Cloning and Characterization of Limonoid Glucosyltransferase from Kinnow Mandarin (Citrus reticulata Blanco).

Kinnow mandarin (Citrus reticulata Blanco) is a popular citrus crop of northwestern India and it occupies maximum fruit area in Punjab. However, citrus juice processing industry is still suffering from delayed bitterness problem caused mainly by limonoid aglycones such as limonin. In order to study citrus limonoid metabolism, limonoid glucosyltransferase (LGT) gene, which encodes a natural debittering enzyme, was isolated from the fruit tissues of Kinnow mandarin. After confirmation and characterization, its full-length gene sequence (1533 bp) was submitted to National Centre for Biotechnology Information. Citrus reticulata limonoid glucosyltransferase (CrLGT) occupies a position on an independent branch in the largest subgroup and is phylogenetically different from those in other mandarin species like C. unshiu, showing its uniqueness in several features. The transcript expression of CrLGT, evaluated in different tissues such as young leaf, flavedo, albedo, sac covering and seed of Kinnow mandarin during early (90 days after flowering (DAF)), mid (150-210 DAF) and late (240 DAF) fruit developmental stages using semi-quantitative method, showed the highest expression in flavedo. Thus, it was concluded that the isolated LGT gene has an effect on limonoid metabolic engineering in citrus. Overexpression of this gene can reduce the delayed bitterness problem in citrus juice and enhance the accumulation of specific glucosides that have anticancer effects.

In Silico Identification and Validation of Potential microRNAs in Kinnow Mandarin (Citrus reticulata Blanco).

MicroRNAs (miRNAs) are a large family of 19-25 nucleotides, regulatory, non-coding RNA molecules that control gene expression by cleaving or inhibiting the translation of target gene transcripts in animals and plants. Despite the important functions of miRNAs related to regulation of plant growth and development processes, metabolism, and abiotic and biotic stresses, little is known about the disease-related miRNA. Here, we present a new pipeline for miRNA analysis using expressed sequence tags (ESTs)-based bioinformatics approach in Kinnow mandarin, a commercially important citrus fruit crop. For this, 56,041 raw EST sequences of Citrus reticulata Blanco were retrieved from EST database in NCBI through step-by-step filtering and processing methods and 130 miRNAs were predicted. Upon blast with Citrus sinensis transcriptome data, these produced potential targets related to disease resistance proteins, pectin lyase-like superfamily proteins, lateral organ boundaries (LOB) domain-containing proteins 11, and protein phosphatase 2C family proteins, protein kinases, dehydrogenases, and methyltransferases. Majority of the predicted miRNAs were of 22, 23, and 24 nucleotides in length. To validate these computationally predicted miRNA, poly(A)-tailed Reverse Transcription-PCR was applied to detect the expression of seven miRNA which showed disease-related potential targets, in citrus greening diseased leaf tissues in comparison to the healthy tissues of Kinnow mandarin. Our study provides information on regulatory roles of these potential miRNAs for the citrus greening disease development, miRNA targets, and would be helpful for future research of miRNA function in citrus.

Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality.

Drought is a major stress that affects the yield and quality of tea, a widely consumed beverage crop grown in more than 20 countries of the world. Therefore, osmotin gene-expressing transgenic tea plants produced using earlier optimized conditions were evaluated for their tolerance of drought stress and their quality. Improved tolerance of polyethylene glycol-induced water stress and faster recovery from stress were evident in transgenic lines compared with the normal phenotype. Significant improvements in growth under in-vitro conditions were also observed. Besides enhanced reactive oxygen species-scavenging enzyme activity, the transgenic lines contained significantly higher levels of flavan-3-ols and caffeine, key compounds that govern quality and commercial yield of the beverage. The selected transgenic lines have the potential to meet the demands of the tea industry for stress-tolerant plants with higher yield and quality. These traits of the transgenic lines can be effectively maintained for generations because tea is commercially cultivated through vegetative propagation only.

Characterization of novel small RNAs from tea (Camellia sinensis L.).

