Runxuan Zhang

Identification and Characterization of miRNA Transcriptome in Potato by High-Throughput Sequencing

Micro RNAs (miRNAs) represent a class of short, non-coding, endogenous RNAs which play important roles in post-transcriptional regulation of gene expression. While the diverse functions of miRNAs in model plants have been well studied, the impact of miRNAs in crop plant biology is poorly understood. Here we used high-throughput sequencing and bioinformatics analysis to analyze miRNAs in the tuber bearing crop potato (Solanum tuberosum). Small RNAs were analysed from leaf and stolon tissues. 28 conserved miRNA families were found and potato-specific miRNAs were identified and validated by RNA gel blot hybridization. The size, origin and predicted targets of conserved and potato specific miRNAs are described. The large number of miRNAs and complex population of small RNAs in potato suggest important roles for these non-coding RNAs in diverse physiological and metabolic pathways.

Phytophthora capsici-tomato interaction features dramatic shifts in gene expression associated with a hemi-biotrophic lifestyle

BackgroundPlant-microbe interactions feature complex signal interplay between pathogens and their hosts. Phytophthora species comprise a destructive group of fungus-like plant pathogens, collectively affecting a wide range of plants important to agriculture and natural ecosystems. Despite the availability of genome sequences of both hosts and microbes, little is known about the signal interplay between them during infection. In particular, accurate descriptions of coordinate relationships between host and microbe transcriptional programs are lacking.ResultsHere, we explore the molecular interaction between the hemi-biotrophic broad host range pathogen Phytophthora capsici and tomato. Infection assays and use of a composite microarray allowed us to unveil distinct changes in both P. capsici and tomato transcriptomes, associated with biotrophy and the subsequent switch to necrotrophy. These included two distinct transcriptional changes associated with early infection and the biotrophy to necrotrophy transition that may contribute to infection and completion of the P. capsici lifecycleConclusionsOur results suggest dynamic but highly regulated transcriptional programming in both host and pathogen that underpin P. capsici disease and hemi-biotrophy. Dynamic expression changes of both effector-coding genes and host factors involved in immunity, suggests modulation of host immune signaling by both host and pathogen. With new unprecedented detail on transcriptional reprogramming, we can now explore the coordinate relationships that drive host-microbe interactions and the basic processes that underpin pathogen lifestyles. Deliberate alteration of lifestyle-associated transcriptional changes may allow prevention or perhaps disruption of hemi-biotrophic disease cycles and limit damage caused by epidemics.

Physiological, biochemical and molecular responses of the potato (Solanum tuberosum L.) plant to moderately elevated temperature

Although significant work has been undertaken regarding the response of model and crop plants to heat shock during the acclimatory phase, few studies have examined the steady-state response to the mild heat stress encountered in temperate agriculture. In the present work, we therefore exposed tuberizing potato plants to mildly elevated temperatures (30/20 °C, day/night) for up to 5 weeks and compared tuber yield, physiological and biochemical responses, and leaf and tuber metabolomes and transcriptomes with plants grown under optimal conditions (22/16 °C). Growth at elevated temperature reduced tuber yield despite an increase in net foliar photosynthesis. This was associated with major shifts in leaf and tuber metabolite profiles, a significant decrease in leaf glutathione redox state and decreased starch synthesis in tubers. Furthermore, growth at elevated temperature had a profound impact on leaf and tuber transcript expression with large numbers of transcripts displaying a rhythmic oscillation at the higher growth temperature. RT-PCR revealed perturbation in the expression of circadian clock transcripts including StSP6A, previously identified as a tuberization signal. Our data indicate that potato plants grown at moderately elevated temperatures do not exhibit classic symptoms of abiotic stress but that tuber development responds via a diversity of biochemical and molecular signals.

Improved GAP-RBF network for classification problems

This paper presents the performance evaluation of the recently developed Growing and Pruning Radial Basis Function (GAP-RBF) algorithm for classification problems. Earlier GAP-RBF was evaluated only for function approximation problems. Improvements to GAP-RBF for enhancing its performance in both accuracy and speed are also described and the resulting algorithm is referred to as Fast GAP-RBF (FGAP-RBF). Performance comparison of FGAP-RBF algorithm with GAP-RBF and the Minimal Resource Allocation Network (MRAN) algorithm based on four benchmark classification problems, viz. Phoneme, Segment, Satimage and DNA are presented. The results indicate that FGAP-RBF produces higher classification accuracy with reduced computational complexity.

A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing

Abstract Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.

