R-loops act as regulatory switches modulating transcription of COLD-responsive genes in rice.

COLD is a major naturally occurring stress that usually causes complex symptoms and severe yield loss in crops. R-loops function in various cellular processes, including development and stress responses, in plants. However, how R-loops function in COLD responses is largely unknown in COLD susceptible crops like rice (Oryza sativa L.). We conducted DRIP-Seq along with other omics data (RNA-Seq, DNase-Seq and ChIP-Seq) in rice with or without COLD treatment. COLD treatment caused R-loop reprogramming across the genome. COLD-biased R-loops had higher GC content and novel motifs for the binding of distinct transcription factors (TFs). Moreover, R-loops can directly/indirectly modulate the transcription of a subset of COLD-responsive genes, which can be mediated by R-loop overlapping TF-centered or cis-regulatory element-related regulatory networks and lncRNAs, accounting for c. 60% of COLD-induced expression of differential genes in rice, which is different from the findings in Arabidopsis. We validated two R-loop loci with contrasting (negative/positive) roles in the regulation of two individual COLD-responsive gene expression, as potential targets for enhanced COLD resistance. Our study provides detailed evidence showing functions of R-loop reprogramming during COLD responses and provides some potential R-loop loci for genetic and epigenetic manipulation toward breeding of rice varieties with enhanced COLD tolerance.

Brassinosteroids participate in the control of basal and acquired freezing tolerance of plants.

Brassinosteroids (BRs) are growth-promoting plant hormones that play a role in abiotic stress responses, but molecular modes that enable this activity remain largely unknown. Here we show that BRs participate in the regulation of freezing tolerance. BR signaling-defective mutants of Arabidopsis thaliana were hypersensitive to freezing before and after cold acclimation. The constitutive activation of BR signaling, in contrast, enhanced freezing resistance. Evidence is provided that the BR-controlled basic helix-loop-helix transcription factor CESTA (CES) can contribute to the constitutive expression of the C-REPEAT/DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR (CBF) transcriptional regulators that control cold responsive (COR) gene expression. In addition, CBF-independent classes of BR-regulated COR genes are identified that are regulated in a BR- and CES-dependent manner during cold acclimation. A model is presented in which BRs govern different cold-responsive transcriptional cascades through the posttranslational modification of CES and redundantly acting factors. This contributes to the basal resistance against freezing stress, but also to the further improvement of this resistance through cold acclimation.

Cold stress-induced changes in metabolism of carbonyl compounds and membrane fatty acid composition in chickpea.

In this study, changes in membrane fatty acid (FA) composition and damage indices contents as well as the transcript patterns of carbonyl-detoxifying genes were evaluated in two chickpea (Cicer arietinum L.) genotypes, cold-tolerant Sel96th11439 and cold-sensitive ILC533 under cold stress (CS; 4 °C). During CS, H2O2 and malondialdehyde (MDA) contents increased (by 47% and 57%, respectively) in the sensitive genotype, while these contents remained unchanged in the tolerant genotype. In tolerant plants, higher content of linoleic, linolenic, unsaturated FAs (UFAs), total FAs and double bond index (DBI) (by 23, 21, 19, 17 and 9%, respectively) was observed at 6 days after stress (DAS) compared to sensitive plants, which, along with alterations of the damage indices, indicate their enhanced tolerance to CS. Compared with the sensitive genotype, less lipoxygenase (LOX) activity (by 59%) in the tolerant genotype was accompanied by decreased MDA and increased levels of UFAs and DBI during CS, particularly at 6 DAS. Upregulation of aldehyde dehydrogenase and aldo-keto reductase genes (by 9- and 10-fold, respectively) at 1 DAS, along with the enhanced transcript levels of aldehyde reductase and 2-alkenal reductase (by 3- and 14.7-fold, respectively) at 6 DAS were accompanied by increased UFAs and reduced MDA contents in the tolerant genotype. Overall, the results suggest that cold tolerance in chickpea was partly associated with regulation of membrane FA compositions and the potential metabolic networks involved in synthesis and degradation of carbonyl compounds.

Biochemical responses of rice roots to cold stress.

