Asosii Paul

An RNA isolation system for plant tissues rich in secondary metabolites

BackgroundSecondary 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.FindingsAn 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.ConclusionsThe 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.

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 (GA3) modulated expression of selected genes, depending upon if the tissue was harvested during PAG or WD. Tissue preparedness was critical for ABA- and GA3-mediated response, particularly for stress-responsive genes/chaperons. Data identified the common gene networks for winter dormancy, temperature, and plant hormone responses.

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.

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

The QMlike 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.

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.

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.