AtRTD – a comprehensive reference transcript dataset resource for accurate quantification of transcript‐specific expression in Arabidopsis thaliana

Summary RNA‐sequencing (RNA‐seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA‐seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA‐seq data using the transcriptome‐based alignment‐free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA‐seq by high resolution reverse transcription polymerase chain reaction (HR RT‐PCR). Good correlations between splicing ratios from RNA‐seq and HR RT‐PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA‐seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA‐seq data to quantify differential transcript abundance and expression.

Barley SIX-ROWED SPIKE3 encodes a putative Jumonji C-type H3K9me2/me3 demethylase that represses lateral spikelet fertility

The barley inflorescence (spike) comprises a multi-noded central stalk (rachis) with tri-partite clusters of uni-floretted spikelets attached alternately along its length. Relative fertility of lateral spikelets within each cluster leads to spikes with two or six rows of grain, or an intermediate morphology. Understanding the mechanisms controlling this key developmental step could provide novel solutions to enhanced grain yield. Classical genetic studies identified five major SIX-ROWED SPIKE (VRS) genes, with four now known to encode transcription factors. Here we identify and characterise the remaining major VRS gene, VRS3, as encoding a putative Jumonji C-type H3K9me2/me3 demethylase, a regulator of chromatin state. Exploring the expression network modulated by VRS3 reveals specific interactions, both with other VRS genes and genes involved in stress, hormone and sugar metabolism. We show that combining a vrs3 mutant allele with natural six-rowed alleles of VRS1 and VRS5 leads to increased lateral grain size and greater grain uniformity.The VRS genes of barley control the fertility of the lateral spikelets on the barley inflorescence. Here, Bull et al. show that VRS3 encodes a putative Jumonji C-type histone demethylase that regulates expression of other VRS genes, and genes involved in stress, hormone and sugar metabolism.

The Dynamics of Transcript Abundance during Cellularization of Developing Barley Endosperm

Gene transcript profiles during barley endosperm cellularization reveal functional modules, coexpression networks, and regulatory genes, all of which align with cell wall changes in developing grain. Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would help predict end-use properties such as yield, quality, and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm 3 d to 8 d after pollination (DAP). Comparisons of transcript abundance over time revealed 47 gene expression modules that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the datasets could be mined to explore and define the processes of cell wall biosynthesis, remodeling, and degradation. Using a combination of spatial molecular network and gene ontology enrichment analyses, it is shown that genes involved in cell wall metabolism are found in multiple modules, but cluster into two main groups that exhibit peak expression at 3 DAP to 4 DAP and 5 DAP to 8 DAP. The presence of transcription factor genes in these modules allowed candidate genes for the control of wall metabolism during early barley grain development to be identified. The data are publicly available through a dedicated web interface (https://ics.hutton.ac.uk/barseed/), where they can be used to interrogate co- and differential expression for any other genes, groups of genes, or transcription factors expressed during early endosperm development.

Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection

Potato (Solanum tuberosum L) is a natural host of Potato spindle tuber viroid (PSTVd) which can cause characteristic symptoms on developing plants including stunting phenotype and distortion of leaves and tubers. PSTVd is the type species of the family Pospiviroidae, and can replicate in the nucleus and move systemically throughout the plant. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. Our primary focus in this study is the identification of genes which can affect tuber formation since viroid infection can strongly influence tuber development and especially tuber shape. In this study, we used a large-scale method to identify differentially expressed genes in potato. We have identified defence, stress and sugar metabolism related genes having altered expression levels upon infection. Additionally, hormone pathway related genes showed significant up- or down-regulation. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 transcripts were identified and validated showing differential expression in viroid infected tissues. Our study suggests that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection.

High-quality reference transcript datasets hold the key to transcript-specific RNA-sequencing analysis in plants.

525 I. 525 II. 526 III. 527 IV. 527 V. 529 VI. 529 529 References 529 SUMMARY: Re-programming of the transcriptome involves both transcription and alternative splicing (AS). Some genes are regulated only at the AS level with no change in expression at the gene level. AS data must be incorporated as an essential aspect of the regulation of gene expression. RNA-sequencing (RNA-seq) can deliver both transcriptional and AS information, but accurate methods to analyse the added complexity in RNA-seq data are needed. The construction of a comprehensive reference transcript dataset (RTD) for a specific plant species, variety or accession, from all available sequence data, will immediately allow more robust analysis of RNA-seq data. RTDs will continually evolve and improve, a process that will be more efficient if resources across a community are shared and pooled.