Functional characterization of a serine-threonine protein kinase from Bambusa balcooa that implicates in cellulose overproduction and superior quality fiber formation

BACKGROUND: Molecular markers allow rapid identification of biologically important germplasm/s having desired character. Previously we have reported a genotype specific molecular marker, Balco(1128) [GenBank ID EU258678] of Bambusa balcooa containing an ORF (375 bp) having high similarity with receptor like cytoplasmic kinase of Arabidopsis and Oryza. Balco(1128) was found to be associated only with bamboo genotypes endowed with high cellulose and low lignin contents of fibers. Under the above backdrop, it was necessitated to characterize this genetic marker for better understanding of its biological significance in context of superior quality fiber development. RESULTS: The full length cDNA (3342 bp) of BbKst, a serine-threonine protein kinase was isolated from B. balcooa comprising of six LRR domains at the N-terminal end and a kinase domain at the C-terminal end. Bacteria-expressed BbKst-kinase domain (3339 bp long) showed Mg(2+) dependent kinase activity at pH 7.0, 28°C. Bioinformatics study followed by phospho-amino analysis further confirmed that BbKst-kinase belongs to the serine/threonine protein kinase family. Transcript analysis of the BbKst gene following RNA slot blot hybridization and qPCR revealed higher expression of BbKst during initiation and elongation stages of fiber development. Tissue specific expression studies showed much higher expression of BbKst transcript in stems and internodes of B. balcooa than in leaves and rhizomes. Southern analysis revealed single copy insertion of BbKst in most of the Agrobacterium mediated transgenic tobacco plants. Real-time PCR detected 150-200 fold enhanced expression of BbKst in different T(1) tobacco lines than that of the vector transformed plants. Heterologous expression of BbKst under control of 35S promoter in transgenic tobacco showed high cellulose deposition in the xylem fibers. Number of xylary fibers was higher in transgenic T(0) and T(1) plants than that of empty-vector transformed tobacco plants offering enhanced mechanical strength to the transgenic plants, which was also substantiated by their strong upright phenotypes, significantly higher cellulose contents, flexibility coefficient, slenderness ratio, and lower Runkel ratio of the fibers. CONCLUSIONS: This finding clearly demonstrated that BbKst gene (GenBank ID JQ432560) encodes a serine/threonine protein kinase. BbKst induced higher cellulose deposition/synthesis in transgenic tobacco plants, an important attribute of fiber quality bestowing additional strength to the plant

Histone H3 Lys79 methylation is required for efficient nucleotide excision repair in a silenced locus of Saccharomyces cerevisiae

Methylation of specific histone lysine residues regulates gene expression and heterochromatin function, but little is known about its role in DNA repair. To examine how changes in conserved methylated residues of histone H3 affect nucleotide excision repair (NER), viable H3K4R and H3K79R mutants were generated in Saccharomyces cerevisiae . These mutants show decreased UV survival and impaired NER at the transcriptionally silent HML locus, while maintaining normal NER in the constitutively expressed RPB2 gene and transcriptionally repressed, nucleosome loaded GAL10 gene. Moreover, the HML chromatin in these mutants has reduced accessibility to Micrococcal nuclease (MNase). Importantly, chromatin immunoprecipitation analysis demonstrates there is enhanced recruitment of the Sir complex at the HML locus of these mutants, and deletion of the SIR2 or SIR3 genes restores the MNase accessibility and DNA repair efficiency at this locus. Furthermore, following UV irradiation expression of NER genes in these mutants remains at wild type levels, with the exception of RAD16 which decreases by more than 2-fold. These results indicate that impaired NER occurs in the silenced chromatin of H3K79R and H3K4,79R mutants as a result of increased binding of Sir complexes, which may reduce DNA lesion accessibility to repair enzymes

Domain analysis of a groundnut calcium-dependent protein kinase: nuclear localization sequence in the junction domain is coupled with nonconsensus calcium binding domains

