Alternative splicing as a driver of natural variation in abscisic acid response.

Abscisic acid (ABA) is a crucial player in plant responses to the environment. It accumulates under stress, activating downstream signaling to implement molecular responses that restore homeostasis. Natural variance in ABA sensitivity remains barely understood, and the ABA pathway has been mainly studied at the transcriptional level, despite evidence that posttranscriptional regulation, namely, via alternative splicing, contributes to plant stress tolerance. Here, we identified the Arabidopsis accession Kn-0 as less sensitive to ABA than the reference Col-0, as shown by reduced effects of the hormone on seedling establishment, root branching, and stomatal closure, as well as by decreased induction of ABA marker genes. An in-depth comparative transcriptome analysis of the ABA response in the two variants revealed lower expression changes and fewer genes affected for the least ABA-sensitive ecotype. Notably, Kn-0 exhibited reduced levels of the ABA-signaling SnRK2 protein kinases and lower basal expression of ABA-reactivation genes, consistent with our finding that Kn-0 contains less endogenous ABA than Col-0. ABA also markedly affected alternative splicing, primarily intron retention, with Kn-0 being less responsive regarding both the number and magnitude of alternative splicing events, particularly exon skipping. We find that alternative splicing introduces a more ecotype-specific layer of ABA regulation and identify ABA-responsive splicing changes in key ABA pathway regulators that provide a functional and mechanistic link to the differential sensitivity of the two ecotypes. Our results offer new insight into the natural variation of ABA responses and corroborate a key role for alternative splicing in implementing ABA-mediated stress responses.

ABA signaling prevents phosphodegradation of the SR45 splicing factor to alleviate inhibition of early seedling development in Arabidopsis.

Serine/arginine-rich (SR) proteins are conserved splicing regulators that play important roles in plant stress responses, namely those mediated by the abscisic acid (ABA) hormone. The Arabidopsis thaliana SR-like protein SR45 is a described negative regulator of the ABA pathway during early seedling development. How the inhibition of growth by ABA signaling is counteracted to maintain plant development under stress conditions remains largely unknown. Here, we show that SR45 overexpression reduces Arabidopsis sensitivity to ABA during early seedling development. Biochemical and confocal microscopy analyses of transgenic plants expressing fluorescently tagged SR45 revealed that exposure to ABA dephosphorylates the protein at multiple amino acid residues and leads to its accumulation, due to SR45 stabilization via reduced ubiquitination and proteasomal degradation. Using phosphomutant and phosphomimetic transgenic Arabidopsis lines, we demonstrate the functional relevance of ABA-mediated dephosphorylation of a single SR45 residue, T264, in antagonizing SR45 ubiquitination and degradation to promote its function as a repressor of seedling ABA sensitivity. Our results reveal a mechanism that negatively autoregulates ABA signaling and allows early plant growth under stress via posttranslational control of the SR45 splicing factor.

Assessing Postgermination Development in Arabidopsis thaliana Under Abiotic Stress.

The abscisic acid (ABA) phytohormone is well known to regulate responses to abiotic stress, particularly tolerance to osmotic stress. Screening for phenotypes at the early plant development stages is fundamental to identify new regulators of the ABA pathway, which in turn is extremely relevant for agriculture in a global climate change context. Typically, under experimental conditions, seeds are germinated in hormone-containing plates, and postgermination development is then assessed through scoring of the appearance of green or expanded cotyledons. However, postgermination phenotypes may be either masked or exacerbated by prior defects in seed germination. To circumvent this, we propose a transfer assay to screen specifically and quickly for postgermination phenotypes affected by exogenous ABA. The assay can be applied to different forms of abiotic stress, and we provide tips to score for postgermination phenotypes in genotypes exhibiting differential development.

Alternative Splicing as a Regulator of Early Plant Development.

Most plant genes are interrupted by introns and the corresponding transcripts need to undergo pre-mRNA splicing to remove these intervening sequences. Alternative splicing (AS) is an important posttranscriptional process that creates multiple mRNA variants from a single pre-mRNA molecule, thereby enhancing the coding and regulatory potential of genomes. In plants, this mechanism has been implicated in the response to environmental cues, including abiotic and biotic stresses, in the regulation of key developmental processes such as flowering, and in circadian timekeeping. The early plant development steps - from embryo formation and seed germination to skoto- and photomorphogenesis - are critical to both execute the correct body plan and initiate a new reproductive cycle. We review here the available evidence for the involvement of AS and various splicing factors in the initial stages of plant development, while highlighting recent findings as well as potential future challenges.

The Arabidopsis SR45 Splicing Factor, a Negative Regulator of Sugar Signaling, Modulates SNF1-Related Protein Kinase 1 Stability.