Small RNAs play important roles in plant development, metabolism, signal transduction and responses to biotic and abiotic stresses by affecting gene expression. Tea (Camellia sinensis L.) is an important commercial crop in the world. To understand the regulatory mechanisms involving small RNAs in tea metabolism, we constructed a small RNA (sRNA) library from its tea drink manufacturing tissue part i.e. topmost two leaves and a bud. For the first time, we isolated and cloned six novel small RNAs candidates from tea. These were predicted to target 67 genes responsible for various important plant functions. Isolated small RNAs were validated through expression analysis in young leaf and old leaf during non-dormant and dormant growth phases of tea. Results suggest the probable role of isolated small RNAs in development and seasonal variations of tea.

Producing low-caffeine tea through post-transcriptional silencing of caffeine synthase mRNA.

In this study, attempt has been made to produce a selected cultivar of tea with low-caffeine content using RNAi technology. The caffeine biosynthetic pathway in tea has been proposed to involve three N-methyltransferases such as xanthosine methyltransferase, 7-N-methylxanthine methyltransferase and 3, 7-dimethylxanthine methyltransferase. Last two steps of caffeine biosynthesis in tea have been known to be catalyzed by a bifunctional enzyme known as caffeine synthase. To suppress the caffeine synthesis in the selected tea [Camellia sinensis (L.) O. Kuntze] cv. Kangra jat, we isolated a partial fragment of caffeine synthase (CS) from the same cultivar and used to design RNAi construct (pFGC1008-CS). Somatic embryos were transformed with the developed construct using biolistic method. Transformed somatic embryos showed reduction in the levels of CS transcript expression as well as in caffeine content. Plants were regenerated from the transformed somatic embryos. Transgenic plants showed a significant suppression of CS transcript expression and also showed a reduction of 44-61% in caffeine and 46-67% in theobromine contents as compared to the controls. These results suggest that the RNAi construct developed here using a single partial fragment of CS gene reduced the expression of the targeted endogenous gene significantly. However, the reduction in theobromine content in addition to caffeine documented the involvement of this single CS in the catalysis of last two methyl transfer steps in caffeine biosynthesis of tea.

Agrobacterium-mediated silencing of caffeine synthesis through root transformation in Camellia sinensis L.

Tea [Camellia sinensis (L.) O. Kuntze] is a perennial and most popular non-alcoholic caffeine-containing beverage crop. Tea has several constraints for its genetic improvement such as its high polyphenolic content and woody perennial nature. The development of transgenic tea is very difficult, laborious, and time taking process. In tea, regeneration requires minimum 8-12 months. In view of this, attempt has been made in this article to develop a rapid, efficient, and quite economical Agrobacterium-mediated root transformation system for tea. The feasibility of the developed protocol has been documented through silencing caffeine biosynthesis. For this, one-month-old tea seedlings were exposed to fresh wounding at the elongation zone of roots and were inoculated with Agrobacterium tumefaciens cultures carrying a RNAi construct (pFGC1008-CS). The pFGC1008-CS contained 376 bp of caffeine synthase (CS) cDNA fragment in sense and antisense direction with an intron in between. This has made the RNAi construct to produce a hairpin RNA (ihpRNA). The suppressed expression of CS gene and a marked reduction in caffeine and theobromine contents in young shoots of tea seedlings were obtained after root transformation through Agrobacterium infiltration. Such transformation system could be useful for functional analysis of genes in tea like woody and perennial plants.

A CsGS is regulated at transcriptional level during developmental stages and nitrogen utilization in Camellia sinensis (L.) O. Kuntze.

Glutamine synthetase is a very important enzyme of ammonium assimilation in plants. Here, we report on the regulation of a cytosolic glutamine synthetase (CsGS) from Camellia sinensis (L.) O. Kuntze during developmental stages and light/dark conditions on the utilization of nitrate and ammonia. The CsGS expression levels decreased during dormancy compared to non-dormancy phase of growth. Different leaf positions present different ages of the leaf and CsGS expression level was highest in apical bud (youngest leaf) and lower in mature fourth leaf, suggesting transcriptional regulation of CsGS during developmental stages. The CsGS enzyme activity showed similar trend to that of expression during developmental stages. The nitrate, ammonium and total amino acid contents were increased upon exposure to both N-sources during light and dark conditions. During light conditions, expression of CsGS in apical bud increased upon exposure to both N-sources nitrate and ammonium. While during dark conditions expression was increased only by ammonium and nitrate had no influence. Exposure to both N-sources also showed enhancing effect on CsGS enzyme activity during light. Under dark, ammonium application increased CsGS enzyme activity and nitrate had inhibitory effect on the activity. Results suggest the transcriptional regulation of CsGS on N-utilization during light/dark conditions.