The signature of calcium-dependent protein kinases (CDPKs) is a C-terminal calmodulin-like domain (CaMLD) with four consensus calcium-binding sites. A junction domain (JD) joins the kinase with CaMLD and interacts with them through its autoinhibitory and CaMLD binding subdomains, respectively. We noted several CDPKs additionally have a bipartite nuclear localization signal (NLS) sequence as a subdomain in their JD, and this feature is obligatorily coupled with the absence of consensus calcium-binding sites in their respective CaMLDs. These predicted features are substantiated by undertaking investigations on a CDPK (gi:67479988) isolated from cultured groundnut (Arachis hypogea) cells. This kinase can bind 3.1 mol of Ca2+ under saturating conditions with a considerably high Kd of 392 μm as compared with its canonical counterparts. CD spectroscopic analysis, however, indicates the intramolecular structural changes accompanied with calcium binding to be similar to canonical CDPKs. Attesting to the presence of NLS in the JD, the endogenous kinase is localized in the nucleus of osmotically stressed Arachis cells, and in vitro binding assays indicate the NLS in the JD to interact with nuclear transport factors of the importin family. Homology modeling also indicates the feasibility of interaction of importins with the NLS present in the JD of such CDPKs in their activated form. The possible significance of obligatory coupling between the presence of NLS in the junction domain and atypical calcium binding properties of these CDPKs is discussed in the light of the known mechanisms of activation of these kinases

A Calcium/Calmodulin-regulated Member of the Receptor-like Kinase Family Confers Cold Tolerance in Plants

Cold is a limiting environmental factor that adversely affects plant growth and productivity. Calcium/calmodulin-mediated signaling is believed to play a pivotal role in plant response to cold stress, but its exact role is not clearly understood. Here, we report that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase, is crucial for cold tolerance in plants. CRLK1 has two calmodulin-binding sites with different affinities as follows: one located at residues 369–390 with a K d of 25 n m , and the other located at residues 28–112 with a K d of 160 n m . Calcium/calmodulin stimulated the kinase activity, but the addition of chlorpromazine, a calmodulin antagonist, blocked its stimulation. CRLK1 is mainly localized in the plasma membrane, and its expression is stimulated by cold and hydrogen peroxide treatments. Under normal growth conditions, there is no noticeable phenotypic difference between wild-type and crlk1 knock-out mutant plants. However, as compared with wild-type plants, the crlk1 knock-out mutants exhibited an increased sensitivity to chilling and freezing temperatures. Northern analysis showed that the induction of cold-responsive genes, including CBF1, RD29A, COR15a , and KIN1 in crlk1 mutants, is delayed as compared with wild-type plants. These results indicate that CRLK1 is a positive regulator of cold tolerance in plants. Furthermore, our results suggest that CRLK1 plays a role in bridging calcium/calmodulin signaling and cold signaling

Differential Acetylation of Histone H3 at the Regulatory Region of <i>OsDREB1b</i> Promoter Facilitates Chromatin Remodelling and Transcription Activation during Cold Stress

<div><p>The rice ortholog of DREB1, <i>OsDREB1b</i>, is transcriptionally induced by cold stress and over-expression of <i>OsDREB1b</i> results in increase tolerance towards high salt and freezing stress. This spatio-temporal expression of <i>OsDREB1b</i> is preceded by the change in chromatin structure at the promoter and the upstream region for gene activation. The promoter and the upstream region of <i>OsDREB1b</i> genes appear to be arranged into a nucleosome array. Nucleosome mapping of ∼700bp upstream region of <i>OsDREB1b</i> shows two positioned nucleosomes between −610 to −258 and a weakly positioned nucleosome at the core promoter and the TSS. Upon cold stress, there is a significant change in the nucleosome arrangement at the upstream region with increase in DNaseI hypersensitivity or MNase digestion in the vicinity of <i>cis</i> elements and TATA box at the core promoter. ChIP assays shows hyper-acetylation of histone H3K9 throughout the locus whereas region specific increase was observed in H3K14ac and H3K27ac. Moreover, there is an enrichment of RNA PolII occupancy at the promoter region during transcription activation. There is no significant change in the H3 occupancy in <i>OsDREB1b</i> locus negating the possibility of nucleosome loss during cold stress. Interestingly, cold induced enhanced transcript level of <i>OsDREB1b</i> as well as histone H3 acetylation at the upstream region was found to diminish when stressed plants were returned to normal temperature. The result indicates absolute necessity of changes in chromatin conformation for the transcription up-regulation of <i>OsDREB1b</i> gene in response to cold stress. The combined results show the existence of closed chromatin conformation at the upstream and promoter region of <i>OsDREB1b</i> in the transcription “off” state. During cold stress, changes in region specific histone modification marks promote the alteration of chromatin structure to facilitate the binding of transcription machinery for proper gene expression.</p></div

