Membrane localized thaumatin-like protein from tea (CsTLP) enhanced seed yield and the plant survival under drought stress in Arabidopsis thaliana.

Thaumatin-like proteins (TLPs) are pathogenesis-related (PR5) proteins, which are induced in response to various biotic and abiotic stresses. The present work was carried out to clone TLP of Camellia sinensis (CsTLP) and to evaluate the response of transgenic lines of Arabidopsis constitutively expressing CsTLP under drought conditions. Data showed that transgenic lines exhibited lower relative electrolyte leakage and higher water retention capacity as compared to the wild-type (WT) plants under drought stress. In addition, results with confocal microscopy showed CsTLP + GFP fusion protein to be localized in the cell membrane which moved to the intercellular spaces under prolonged drought stress. Expression of CsTLP enhanced seed yield and the plant survival in transgenic lines as compared to the WT plants under drought stress. Results suggested the importance of CsTLP in improving drought tolerance in Arabidopsis.

The α-Crystallin Domain Containing Genes: Identification, Phylogeny and Expression Profiling in Abiotic Stress, Phytohormone Response and Development in Tomato (Solanum lycopersicum).

The α-crystallin domain (ACD) is an ancient domain conserved among all kingdoms. Plant ACD proteins have roles in abiotic stresses, transcriptional regulation, inhibiting virus movement, and DNA demethylation. An exhaustive in-silico analysis using Hidden Markov Model-based conserved motif search of the tomato proteome yielded a total of 50 ACD proteins that belonged to four groups, sub-divided further into 18 classes. One of these groups belongs to the small heat shock protein (sHSP) class of proteins, molecular chaperones implicated in heat tolerance. Both tandem and segmental duplication events appear to have shaped the expansion of this gene family with purifying selection being the primary driving force for evolution. The expression profiling of the Acd genes in two different heat stress regimes suggested that their transcripts are differentially regulated with roles in acclimation and adaptive response during recovery. The co-expression of various genes in response to different abiotic stresses (heat, low temperature, dehydration, salinity, and oxidative stress) and phytohormones (abscisic acid and salicylic acid) suggested possible cross-talk between various members to combat a myriad of stresses. Further, several genes were highly expressed in fruit, root, and flower tissues as compared to leaf signifying their importance in plant development too. Evaluation of the expression of this gene family in field grown tissues highlighted the prominent role they have in providing thermo-tolerance during daily temperature variations. The function of three putative sHSPs was established as holdase chaperones as evidenced by protection to malate-dehydrogenase against heat induced protein-aggregation. This study provides insights into the characterization of the Acd genes in tomato and forms the basis for further functional validation in-planta.

RNA-seq-mediated transcriptome analysis of actively growing and winter dormant shoots identifies non-deciduous habit of evergreen tree tea during winters.

Tea [Camellia sinensis (L.) O. Kuntze] is a perennial tree which undergoes winter dormancy and unlike deciduous trees, the species does not shed its leaves during winters. The present work dissected the molecular processes operating in the leaves during the period of active growth and winter dormancy through transcriptome analysis to understand a long-standing question: why should tea be a non-deciduous species? Analyses of 24,700 unigenes obtained from 57,767 primarily assembled transcripts showed (i) operation of mechanisms of winter tolerance, (ii) down-regulation of genes involved in growth, development, protein synthesis and cell division, and (iii) inhibition of leaf abscission due to modulation of senescence related processes during winter dormancy in tea. These senescence related processes exhibited modulation to favour leaf abscission (i) in deciduous Populus tremula during winters, and (ii) also in tea but under osmotic stress during which leaves also abscise. These results validated the relevance of the identified senescence related processes for leaf abscission and suggested their operation when in need in tea.

A stress-responsive late embryogenesis abundant protein 7 (CsLEA7) of tea [Camellia sinensis (L.) O. Kuntze] encodes for a chaperone that imparts tolerance to Escherichia coli against stresses.

