Proteomic identification of putative microRNA394 target genes in <em>Arabidopsis thaliana</em> identifies major latex protein family members critical for normal development

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in <i>Arabidopsis thaliana</i> produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development

DRB2 Is Required for MicroRNA Biogenesis in Arabidopsis thaliana

Background The Arabidopsis thaliana (Arabidopsis) DOUBLE-STRANDED RNA BINDING (DRB) protein family consists of five members, DRB1 to DRB5. The biogenesis of two developmentally important small RNA (sRNA) species, the microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) by DICER-LIKE (DCL) endonucleases requires the assistance of DRB1 and DRB4 respectively. The importance of miRNA-directed target gene expression in plant development is exemplified by the phenotypic consequence of loss of DRB1 activity (drb1 plants). Principal Findings Here we report that the developmental phenotype of the drb235 triple mutant plant is the result of deregulated miRNA biogenesis in the shoot apical meristem (SAM) region. The expression of DRB2, DRB3 and DRB5 in wild-type seedlings is restricted to the SAM region. Small RNA sequencing of the corresponding tissue of drb235 plants revealed altered miRNA accumulation. Approximately half of the miRNAs detected remained at levels equivalent to those of wild-type plants. However, the accumulation of the remaining miRNAs was either elevated or reduced in the triple mutant. Examination of different single and multiple drb mutants revealed a clear association between the loss of DRB2 activity and altered accumulation for both the elevated and reduced miRNA classes. Furthermore, we show that the constitutive over-expression of DRB2 outside of its wild-type expression domain can compensate for the loss of DRB1 activity in drb1 plants. Conclusions/Significance Our results suggest that in the SAM region, DRB2 is both antagonistic and synergistic to the role of DRB1 in miRNA biogenesis, adding an additional layer of gene regulatory complexity in this developmentally important tissue

Viral Small Interfering RNAs Target Host Genes to Mediate Disease Symptoms in Plants

The Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco). How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA)-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide (nt) complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants, CHLI expression is dramatically down-regulated. Small RNA sequencing and 5′ RACE analyses confirmed that this 22-nt sequence was targeted for mRNA cleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNA interference (RNAi) vector targeting CHLI induced Y-Sat-like symptoms. In addition, the symptoms of Y-Sat infection can be completely prevented by transforming tobacco with a silencing-resistant variant of the CHLI gene. These results suggest that siRNA-directed silencing of CHLI is solely responsible for the Y-Sat-induced symptoms. Furthermore, we demonstrate that two Nicotiana species, which do not develop yellowing symptoms upon Y-Sat infection, contain a single nucleotide polymorphism within the siRNA-targeted CHLI sequence. This suggests that the previously observed species specificity of Y-Sat-induced symptoms is due to natural sequence variation in the CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-Sat siRNA. Taken together, these findings provide the first demonstration of small RNA-mediated viral disease symptom production and offer an explanation of the species specificity of the viral disease

Profiling of the Differential Abundance of Drought and Salt Stress-Responsive MicroRNAs Across Grass Crop and Genetic Model Plant Species

In recent years, it has become readily accepted among interdisciplinary agriculturalists that the current global crop yield to land capability ratio is significantly insufficient to achieve food security for the predicted population of 9.5 billion individuals by the year 2050. This issue is further compounded by the: (1) food versus biofuel debate; (2) decreasing availability of arable land; (3) required reductions to the extensive and ongoing environmental damage caused by either poor agricultural practices or agriculture expansion, and; (4) increasingly unfavorable (duration and severity) crop cultivation conditions that accompany man-made climate change, driven by ever-expanding urbanization and its associated industrial practices. Mounting studies are repeatedly highlighting the critical importance of linking genotypes to agronomically beneficial phenotypes and/or using a molecular approach to help address this global crisis, as &ldquo;simply&rdquo; clearing the remaining natural ecosystems of the globe for the cultivation of additional, non-modified crops is not efficient, nor is this practice sustainable. The majority of global food crop production is sourced from a small number of members of the Poaceae family of grasses, namely; maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.). It is, therefore, of significant concern that all three of these Poaceae grass species are susceptible to a range of abiotic stresses, including drought and salt stress. Highly conserved among monocotyledonous and dicotyledonous plant species, microRNAs (miRNAs) are now well-established master regulators of gene expression, influencing all aspects of plant development, mediating defense responses against pathogens and adaptation to environmental stress. Here we investigate the variation in the abundance profiles of six known abiotic stress-responsive miRNAs, following exposure to salt and drought stress across these three key Poaceae grass crop species as well as to compare these profiles to those obtained from the well-established genetic model plant species, Arabidopsis thaliana (L.) Heynh. Additionally, we outline the variables that are the most likely primary contributors to instances of differential miRNA abundance across the assessed species following drought or salt stress exposure, specifically; (1) identifying variations in the experimental conditions and/or methodology used to assess miRNA abundance, and; (2) the distribution of regulatory transcription factor binding sites within the putative promoter region of a MICRORNA (MIR) gene that encodes the highly conserved, stress-responsive miRNA. We also discuss the emerging role that non-conserved, species-specific miRNAs play in mediating a plant&rsquo;s response to drought or salt stress

Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis

Abstract Background Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail (Setaria viridis) has recently been proposed as a potential experimental model for the study of C4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens-mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis (S. viridis). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data. Results Eleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE/THERONINE-PROTEIN PHOSPHATASE 2A (PP2A), 5′-ADENYLYLSULFATE REDUCTASE 6 (ASPR6) and DUAL SPECIFICITY PHOSPHATASE (DUSP) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A, ASPR6 and DUSP, were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes across the same tissues. Conclusions This approach readily demonstrated the suitably of the three selected reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. Further, the work reported here forms a highly useful platform for future gene expression quantification in S. viridis and can also be potentially directly translatable to other closely related and agronomically important C4 crop species

The presence of high-molecular-weight viral RNAs interferes with the detection of viral small RNAs

Viral small interfering RNA (siRNA) accumulation in plants is reported to exhibit a strong strand polarity bias, with plus (+) strand siRNAs dominating over minus (−) strand populations. This is of particular interest, as siRNAs processed from double-stranded RNA would be expected to accumulate equivalent amounts of both species. Here, we show that, as reported, (−) strand viral siRNAs are detected at much lower levels than (+) strand-derived species using standard Northern hybridization approaches. However, when total RNA is spiked with in vitro-transcribed antisense viral genomic RNA, (−) strand viral siRNAs are detected at increased levels equivalent to those of (+) strand siRNA. Our results suggest that (+) and (−) strand viral siRNAs accumulate to equivalent levels; however, a proportion of the (−) strand siRNAs are sequestered from the total detectable small RNA population during gel electrophoresis by hybridizing to the high-molecular-weight sense strand viral genomic RNA. Our findings provide a plausible explanation for the observed strand bias of viral siRNA accumulation, and could have wider implications in the analysis of both viral and nonviral small RNA accumulation

Profiling the Abiotic Stress Responsive microRNA Landscape of Arabidopsis thaliana

It is well established among interdisciplinary researchers that there is an urgent need to address the negative impacts that accompany climate change. One such negative impact is the increased prevalence of unfavorable environmental conditions that significantly contribute to reduced agricultural yield. Plant microRNAs (miRNAs) are key gene expression regulators that control development, defense against invading pathogens and adaptation to abiotic stress. Arabidopsis thaliana (Arabidopsis) can be readily molecularly manipulated, therefore offering an excellent experimental system to alter the profile of abiotic stress responsive miRNA/target gene expression modules to determine whether such modification enables Arabidopsis to express an altered abiotic stress response phenotype. Towards this goal, high throughput sequencing was used to profile the miRNA landscape of Arabidopsis whole seedlings exposed to heat, drought and salt stress, and identified 121, 123 and 118 miRNAs with a greater than 2-fold altered abundance, respectively. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was next employed to experimentally validate miRNA abundance fold changes, and to document reciprocal expression trends for the target genes of miRNAs determined abiotic stress responsive. RT-qPCR also demonstrated that each miRNA/target gene expression module determined to be abiotic stress responsive in Arabidopsis whole seedlings was reflective of altered miRNA/target gene abundance in Arabidopsis root and shoot tissues post salt stress exposure. Taken together, the data presented here offers an excellent starting platform to identify the miRNA/target gene expression modules for future molecular manipulation to generate plant lines that display an altered response phenotype to abiotic stress

Profiling of the Salt Stress Responsive MicroRNA Landscape of C4 Genetic Model Species Setaria viridis (L.) Beauv

Setaria viridis has recently emerged as an ideal model species to genetically characterize the C4 monocotyledonous grasses via a molecular modification approach. Soil salinization has become a compelling agricultural problem globally with salinity adversely impacting the yield potential of many of the major cereals. Small regulatory molecules of RNA, termed microRNAs (miRNAs), were originally demonstrated crucial for developmental gene expression regulation in plants, however, miRNAs have since been shown to additionally command a central regulatory role in abiotic stress adaptation. Therefore, a small RNA sequencing approach was employed to profile the salt stress responsive miRNA landscapes of the shoot and root tissues of two Setaria viridis accessions (A10 and ME034V) amenable to molecular modification. Small RNA sequencing-identified abundance alterations for miRNAs, miR169, miR395, miR396, miR397, miR398 and miR408, were experimentally validated via RT-qPCR. RT-qPCR was further applied to profile the molecular response of the miR160 and miR167 regulatory modules to salt stress. This analysis revealed accession- and tissue-specific responses for the miR160 and miR167 regulatory modules in A10 and ME034V shoot and root tissues exposed to salt stress. The findings reported here form the first crucial step in the identification of the miRNA regulatory modules to target for molecular manipulation to determine if such modification provides S. viridis with an improved tolerance to salt stress

A Vernalization Response in a Winter Safflower (Carthamus tinctorius) Involves the Upregulation of Homologs of FT, FUL, and MAF

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