The SCL30a SR protein regulates ABA-dependent seed traits and germination under stress.

SR proteins are conserved RNA-binding proteins best known as splicing regulators that have also been implicated in other steps of gene expression. Despite mounting evidence for a role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain poorly understood. Here we show that the plant-specific SCL30a SR protein negatively regulates ABA signaling to control seed traits and stress responses during germination in Arabidopsis. Transcriptome-wide analyses revealed that loss of SCL30a function barely affects splicing, but largely induces ABA-responsive gene expression and genes repressed during germination. Accordingly, scl30a mutant seeds display delayed germination and hypersensitivity to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA and salt stress sensitivity. An ABA biosynthesis inhibitor rescues the enhanced mutant seed stress sensitivity, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the gene promotes seed germination under stress by reducing sensitivity to the phytohormone. Our results reveal a new player in ABA-mediated control of early development and stress response.

Data Entry Automation Improves Cost, Quality, Performance, and Job Satisfaction in a Hospital Nursing Unit.

OBJECTIVE: An Automated Data Entry Process Technology tool was developed to free nurses from data entry tasks, thus creating time for patient care and other activities associated with improvements in performance and job satisfaction. BACKGROUND: Manually transferring data from patient measurement devices to electronic health records (EHRs) is an intensive, error-prone task that diverts nurses from patient care while adversely affecting job performance and employee satisfaction. METHODS: Performance improvement analytics were used to compare matched sets of manual and automated EHR data entries for 1933 consecutive vital signs records created by 49 RNs and certified nursing assistants in a 23-bed medical-surgical unit at a large tertiary hospital. Performance and quality effects were evaluated via nurses' responses to a postintervention survey. RESULTS: Data errors decreased from approximately 20% to 0; data transfer times were reduced by 5 minutes to 2 hours per measurement event; nurses had more time for direct patient care; and job satisfaction improved. CONCLUSION: Data entry automation eliminates data errors, substantially reduces delays in getting data into EHRs, and improves job satisfaction by giving nurses more time for direct patient care. Findings are associated with improvements in quality, work performance, and job satisfaction, key goals of nursing leaders.

More Transporters, More Substrates: The Arabidopsis Major Facilitator Superfamily Revisited.

The Major Facilitator Superfamily (MFS) is ubiquitous in living organisms and represents the largest group of secondary active membrane transporters. In plants, significant research efforts have focused on the role of specific families within the MFS, particularly those transporting macronutrients (C, N, and P) that constitute the vast majority of the members of this superfamily. Other MFS families remain less explored, although a plethora of additional substrates and physiological functions have been uncovered. Nevertheless, the lack of a systematic approach to analyzing the MFS as a whole has obscured the high diversity and versatility of these transporters. Here, we present a phylogenetic analysis of all annotated MFS domain-containing proteins encoded in the Arabidopsis thaliana genome and propose that this superfamily of transporters consists of 218 members, clustered in 22 families. In reviewing the available information regarding the diversity in biological functions and substrates of Arabidopsis MFS members, we provide arguments for intensified research on these membrane transporters to unveil the breadth of their physiological relevance, disclose the molecular mechanisms underlying their mode of action, and explore their biotechnological potential.

Pre-mRNA splicing repression triggers abiotic stress signaling in plants.

Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants.

Abscisic acid (ABA) regulation of Arabidopsis SR protein gene expression.

Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses.

Deciphering the plant splicing code: experimental and computational approaches for predicting alternative splicing and splicing regulatory elements.

Extensive alternative splicing (AS) of precursor mRNAs (pre-mRNAs) in multicellular eukaryotes increases the protein-coding capacity of a genome and allows novel ways to regulate gene expression. In flowering plants, up to 48% of intron-containing genes exhibit AS. However, the full extent of AS in plants is not yet known, as only a few high-throughput RNA-Seq studies have been performed. As the cost of obtaining RNA-Seq reads continues to fall, it is anticipated that huge amounts of plant sequence data will accumulate and help in obtaining a more complete picture of AS in plants. Although it is not an onerous task to obtain hundreds of millions of reads using high-throughput sequencing technologies, computational tools to accurately predict and visualize AS are still being developed and refined. This review will discuss the tools to predict and visualize transcriptome-wide AS in plants using short-reads and highlight their limitations. Comparative studies of AS events between plants and animals have revealed that there are major differences in the most prevalent types of AS events, suggesting that plants and animals differ in the way they recognize exons and introns. Extensive studies have been performed in animals to identify cis-elements involved in regulating AS, especially in exon skipping. However, few such studies have been carried out in plants. Here, we review the current state of research on splicing regulatory elements (SREs) and briefly discuss emerging experimental and computational tools to identify cis-elements involved in regulation of AS in plants. The availability of curated alternative splice forms in plants makes it possible to use computational tools to predict SREs involved in AS regulation, which can then be verified experimentally. Such studies will permit identification of plant-specific features involved in AS regulation and contribute to deciphering the splicing code in plants.

Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing.