Change in RNA polymerase and nucleosome occupancy during cold stress.

<p>The relative enrichment of RNA Pol II binding at the promoter and coding region of (A) <i>OsDREB1b</i> and (B) <i>OsDREB2a</i> was determined by ChIP assay using antibody against RNA Pol II CTD (8WG16). The ChIP data was normalised to Actin promoter values. Error bar represent standard error (SE) where number of independent experiments (n) = 3. The significance of the results were analysed by student’s <i>t</i> test and the significant changes (P≤0.05) were marked by *.</p

Change in chromatin structure at the promoter and upstream region of <i>OsDREB1b loci</i> using micrococcal nuclease digestion.

<p>MNase accessibility in control and cold stress treated nuclei (2 Hr and 4 Hr) was detected with quantitative PCR based method. Nuclei were digested with MNase (30 U/ml) for increasing time period as indicated. The isolated DNA was used for PCR reaction with primers specific for promoter and upstream region. The amount of DNA amplified at each time point was normalised to that at time 0 and plotted against time to compare the rate of degradation. The data represented here is a mean of three independent experiments with standard error bars and statistically significant values were marked with *. (A and D) rate of degradation for actin promoter; (B, C, D and E) <i>OsDREB1b locus</i>.</p

Change in DNase I accessibility at the promoter and upstream region of <i>OsDREB1b loci</i>.

<p>Relative DNase I accessibility in control and cold stress treated nuclei (2 Hr and 4 Hr) was detected with PCR based method. Nuclei were digested with DNase I (5 U/ml) for increasing time period (0,3,6,10 min). The isolated DNA was used for PCR reaction with primers specific for promoter and upstream region. The amount of DNA amplified at each time point was normalised to that at time 0 and plotted against time to compare the rate of degradation. The relative rate of accessibility for actin promoter (A and D) and <i>OsDREB1b</i> (B, C, D and E) and The data represented here is a mean of three independent experiments with standard error bars. Statistically significant values were marked with *.</p

Expression profile of <i>OsDREB1b</i> and <i>OsDREB2a</i> gene under cold and high salt stress condition.

<p>The transcript of <i>OsDREB1b</i> and <i>OsDREB2a</i> gene was monitored by northern blot analysis using gene specific probes generated from 3′ end of the gene. The rice actin gene (OSJNBa0005K07) was used as internal control.</p

Alteration of histone H3 modifications during cold stress.

<p>Relative change in Histone H3 acetylation (H3K9ac, H3K14ac, and H3K27ac) during cold stress at (A) region Ia (−232 to −40) and region III (+157 to +307) (B) region Ib (−415 to −246) and region II (−610 to −440) of <i>OsDREB1b</i> gene. (C) Relative change in histone modifications at promoter and upstream region of <i>OsDREB2a</i> during cold stress. Samples were analysed by real time PCR except (A). The mean values for each region were normalised to Actin promoter values. Error bar represent standard error (SE) where number of independent experiments (n) = 3. The significance of the results were analysed by student’s <i>t</i> test and the significant changes (P≤0.05) were marked by *. (C) Western blot showing H3K9ac, H3K14ac and H3K27ac signal in whole cell extract isolated from control and cold stress treated rice seedlings.</p