Caffeine biosynthesis and degradation in tea [Camellia sinensis (L.) O. Kuntze] is under developmental and seasonal regulation.

To study caffeine biosynthesis and degradation, here we monitored caffeine synthase gene expression and caffeine and allantoin content in various tissues of four Camellia sinensis (L.) O. Kuntze cultivars during non-dormant (ND) and dormant (D) growth phases. Caffeine synthase expression as well as caffeine content was found to be higher in commercially utilized tissues like apical bud, 1st leaf, 2nd leaf, young stem, and was lower in old leaf during ND compared to D growth phase. Among fruit parts, fruit coats have higher caffeine synthase expression, caffeine content, and allantoin content. On contrary, allantoin content was found lower in the commercially utilized tissues and higher in old leaf. Results suggested that caffeine synthesis and degradation in tea appears to be under developmental and seasonal regulation.

Cloning and characterization of a cytosolic glutamine synthetase from Camellia sinensis (L.) O. Kuntze that is upregulated by ABA, SA, and H2O2.

A cDNA encoding glutamine synthetase, one of the enzymes of the GS/GOGAT pathway, was cloned from Camellia sinensis (CsGS). The isolated cDNA consists of 1,071 nucleotides encoding a polypeptide of 356 amino acids with an estimated isoelectric point of 6.13. The recombinant protein purified from Escherichia coli using Ni-NTA affinity chromatography showed molecular mass of 39.2 kDa. The purified protein was confirmed by blotting with anti-His antibodies. Catalytic parameters of the protein were determined using glutamate and ATP as substrates. The observed Km was 9 mM and Vmax was 93 U/mg protein with glutamate as substrate, while with ATP Km and Vmax values were 6 mM and 70 U/mg protein, respectively. Purified enzyme showed pH optima at 8. Cations were found to be showing enhancing effect on the activity of GS enzyme and Mg2+ ion exhibited maximum enhancing effect among the various ions used in this study. This enzyme activity increased by 25% in presence of DTT and decreased by 18% when incubated with PMSF. Transcript analysis in tea bud, youngest leaf, showed that CsGS gene expression is stimulated in response to abscisic acid (ABA), salicylic acid (SA), and hydrogen peroxide (H2O2), while gibberellic acid (GA3) has no influence on its expression levels.

Cadmium induced oxidative stress influence on glutathione metabolic genes of Camellia sinensis (L.) O. Kuntze.

Glutathione, a tripeptide with sulfhydryl (-SH) group is a very crucial compound primarily involved in redox balance maintenance of the cellular environment. In this study, we monitored the influence of Cd exposure on the transcript levels of glutathione metabolic genes in bud tissues, the youngest leaf, of Camellia sinensis L. In addition, some physiochemical parameters were also studied. Cd exposure decreased chlorophyll and protein contents, while increase was observed in lipid peroxidation upon Cd treatments. These changes were found to be concentration and duration dependent, indicating the occurrence of oxidative stress upon Cd exposure. The transcript levels of glutathione biosynthetic genes viz. gamma-glutamylcysteine synthetase (gamma-ECS) and glutathione synthetase (GSHS) increased upon Cd exposure. Furthermore, transcript levels of glutathione reductase (GR), an enzyme involved in reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), also showed upregulation on Cd exposure. However, the transcript levels of glutathione-S-transferase (GST), an enzyme involved in forming metal-GSH complex and help in sequestration of high levels of metal ions to vacuole, did not show any change on Cd treatment. This study document that Cd exposure induces oxidative stress in Camellia sinensis and the upregulation in transcript levels of glutathione metabolic genes except GST have suggested the role of these enzymes in the protection of plants from high level Cd exposure.