SR45 is a serine/arginine-rich (SR)-like protein with two arginine/serine-rich (RS) domains. We have previously shown that SR45 regulates alternative splicing (AS) by differential selection of 5' and 3' splice sites. However, it is unknown how SR45 regulates AS. To gain mechanistic insights into the roles of SR45 in splicing, we screened a yeast two-hybrid library with SR45. This screening resulted in the isolation of two spliceosomal proteins, U1-70K and U2AF(35) b that are known to function in 5' and 3' splice site selection, respectively. This screen not only confirmed our prior observation that U1-70K and SR45 interact, but also helped to identify an additional interacting partner (U2AF(35) ). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF(35) . Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF(35) proteins. Interaction studies among U2AF(35) paralogs and between U2AF(35) and U1-70K revealed that U2AF(35) can form homo- or heterodimers and that U2AF(35) proteins can associate with U1-70K. Using RNA probes from SR30 intron 10, whose splicing is altered in the sr45 mutant, we show that SR45 and U2AF(35) b bind to different parts of the intron, with a binding site for SR45 in the 5' region and two binding regions, each ending with a known 3' splice site, for U2AF(35) b. These results suggest that SR45 recruits U1snRNP and U2AF to 5' and 3' splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF(35) and modulates AS.

Comparative analysis of serine/arginine-rich proteins across 27 eukaryotes: insights into sub-family classification and extent of alternative splicing.

Alternative splicing (AS) of pre-mRNA is a fundamental molecular process that generates diversity in the transcriptome and proteome of eukaryotic organisms. SR proteins, a family of splicing regulators with one or two RNA recognition motifs (RRMs) at the N-terminus and an arg/ser-rich domain at the C-terminus, function in both constitutive and alternative splicing. We identified SR proteins in 27 eukaryotic species, which include plants, animals, fungi and "basal" eukaryotes that lie outside of these lineages. Using RNA recognition motifs (RRMs) as a phylogenetic marker, we classified 272 SR genes into robust sub-families. The SR gene family can be split into five major groupings, which can be further separated into 11 distinct sub-families. Most flowering plants have double or nearly double the number of SR genes found in vertebrates. The majority of plant SR genes are under purifying selection. Moreover, in all paralogous SR genes in Arabidopsis, rice, soybean and maize, one of the two paralogs is preferentially expressed throughout plant development. We also assessed the extent of AS in SR genes based on a splice graph approach (http://combi.cs.colostate.edu/as/gmap_SRgenes). AS of SR genes is a widespread phenomenon throughout multiple lineages, with alternative 3' or 5' splicing events being the most prominent type of event. However, plant-enriched sub-families have 57%-88% of their SR genes experiencing some type of AS compared to the 40%-54% seen in other sub-families. The SR gene family is pervasive throughout multiple eukaryotic lineages, conserved in sequence and domain organization, but differs in gene number across lineages with an abundance of SR genes in flowering plants. The higher number of alternatively spliced SR genes in plants emphasizes the importance of AS in generating splice variants in these organisms.

Organ-specific, developmental, hormonal and stress regulation of expression of putative pectate lyase genes in Arabidopsis.

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.

Comprehensive comparative analysis of kinesins in photosynthetic eukaryotes.

BACKGROUND: Kinesins, a superfamily of molecular motors, use microtubules as tracks and transport diverse cellular cargoes. All kinesins contain a highly conserved approximately 350 amino acid motor domain. Previous analysis of the completed genome sequence of one flowering plant (Arabidopsis) has resulted in identification of 61 kinesins. The recent completion of genome sequencing of several photosynthetic and non-photosynthetic eukaryotes that belong to divergent lineages offers a unique opportunity to conduct a comprehensive comparative analysis of kinesins in plant and non-plant systems and infer their evolutionary relationships. RESULTS: We used the kinesin motor domain to identify kinesins in the completed genome sequences of 19 species, including 13 newly sequenced genomes. Among the newly analyzed genomes, six represent photosynthetic eukaryotes. A total of 529 kinesins was used to perform comprehensive analysis of kinesins and to construct gene trees using the Bayesian and parsimony approaches. The previously recognized 14 families of kinesins are resolved as distinct lineages in our inferred gene tree. At least three of the 14 kinesin families are not represented in flowering plants. Chlamydomonas, a green alga that is part of the lineage that includes land plants, has at least nine of the 14 known kinesin families. Seven of ten families present in flowering plants are represented in Chlamydomonas, indicating that these families were retained in both the flowering-plant and green algae lineages. CONCLUSION: The increase in the number of kinesins in flowering plants is due to vast expansion of the Kinesin-14 and Kinesin-7 families. The Kinesin-14 family, which typically contains a C-terminal motor, has many plant kinesins that have the motor domain at the N terminus, in the middle, or the C terminus. Several domains in kinesins are present exclusively either in plant or animal lineages. Addition of novel domains to kinesins in lineage-specific groups contributed to the functional diversification of kinesins. Results from our gene-tree analyses indicate that there was tremendous lineage-specific duplication and diversification of kinesins in eukaryotes. Since the functions of only a few plant kinesins are reported in the literature, this comprehensive comparative analysis will be useful in designing functional studies with photosynthetic eukaryotes.