The present study characterized CsLEA7, a group 7 late embryogenesis abundant (LEA) gene, from tea [Camellia sinensis (L.) O. Kuntze]. The gene had an open reading frame of 462 base pairs encoding 153 amino acids with calculated molecular weight of 16.63 kDa and an isoelectric point (pI) of 4.93. Analysis revealed CsLEA7 to be an intrinsically ordered protein consisting of nine ß-strands and two α-helices. CsLEA7 expressed ubiquitously in all the tissues analyzed with highest level of transcripts in mature leaf as compared to in flower bud, younger leaves, stem and fruit. Expression was the least in root tissue. CsLEA7 exhibited up-regulation in response to low temperature, polyethylene glycol-8000, sodium chloride and hydrogen peroxide in tea. Analysis of the promoter of CsLEA7 revealed a core promoter element and distinct cis-acting regulatory elements regulating gene expression under abiotic stresses. CsLEA7 exhibited chaperonic activity as evinced by protection to malate dehydrogenase against heat denaturation assay. Recombinant Escherichia coli cells producing CsLEA7 exhibited improved tolerance against diverse cues: polyethylene glycol-8000, sodium chloride, hydrogen peroxide and low temperature signifying its role in imparting stress tolerance.

Dehydrin2 is a stress-inducible, whereas Dehydrin1 is constitutively expressed but up-regulated gene under varied cues in tea [Camellia sinensis (L.) O. Kuntze].

Two expressed sequence tags with similarity to dehydrin were cloned to full-length (CsDHN1 and CsDHN2) through rapid amplification of cDNA ends. CsDHN1 and CsDHN2 were 1,027 and 992 base pair long and encoded for predicted polypeptides of 260 and 201 amino acids, respectively. Deduced CsDHN1 protein had a S-segment and three lysine-rich consensus motifs (K-segments). The Y-segment was, however, absent in the deduced CsDHN1. CsDHN2 had three Y motifs, one S-segment and two K-segments. Expression of CsDHN1 was visible at all the time points of study, though up-regulation was observed in response to winter dormancy (WD) as well as abiotic stresses [low temperature, sodium chloride, polyethylene glycol, and hydrogen peroxide]. Expression of CsDHN2 was strongly up-regulated within 1 h of exposure to abiotic stresses as well in the tissue harvested during WD in contrast to the respective "control" for abiotic stresses and in tissue during the period of active growth, when the expression was not detectable.

CsNAM-like protein encodes a nuclear localized protein and responds to varied cues in tea [Camellia sinensis (L.) O. Kuntze].

Abiotic stress possesses serious threat to plant distribution and production. In response to stress, plants induce the expression of many genes that function to protect the cellular machinery from stress-induced damages. These genes are largely regulated by specific transcription factors (TFs). NAC family proteins are plant specific TFs implicated in diverse processes including development, and biotic and abiotic stress responses. The present work described (i) cloning of CsNAM-like protein gene from a tree crop tea [Camellia sinensis (L.) O. Kuntze], (ii) its cellular localization, and (iii) regulation of the gene by external cues. The gene had an open reading frame of 873 base pairs encoding 291 amino acids with calculated molecular weight of 33.4 kDa and an isoelectric point (pI) of 6.72. Expression characterization showed the gene to be induced by drought, osmoticum, salt, heat and hydrogen peroxide. During the period of active growth, CsNAM-like protein showed ubiquitous expression in all the tissues analyzed, with higher level of transcripts in stem, flower bud and mature leaf as compared to the root, young leaf and fruit. The common response of CsNAM-like protein to various cues suggests its important role in imparting tolerance against abiotic stress.

A shared response of thaumatin like protein, chitinase, and late embryogenesis abundant protein3 to environmental stresses in tea [Camellia sinensis (L.) O. Kuntze].

