes. Arabidopsis thaliana Serine/Arginine-rich 45 (SR45), regulates pre-mRNA splicing by interacting with other regulatory proteins and spliceosomal subunits. Although SR45 has orthologs in diverse eukaryotes, including human RNPS1, the sr45-1 null mutant is viable. Narrow flower petals and reduced seed formation suggest that SR45 regulates genes involved in diverse processes, including reproduction. To understand how SR45 is involved in the regulation of reproductive processes, we studied mRNA from the wild-type and sr45-1 inflorescences using RNA-seq, and identified SR45-bound RNAs by immunoprecipitation. RESULTS: Using a variety of bioinformatics tools, we identified a total of 358 SR45 differentially regulated (SDR) genes, 542 SR45-dependent alternative splicing (SAS) events, and 1812 SR45-associated RNAs (SARs). There is little overlap between SDR genes and SAS genes, and neither set of genes is enriched for flower or seed development. However, transcripts from reproductive process genes are significantly overrepresented in SARs. In exploring the fate of SARs, we found that a total of 81 SARs are subject to alternative splicing, while 14 of them are known Nonsense-Mediated Decay (NMD) targets. Motifs related to GGNGG are enriched both in SARs and near different types of SAS events, suggesting that SR45 recognizes this motif directly. Genes involved in plant defense are significantly over-represented among genes whose expression is suppressed by SR45, and sr45-1 plants do indeed show enhanced immunity. CONCLUSION: We find that SR45 is a suppressor of innate immunity. We find that a single motif (GGNGG) is highly enriched in both RNAs bound by SR45 and in sequences near SR45- dependent alternative splicing events in inflorescence tissue. We find that the alternative splicing events regulated by SR45 are enriched for this motif whether the effect of SR45 is activation or repression of the particular event. Thus, our data suggests that SR45 acts to control splice site choice in a way that defies simple categorization as an activator or repressor of splicing.

The Splicing Factor SR45 Negatively Regulates Anthocyanin Accumulation under High-Light Stress in Arabidopsis thaliana.

High-intensity light (HL) greatly induces the accumulation of anthocyanin, a fundamental compound in photoprotection and antioxidation. Many mechanisms regulating anthocyanin biosynthesis are well-characterized across developmental and environmental conditions; however, post-transcriptional regulation of its biosynthesis remains unclear. RNA splicing is one mechanism of post-transcriptional control and reprogramming in response to different developmental cues and stress conditions. The Arabidopsis splicing modulator SR45 regulates a number of developmental and environmental stress responses. Here, we investigated the role of SR45 and its isoforms in HL-induced anthocyanin accumulation. We found that the SR45 promoter contains light-responsive cis-elements, and that light stress significantly increases SR45 expression. Furthermore, we found that mutant plants lacking SR45 function (sr45) accumulate significantly more anthocyanin under HL. SR45 is alternatively spliced to produce two proteins, SR45.1 and SR45.2, which differ by seven amino acids. Intriguingly, these isoforms exhibited distinct functions, with only SR45.1 reversing anthocyanin accumulation in the sr45 plants. We also identified possible SR45 target genes that are involved in anthocyanin synthesis. Consistent with the antioxidant role of anthocyanin, we found that sr45 mutants and SR45.2 overexpression lines accumulate anthocyanin and better tolerate paraquat which induces oxidative stress. Collectively, our results reveal that the Arabidopsis splicing regulator SR45 inhibits anthocyanin accumulation under HL, which may negatively affect oxidative stress tolerance. This study illuminates splicing-level regulation of anthocyanin production in response to light stress and offers a possible target for genetic modification to increase plant stress tolerance.

BioViz Connect: Web Application Linking CyVerse Cloud Resources to Genomic Visualization in the Integrated Genome Browser.

Genomics researchers do better work when they can interactively explore and visualize data. Due to the vast size of experimental datasets, researchers are increasingly using powerful, cloud-based systems to process and analyze data. These remote systems, called science gateways, offer user-friendly, Web-based access to high performance computing and storage resources, but typically lack interactive visualization capability. In this paper, we present BioViz Connect, a middleware Web application that links CyVerse science gateway resources to the Integrated Genome Browser (IGB), a highly interactive native application implemented in Java that runs on the user's personal computer. Using BioViz Connect, users can 1) stream data from the CyVerse data store into IGB for visualization, 2) improve the IGB user experience for themselves and others by adding IGB specific metadata to CyVerse data files, including genome version and track appearance, and 3) run compute-intensive visual analytics functions on CyVerse infrastructure to create new datasets for visualization in IGB or other applications. To demonstrate how BioViz Connect facilitates interactive data visualization, we describe an example RNA-Seq data analysis investigating how heat and desiccation stresses affect gene expression in the model plant Arabidopsis thaliana. The RNA-Seq use case illustrates how interactive visualization with IGB can help a user identify problematic experimental samples, sanity-check results using a positive control, and create new data files for interactive visualization in IGB (or other tools) using a Docker image deployed to CyVerse via the Terrain API. Lastly, we discuss limitations of the technologies used and suggest opportunities for future work. BioViz Connect is available from https://bioviz.org.

Environmental Stresses Modulate Abundance and Timing of Alternatively Spliced Circadian Transcripts in Arabidopsis.

Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad range thermal cycles triggered a sharp increase in the long intron retaining CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or P. syringae infection induced similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out of phase oscillations of CCA1 IR transcripts. Altogether, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of NMD-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs.