Molecular regulation of anthocyanin discoloration under water stress and high solar irradiance in pluckable shoots of purple tea cultivar.

MAIN CONCLUSION: During water-deficit stress, antioxidant enzymes use anthocyanin molecules as co-substrates to scavenge for reactive oxygen species leading to reduced anthocyanin content and ultimately loss of purple leaf pigmentation in tea. Anthocyanins are an important class of flavonoids responsible for liquor color and market acceptability of processed tea from the anthocyanin-rich purple tea cultivar 'TRFK 306'. However, the color in pluckable shoots fade and turn green during the dry and hot season, before rapidly reverting back to purple when weather is favorably wet and cool/cold. Our study revealed that loss of purple leaf pigmentation correlated well with reduced precipitation, high soil water-deficit, increased intensity and duration of sunlight and temperature. Richly purple pigmented leaves harvested during the cool, wet conditions recorded significantly higher anthocyanin content compared to faded samples harvested during the dry season. Similarly, individual anthocyanins were affected by seasonal changes with malvidin being the most abundant. Comparative transcriptomics of two RNA-seq libraries, dry/discolored and wet/colored seasons, revealed depression of most metabolic processes related to anthocyanin accumulation in dry conditions. Specifically, transcripts encoding pathway regulators, MYB-bHLH-WD40 (MBW) complex, were repressed possibly contributing to the suppression of late biosynthetic genes of the pathway. Further, suppression of anthocyanin transport genes could be linked to reduced accumulation of anthocyanin in the vacuole during the dry season. However, slight increase in expression of some transporter and reactive oxygen species (ROS) antioxidant genes in the discolored leaf suggests non-enzymatic degradation of anthocyanin, ultimately leading to loss of purple color during the dry season. Based on increased expression of ROS antioxidant genes (especially catalase and superoxide dismutase) in the discolored leaf, we speculate that anthocyanins are used as co-substrates by antioxidant enzymes to scavenge for ROS (especially hydrogen peroxide) that escape from organelles, leading to reduced anthocyanins and loss of pigmentation during the dry season.

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.

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.

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.