Differential sRNA regulation in leaves and roots of sugarcane under water depletion.

Plants have developed multiple regulatory mechanisms to respond and adapt to stress. Drought stress is one of the major constraints to agricultural productivity worldwide and recent reports have highlighted the importance of plant sRNA in the response and adaptation to water availability. In order to increase our understanding of the roles of sRNA in response to water depletion, cultivars of sugarcane were submitted to treatment of ceasing drip irrigation for 24 hours. Deep sequencing analysis was carried out to identify the sRNA regulated in leaves and roots of sugarcane cultivars with different drought sensitivities. The pool of sRNA selected allowed the analysis of different sRNA classes (miRNA and siRNA). Twenty-eight and 36 families of conserved miRNA were identified in leaf and root libraries, respectively. Dynamic regulation of miRNA was observed and the expression profiles of eight miRNA were verified in leaf samples from three biological replicates by stem-loop qRT-PCR assay using the cultivars: SP90-1638--sensitive cultivar--and SP83-2847 and SP83-5073--tolerant cultivars. Altered miRNA regulation was correlated with changes in mRNA levels of specific targets. Two leaf libraries from individual sugarcane cultivars with contrasting drought-tolerance properties were also analyzed. An enrichment of 22-nt sRNA species was observed in leaf libraries. 22-nt miRNA triggered siRNA production by cleavage of their targets in response to water depletion. A number of genes of the sRNA biogenesis pathway were down-regulated in tolerant genotypes and up-regulated in sensitive in response to water depletion treatment. Our analysis contributes to increase the knowledge on the roles of sRNA in sugarcane submitted to water depletion.

Integrated RNA-seq and sRNA-seq analysis identifies novel nitrate-responsive genes in Arabidopsis thaliana roots.

BACKGROUND: Nitrate and other nitrogen metabolites can act as signals that regulate global gene expression in plants. Adaptive changes in plant morphology and physiology triggered by changes in nitrate availability are partly explained by these changes in gene expression. Despite several genome-wide efforts to identify nitrate-regulated genes, no comprehensive study of the Arabidopsis root transcriptome under contrasting nitrate conditions has been carried out. RESULTS: In this work, we employed the Illumina high throughput sequencing technology to perform an integrated analysis of the poly-A + enriched and the small RNA fractions of the Arabidopsis thaliana root transcriptome in response to nitrate treatments. Our sequencing strategy identified new nitrate-regulated genes including 40 genes not represented in the ATH1 Affymetrix GeneChip, a novel nitrate-responsive antisense transcript and a new nitrate responsive miRNA/TARGET module consisting of a novel microRNA, miR5640 and its target, AtPPC3. CONCLUSIONS: Sequencing of small RNAs and mRNAs uncovered new genes, and enabled us to develop new hypotheses for nitrate regulation and coordination of carbon and nitrogen metabolism.

Computational identification and analysis of novel sugarcane microRNAs.

BACKGROUND: MicroRNA-regulation of gene expression plays a key role in the development and response to biotic and abiotic stresses. Deep sequencing analyses accelerate the process of small RNA discovery in many plants and expand our understanding of miRNA-regulated processes. We therefore undertook small RNA sequencing of sugarcane miRNAs in order to understand their complexity and to explore their role in sugarcane biology. RESULTS: A bioinformatics search was carried out to discover novel miRNAs that can be regulated in sugarcane plants submitted to drought and salt stresses, and under pathogen infection. By means of the presence of miRNA precursors in the related sorghum genome, we identified 623 candidates of new mature miRNAs in sugarcane. Of these, 44 were classified as high confidence miRNAs. The biological function of the new miRNAs candidates was assessed by analyzing their putative targets. The set of bona fide sugarcane miRNA includes those likely targeting serine/threonine kinases, Myb and zinc finger proteins. Additionally, a MADS-box transcription factor and an RPP2B protein, which act in development and disease resistant processes, could be regulated by cleavage (21-nt-species) and DNA methylation (24-nt-species), respectively. CONCLUSIONS: A large scale investigation of sRNA in sugarcane using a computational approach has identified a substantial number of new miRNAs and provides detailed genotype-tissue-culture miRNA expression profiles. Comparative analysis between monocots was valuable to clarify aspects about conservation of miRNA and their targets in a plant whose genome has not yet been sequenced. Our findings contribute to knowledge of miRNA roles in regulatory pathways in the complex, polyploidy sugarcane genome.

ABAP1 is a novel plant Armadillo BTB protein involved in DNA replication and transcription.

In multicellular organisms, organogenesis requires a tight control of the balance between cell division and cell differentiation. Distinct signalling pathways that connect both cellular processes with developmental cues might have evolved to suit different developmental plans. Here, we identified and characterized a novel protein that interacts with pre-replication complex (pre-RC) subunits, designated Armadillo BTB Arabidopsis protein 1 (ABAP1). Overexpression of ABAP1 in plants limited mitotic DNA replication and decreased cell proliferation in leaves, whereas ABAP1 downregulation increased cell division rates. Activity of ABAP1 in transcription was supported by its association with the transcription factor AtTCP24. The ABAP1-AtTCP24 complex bound specifically to the promoters of AtCDT1a and AtCDT1b in vitro and in vivo. Moreover, expression levels of AtCDT1a and AtCDT1b were reduced in ABAP1-overexpressing plants and they were increased in plants with reduced levels of ABAP1. We propose that ABAP1 participates in a negative feedback loop regulating mitotic DNA replication during leaf development, either by repressing transcription of pre-RC genes and possibly by regulating pre-RC utilization through direct association with pre-RC components.