The ability to sense and respond to sugar signals allows plants to cope with environmental and metabolic changes by adjusting growth and development accordingly. We previously reported that the SR45 splicing factor negatively regulates glucose signaling during early seedling development in Arabidopsis thaliana Here, we show that under glucose-fed conditions, the Arabidopsis sr45-1 loss-of-function mutant contains higher amounts of the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1) despite unaffected SnRK1 transcript levels. In agreement, marker genes for SnRK1 activity are upregulated in sr45-1 plants, and the glucose hypersensitivity of sr45-1 is attenuated by disruption of the SnRK1 gene. Using a high-resolution RT-PCR panel, we found that the sr45-1 mutation broadly targets alternative splicing in vivo, including that of the SR45 pre-mRNA itself. Importantly, the enhanced SnRK1 levels in sr45-1 are suppressed by a proteasome inhibitor, indicating that SR45 promotes targeting of the SnRK1 protein for proteasomal destruction. Finally, we demonstrate that SR45 regulates alternative splicing of the Arabidopsis 5PTase13 gene, which encodes an inositol polyphosphate 5-phosphatase previously shown to interact with and regulate the stability of SnRK1 in vitro, thus providing a mechanistic link between SR45 function and the modulation of degradation of the SnRK1 energy sensor in response to sugars.

LEAFDATA: a literature-curated database for Arabidopsis leaf development.

BACKGROUND: In the post-genomic era, biological databases provide an easy access to a wide variety of scientific data. The vast quantity of literature calls for curated databases where existing knowledge is carefully organized in order to aid novel discoveries. Leaves, the main photosynthetic organs are not only vital for plant growth but also essential for maintaining the global ecosystem by producing oxygen and food. Therefore, studying and understanding leaf formation and growth are key objectives in biology. Arabidopsis thaliana to this date remains the prime experimental model organism in plant science. DESCRIPTION: LEAFDATA was created as an easily accessible and searchable web tool to assemble a relevant collection of Arabidopsis leaf literature. LEAFDATA currently contains 13,553 categorized statements from 380 processed publications. LEAFDATA can be searched for genes of interest using Arabidopsis Genome Initiative identifiers, for selected papers by means of PubMed IDs, authors and specific keywords. The results page contains details of the original publications, text fragments from the curated literature grouped according to information types and direct links to PubMed pages of the original papers. CONCLUSIONS: The LEAFDATA database offers access to searchable entries curated from a large number of scientific publications. Due to the unprecedented details of annotations and the fact that LEAFDATA already provides records about approximately 1600 individual loci, this database is useful for the entire plant research community.

PRL1 modulates root stem cell niche activity and meristem size through WOX5 and PLTs in Arabidopsis.

The stem cell niche in the root meristem maintains pluripotent stem cells to ensure a constant supply of cells for root growth. Despite extensive progress, the molecular mechanisms through which root stem cell fates and stem cell niche activity are determined remain largely unknown. In Arabidopsis thaliana, the Pleiotropic Regulatory Locus 1 (PRL1) encodes a WD40-repeat protein subunit of the spliceosome-activating Nineteen Complex (NTC) that plays a role in multiple stress, hormone and developmental signaling pathways. Here, we show that PRL1 is involved in the control of root meristem size and root stem cell niche activity. PRL1 is strongly expressed in the root meristem and its loss of function mutation results in disorganization of the quiescent center (QC), premature stem cell differentiation, aberrant cell division, and reduced root meristem size. Our genetic studies indicate that PRL1 is required for confined expression of the homeodomain transcription factor WOX5 in the QC and acts upstream of the transcription factor PLETHORA (PLT) in modulating stem cell niche activity and root meristem size. These findings define a role for PRL1 as an important determinant of PLT signaling that modulates maintenance of the stem cell niche and root meristem size.

Ribosomal Protein RPL27a Promotes Female Gametophyte Development in a Dose-Dependent Manner.

Ribosomal protein mutations in Arabidopsis (Arabidopsis thaliana) result in a range of specific developmental phenotypes. Why ribosomal protein mutants have specific phenotypes is not fully known, but such defects potentially result from ribosome insufficiency, ribosome heterogeneity, or extraribosomal functions of ribosomal proteins. Here, we report that ovule development is sensitive to the level of Ribosomal Protein L27a (RPL27a) and is disrupted by mutations in the two paralogs RPL27aC and RPL27aB. Mutations in RPL27aC result in high levels of female sterility, whereas mutations in RPL27aB have a significant but lesser effect on fertility. Progressive reduction in RPL27a function results in increasing sterility, indicating a dose-dependent relationship between RPL27a and female fertility. RPL27a levels in both the sporophyte and gametophyte affect female gametogenesis, with different developmental outcomes determined by the dose of RPL27a. These results demonstrate that RPL27aC and RPL27aB act redundantly and reveal a function for RPL27a in coordinating complex interactions between sporophyte and gametophyte during ovule development.

The spliceosome-activating complex: molecular mechanisms underlying the function of a pleiotropic regulator.