Drought poses a significant threat to tree plants including tea [Camellia sinensis (L.) O. Kuntze] that yields a popular beverage "tea." Consequence of drought is heat and salt stress, for which data on molecular response in tree species are not available. The present work analyzed drought-responsive subtracted cDNA libraries of tea to identify drought-responsive genes. Temporal and spatial gene expression suggested the involvement of chaperones as one of the major mechanisms to protect the plant against drought-related damages. A common response of thaumatin like protein, chitinase, and late embryogenesis abundant protein3 across four stresses suggests these to be useful targets to generate "drought stress proof" tea.

Alpha-tubulin (CsTUA) up-regulated during winter dormancy is a low temperature inducible gene in tea [Camellia sinensis (L.) O. Kuntze].

The present manuscript describes cloning and expression characterization of alpha-tubulin (CsTUA) gene in an evergreen tree tea [Camellia sinensis (L.) O. Kuntze] in response to winter dormancy (WD), abiotic stresses (sodium chloride, polyethylene glycol, and hydrogen peroxide) and plant growth regulators [abscisic acid (ABA), gibberellic acid (GA(3)), indole-3-butyric acid (IBA), and 6-benzylaminopurine (BA)]. CsTUA encoded a putative protein of 449 amino acids with a calculated molecular weight of 49.6 kDa and an isoelectric point (pI) of 5.09. CsTUA shared 76-84 and 90-95% identity at nucleotide and amino acid level, respectively with TUA genes from other plant species. During the period of active growth (PAG), CsTUA showed maximum expression in floral buds as compared to leaf, stem, fruit and root. Though the transcript was not detectable in the younger leaf tissue during the PAG, the expression was induced within 24 h of the low temperature (LT) treatment. The expression was not modulated by the plant growth regulators either in the tissue harvested during PAG or during WD. It was interesting to record that the expression of CsTUA was up-regulated in response to sodium chloride, polyethylene glycol, and hydrogen peroxide. Data has been discussed on the possible role of CsTUA in imparting tolerance to stresses including to LT so that the tea does not exhibit deciduous nature during winters.

An improved protocol for the isolation of RNA from roots of tea (Camellia sinensis (L.) O. Kuntze).

Tea, a beverage crop, is a rich source of polyphenols and polysaccharides which greatly attribute to its importance. However, oxidation and precipitation of these compounds during nucleic acids extraction is a limitation to molecular biology and genomic studies. On isolation of total RNA from root tissue using established protocols, difficulties were encountered in terms of purity and quantity of isolated RNA or some of the methods were time-consuming and also yields were low. The present communication combines a phenol-based RNA isolation protocol with a cetyltrimethylammonium bromide-based procedure with appropriate modifications. This protocol successfully isolated RNA from tap root tissue in 2-3 h as compared with 16 h reported by the previous method. Also, RNA yield was higher by more than fourfold. The RNA isolated by this protocol was successfully used for downstream applications such as RT-PCR and the construction of suppression subtractive hybridization library. The developed protocol worked well with other plant tissue with high polyphenols and polysaccharides contents.

Responses to winter dormancy, temperature, and plant hormones share gene networks.

Gene networks modulated in winter dormancy (WD) in relation to temperature and hormone responses were analyzed in tea [Camellia sinensis (L.) O. Kuntze]. Analysis of subtracted cDNA libraries prepared using the RNA isolated from the apical bud and the associated two leaves (two and a bud, TAB) of actively growing (AG) and winter dormant plant showed the downregulation of genes involved in cell cycle/cell division and upregulation of stress-inducible genes including those encoding chaperons during WD. Low temperature (4°C) modulated gene expression in AG cut-shoots in similar fashion as observed in TAB during WD. In tissue harvested during WD, growth temperature (25°C) modulated gene expression in the similar way as observed during the period of active growth (PAG). Abscisic acid (ABA) and gibberellic acid (GA(3)) modulated expression of selected genes, depending upon if the tissue was harvested during PAG or WD. Tissue preparedness was critical for ABA- and GA(3)-mediated response, particularly for stress-responsive genes/chaperons. Data identified the common gene networks for winter dormancy, temperature, and plant hormone responses.

