Reference genes for accurate gene expression analyses across different tissues, developmental stages and genotypes in rice for drought tolerance.

BACKGROUND: Quantitative reverse transcription PCR (qRT-PCR) has been routinely used to quantify gene expression level. This technique determines the expression of a target gene by comparison to an internal control gene uniformly expressed among the samples analyzed. The reproducibility and reliability of the results depend heavily on the reference genes used. To achieve successful gene expression analyses for drought tolerance studies in rice, reference gene selection should be based on consistency in expression across variables. We aimed to provide reference genes that would be consistent across different tissues, developmental stages and genotypes of rice and hence improve the quality of data in qRT-PCR analysis. FINDINGS: Ten candidate reference genes were screened from four ubiquitously expressed gene families by analyzing public microarray data sets that included profiles of multiple organs, developmental stages, and water availability status in rice. These genes were evaluated through qRT-PCR experiments with a rigorous statistical analysis to determine the best reference genes. A ubiquitin isogene showed the best gene expression stability as a single reference gene, while a 3-gene combination of another ubiquitin and two cyclophilin isogenes was the best reference gene combination. Comparison between the qRT-PCR and in-house microarray data on roots demonstrated reliability of the identified reference genes to monitor the differential expression of drought-related candidate genes. CONCLUSIONS: Specific isogenes from among the regularly used gene families were identified for use in qRT-PCR-based analyses for gene expression in studies on drought tolerance in rice. These were stable across variables of treatment, genotype, tissue and growth stage. A single gene and/or a three gene set analysis is recommended, based on the resources available.

Action of multiple intra-QTL genes concerted around a co-localized transcription factor underpins a large effect QTL.

Sub-QTLs and multiple intra-QTL genes are hypothesized to underpin large-effect QTLs. Known QTLs over gene families, biosynthetic pathways or certain traits represent functional gene-clusters of genes of the same gene ontology (GO). Gene-clusters containing genes of different GO have not been elaborated, except in silico as coexpressed genes within QTLs. Here we demonstrate the requirement of multiple intra-QTL genes for the full impact of QTL qDTY12.1 on rice yield under drought. Multiple evidences are presented for the need of the transcription factor 'no apical meristem' (OsNAM12.1) and its co-localized target genes of separate GO categories for qDTY12.1 function, raising a regulon-like model of genetic architecture. The molecular underpinnings of qDTY12.1 support its effectiveness in further improving a drought tolerant genotype and for its validity in multiple genotypes/ecosystems/environments. Resolving the combinatorial value of OsNAM12.1 with individual intra-QTL genes notwithstanding, identification and analyses of qDTY12.1has fast-tracked rice improvement towards food security.

Variation in primary metabolites in parental and near-isogenic lines of the QTL qDTY12.1 : altered roots and flag leaves but similar spikelets of rice under drought.

There is a widespread consensus that drought will mostly affect present and future agriculture negatively. Generating drought-tolerant crops is thus a high priority. However complicated the underlying genetic and regulatory networks for differences in plant performance under stress are, they would be reflected in straightforward differences in primary metabolites. This is because primary metabolites such as amino acids and sugars form the building blocks of all pathways and processes for growth, development, reproduction, and environmental responses. Comparison of such differences was undertaken between the parental line and a near-isogenic line of qDTY12.1 , a QTL for rice yield under drought. The comparison was informative regarding the effect of the QTL in three genetic backgrounds: donor, recipient, and improved recipient, thus illustrating the gene × gene (G × G) interactions. Such a comparison when extended to well-watered and drought conditions illustrated the gene × environment (G × E) interactions. Assessment of such G × G and G × E responses in roots, flag leaves, and spikelets added a yet more informative dimension of tissue-specific responses to drought, mediated by qDTY12.1 . Data on variation in primary metabolites subjected to ANOVA, Tukey's test, Welch's t test, and PCA underscored the importance of the roots and demonstrated concordance between variation in metabolites and morpho-physiological responses to drought. Results suggested that for gainful insights into rice yield under drought, rather than vegetative stage drought tolerance, multiple tissues and genotypes must be assessed at the reproductive stage to avoid misleading conclusions about using particular metabolites or related genes and proteins as candidates or markers for drought tolerance.

Protein SUMOylation and plant abiotic stress signaling: in silico case study of rice RLKs, heat-shock and Ca(2+)-binding proteins.

Plants respond to stress conditions through early stress-response factors (ESRF), which serve the function of stress sensing and/or signal transduction. These mainly comprise qualitative and/or quantitative flux in the redox molecules, calcium ions (Ca(2+)), phosphatidic acid, hexose sugars and phytohormones. The role of resident proteins such as phytohormone receptors and G-proteins as first messengers under stress is well established. Yet, within the modern omics context, most of the stress response at the protein level is injudiciously attributed to substantial up- or down-regulation of expression measured at the RNA or protein level. Proteins such as kinases and transcription factors (TFs) that exhibit cascade effects are primary candidates for studies in plant stress tolerance. However, resident-protein post-translational modification (PTM), specifically in response to particular conditions such as stress, is a candidate for immediate and potent 'quick reaction force' (QRF) kind of effects. Stress-mediated SUMOylation of TFs and other proteins have been observed. SUMOylation can change the rate of activity, function or location of the modified protein. Early SUMOylation of resident proteins can act in the stress signal transduction or in adaptive response. Here, we consider brief background information on ESRFs to establish the crosstalk between these factors that impinge on PTMs. We then illustrate connections of protein SUMOylation to phytohormones and TFs. Finally, we present results of an in silico analysis of rice Receptor-Like Kinases, heat-shock and calcium-binding proteins to identify members of these gene families, whose basal expression under drought but potential SUMOylation presents them as QRF candidates for roles in stress signaling/response.