Correct interpretation of the coding capacity of RNA polymerase II transcribed eukaryotic genes is determined by the recognition and removal of intronic sequences of pre-mRNAs by the spliceosome. Our current knowledge on dynamic assembly and subunit interactions of the spliceosome mostly derived from the characterization of yeast, Drosophila, and human spliceosomal complexes formed on model pre-mRNA templates in cell extracts. In addition to sequential structural rearrangements catalyzed by ATP-dependent DExH/D-box RNA helicases, catalytic activation of the spliceosome is critically dependent on its association with the NineTeen Complex (NTC) named after its core E3 ubiquitin ligase subunit PRP19. NTC, isolated recently from Arabidopsis, occurs in a complex with the essential RNA helicase and GTPase subunits of the U5 small nuclear RNA particle that are required for both transesterification reactions of splicing. A compilation of mass spectrometry data available on the composition of NTC and spliceosome complexes purified from different organisms indicates that about half of their conserved homologs are encoded by duplicated genes in Arabidopsis. Thus, while mutations of single genes encoding essential spliceosome and NTC components lead to cell death in other organisms, differential regulation of some of their functionally redundant Arabidopsis homologs permits the isolation of partial loss of function mutations. Non-lethal pleiotropic defects of these mutations provide a unique means for studying the roles of NTC in co-transcriptional assembly of the spliceosome and its crosstalk with DNA repair and cell death signaling pathways.

Involvement of ribosomal protein RPL27a in meristem activity and organ development.

The plant shoot apical meristem is established early during embryogenesis and subsequently gives rise to a shoot through reiterative generation of lateral organs and axillary meristems. In our recent manuscript we reported identification and characterization of a semi-dominant mutation in ribosomal protein RPL27a, which disrupts plant growth and shoot development.1 rpl27ac-1d effects on the shoot are evident from an early stage of embryo development. During embryogenesis rpl27-1d mutants are slow growing and are defective in apical patterning with a delay in establishment of the shoot meristem and outgrowth of cotyledons. Concomitant with this disturbed patterning, the shoot meristem genes SHOOT MERISTEMLESS (STM) and CUP-SHAPED COTYLEDON2 (CUC2) are misexpressed in outer cell layers of the rpl27ac-1d embryo and there is a delay in expression of the organ-patterning gene FILAMENTOUS FLOWER (FIL). Genetic interactions between rpl27ac-1d and other ribosomal protein mutants indicates rpl27ac-1d has reduced ribosome function. Our results highlight a role for ribosomal proteins in growth and development and we propose that the ribosome regulates specific patterning events during development.

Ribosomal protein L27a is required for growth and patterning in Arabidopsis thaliana.

Ribosomal proteins are integral to ribosome biogenesis, and function in protein synthesis. In higher eukaryotes, loss of cytoplasmic ribosomal proteins results in a reduced growth rate as well as developmental defects. To what extent and how ribosomal proteins affect development is currently not known. Here we describe a semi-dominant mutation in the cytoplasmic ribosomal protein gene RPL27aC that affects multiple aspects of plant shoot development, including leaf patterning, inflorescence and floral meristem function, and seed set. In the embryo, RPL27aC is required to maintain the growth rate and for the transition from radial to bilateral symmetry associated with initiation of cotyledons. rpl27ac-1d embryos undergo stereotypical patterning to establish a globular embryo. However, a temporal delay in initiation and outgrowth of cotyledon primordia leads to development of an enlarged globular embryo prior to apical domain patterning. Defects in embryo development are coincident with tissue-specific ectopic expression of the shoot meristem genes SHOOT MERISTEMLESS (STM) and CUP-SHAPED COTYLEDON2 (CUC2), in addition to delayed expression of the abaxial gene FILAMENTOUS FLOWER (FIL) and mis-regulation of the auxin efflux effector PIN-FORMED1 (PIN1). Genetic interactions with other ribosomal protein mutants indicate that RPL27aC is a component of the ribosome. We propose that RPL27aC regulates discrete developmental events by controlling spatial and temporal expression of developmental patterning genes via an as yet undefined process involving the ribosome.

Perspectives on leaf dorsoventral polarity.

Leaves occur in a vast array of shapes and sizes, with complex diversity contributing to optimization of the principal function of photosynthesis. The program of development from a self-renewing stem cell population to a mature leaf has been of interest to biologists for years. Many genes involved in this process have been identified, particularly in the model eudicot Arabidopsis, so that now we have a greater understanding of mechanisms of stem cell maintenance, cell differentiation and organogenesis. One aspect of leaf development that is of particular interest is the establishment of dorsoventral polarity: the distinct adaxial (upper) and abaxial (lower) sides of the leaf. Early studies postulated conceptual models of how establishment of polarity leads to the development of planar leaves. Studies over the past decade have defined genetic details of this model, and uncovered diverse mechanisms of gene regulation that facilitate development of leaf dorsoventral polarity, including transcriptional regulation, chromatin modification, DNA modification, regulation by short RNAs and translational and post-translational regulation. This review will discuss these regulatory mechanisms in the context of leaf dorsoventrality, and will conclude with unresolved questions and areas of future research.