BACKGROUND: Cold stress is the main factor that reduces rice yield in subtropical areas, especially at the seedling stage. Most of the current studies on cold stress focus the responses of rice shoots to cold stress. Limited studies are available on that of rice roots to cold stress. This study aimed to illustrate the biochemical responses of rice root under cold treatment, and subject to the establishment of cold stress-related biochemical traits for rice breeding or cropping-adjustment. RESULTS: Our results showed that the growth of rice seedling diminished under cold stress with difference extents among eight rice cultivars of most productive in Taiwan. Under cold treatments, the tested cultivars with higher growth rate had a higher level of hydrogen peroxide (H2O2) in the shoots but had a lower level in the roots. In contrast, the tested cultivates with low growth rate had higher levels of H2O2 in the roots but a lower level in the shoots. Meanwhile, higher MDA contents and higher cell-damage related electrolyte leakage were also found in the roots not in the shoots, suggesting that cold stress might induce oxidative stress in the roots, not in the shoots. Furthermore, the activity analysis of four antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), ascorbic peroxidase (APX), and glutathione reductase (GR), revealed that cold stress could increase SOD and CAT activities in the roots. CONCLUSIONS: In summary, low H2O2 and low MDA contents along with lower SOD and CAT activities in rice root could be the biochemical traits of cold responses in rice seedlings. The results are hoping to have a contribution to the rice breeding or cropping-adjustment on cold tolerance.

cDNA-AFLP analysis of transcripts induced in chickpea plants by TiO2 nanoparticles during cold stress.

We evaluated the effect of TiO2 nanoparticles (NPs) on cold tolerance (CT) development in two chickpea (Cicer arietinum L.) genotypes (Sel96Th11439, cold tolerant, and ILC533, cold susceptible) by using cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique during the first and sixth days of cold stress (CS) at 4 °C. Selective amplification by primer combinations generated 4200 transcript-derived fragments (TDFs) while 100 of them (2.62%) were differentially expressed. During CS, 60 differentially expressed TDFs of TiO2 NPs-treated plants were cloned and 10 of them produced successfully readable sequences. These data represented different groups of genes involved in metabolism pathways, cellular defense, cell connections and signaling, transcriptional regulation and chromatin architecture. Two out of 10 TDFs were unknown genes with uncharacterized functions or sequences without homology to known ones. The network-based analysis showed a gene-gene relationship in response to CS. Quantitative reverse-transcriptase polymerase chain reaction (qPCR) confirmed differential expression of identified genes (six out of 10 TDFs) with potential functions in CT and showed similar patterns with cDNA-AFLP results. An increase in transcription level of these TDFs, particularly on the first day of CS, was crucial for developing CT through decreasing electrolyte leakage index (ELI) content in tolerant plants compared to susceptible ones, as well as in TiO2 NPs-treated plants compared to control ones. It could also indicate probable role of TiO2 NPs against CS-induced oxidative stress. Therefore, a new application of TiO2 NPs in CT development is suggested for preventing or controlling the damages in field conditions and increasing crop productivity.

DNA methylation and physio-biochemical analysis of chickpea in response to cold stress.

Cold stress (CS) signals are translated into physiological changes as products of direct and/or indirect of gene expression regulated by different factors like DNA methylation. In this study, some of these factors were comparatively studied in two chickpea (Cicer arietinum L.) genotypes (Sel96Th11439, cold-tolerant genotype, and ILC533, cold susceptible one) under control (23 °C) and days 1, 3, and 6 after exposing the seedlings to CS (4 °C). Under CS, tolerant genotype prevented H2O2 accumulation which led to a decrease in damage indices (malondialdehyde and electrolyte leakage index) compared to susceptible one. The significant activities of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, and polyphenol oxidase) along with a significant proportion of change in DNA methylation/demethylation patterns were often effective factors in preserving cell against cold-induced oxidative stress. Chickpea cells in response to CS changed access to their genome as the number of bands without change from day 1 to day 6 of exposure to CS particularly in tolerant genotype was decreased. During CS, the methylation level was higher compared to demethylation (29.05 vs 19.79 %) in tolerant genotype and (27.92 vs 22.09 %) in susceptible one. However, for prolonged periods of CS, changes in demethylated bands in tolerant genotype were higher than that of in susceptible one (9.24 vs 4.13 %), indicating higher potential for activation of CS responsive genes. Such a status along with higher activity of antioxidants and less damage indices could be related to cold tolerance (CT) mechanisms in chickpea. Sequencing analysis confirmed the important role of some specific DNA sequences in creating CT with possible responsive components involved in CS. Thus, dynamic assessment using multi-dimensional approaches allows us to progressively fill in the gaps between physio-biochemical and molecular events in creating CT, to comprehend better the nature of the plant stress response and molecular mechanisms behind.