An RNA isolation system for plant tissues rich in secondary metabolites.

BACKGROUND: Secondary metabolites are reported to interfere with the isolation of RNA particularly with the recipes that use guanidinium-based salt. Such interference was observed in isolation of RNA with medicinal plants rheum (Rheum australe) and arnebia (Arnebia euchroma). A rapid and less cumbersome system for isolation of RNA was essential to facilitate any study related to gene expression. FINDINGS: An RNA isolation system free of guanidinium salt was developed that successfully isolated RNA from rheum and arnebia. The method took about 45 min and was successfully evaluated on twenty one tissues with varied secondary metabolites. The A260/280 ratio ranged between 1.8 - 2.0 with distinct 28 S and 18 S rRNA bands visible on a formaldehyde-agarose gel. CONCLUSIONS: The present manuscript describes a rapid protocol for isolation of RNA, which works well with all the tissues examined so far. The remarkable feature was the success in isolation of RNA with those tissues, wherein the most commonly used methods failed. Isolated RNA was amenable to downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), differential display (DD), suppression subtractive hybridization (SSH) library construction, and northern hybridization.

Differential display mediated cloning of anthocyanidin reductase gene from tea (Camellia sinensis) and its relationship with the concentration of epicatechins.

Tea [Camellia sinensis (L.) O. Kuntze] leaves are a major source of epicatechin (EC) and its gallolyl derivatives epicatechin gallate, epigallocatechin and epigallocatechin gallate, collectively known as epicatechins (ECs). Epicatechins are important factors determining tea quality, and they also possess many medicinal properties. To gain further information about the regulation of the biosynthesis of ECs, we cloned the gene encoding anthocyanidin reductase from tea (CsANR) by first quantifying changes in the concentrations of ECs in response to drought, gibberellic acid (GA(3)), abscisic acid (ABA) and wounding treatments, followed by differential display of mRNAs and analysis of those bands exhibiting a change in expression paralleling the treatment-induced changes observed in the EC data. Analysis of 133 bands yielded a partial cDNA of CsANR that was later cloned to the full length by rapid amplification of the cDNA ends. The full-length CsANR (Accession No. AY641729) comprised 1233 bp with an ORF of 1014 bp (from 79 to 1092 bp) encoding a polypeptide of 337 amino acids. Expression of CsANR in an Escherichia coli expression vector yielded a functional protein that catalyzed the conversion of cyanidin to EC in the presence of NADPH. Analysis of ECs and gene expression in leaves at different developmental stages and across five tea clones exhibiting variable concentrations of ECs revealed a positive correlation between concentration of ECs and CsANR expression. Expression of CsANR was down-regulated in response to drought, ABA and GA(3) treatments and up-regulated in response to wounding.

Cloning and differential expression of QM like protein homologue from tea [Camellia sinensis (L.) O. Kuntze].

The QM like protein gene encodes for ribosomal protein L10, which is implicated in tumor suppression, transcription factor regulation, and ribosome stability in yeast and mammals. Present study describes cloning of a full-length QM cDNA (CsQM) from tea leaves using differential display of mRNA followed by rapid amplification of cDNA ends. Expression of CsQM was studied in leaves of different stages of development and under various external cues. CsQM contained an open reading frame of 651 bases, encoding 216 amino acids. CsQM shared 71-87% and 85-91% identity at nucleotide and amino acid sequences, respectively with QM genes isolated from other plant species. During active-growth period of tea, higher expression was observed in apical buds that decreased gradually with increasing age of the leaf. During dormancy season, the expression of CsQM gene was severely down-regulated in all the leaves studied. CsQM transcript was found to be down regulated in response to drought stress and abscisic acid treatment but up-regulated by gibberellic acid treatment. A positive association of CsQM transcript abundance with active cellular growth suggested its role in plant growth and development.