Search Results (7)

Our previous study showed that COPPER-CONTAINING AMINE OXIDASE (CuAO) and AMINOALDEHYDE DEHYDROGENASE (AMADH) could regulate the accumulation of γ-aminobutyric acid (GABA) in tea through the polyamine degradation pathway. However, their biological function in drought tolerance has not been determined. In this study, Camellia sinensis (Cs) CsCuAO1 associated with CsAMADH1 conferred drought tolerance, which modulated GABA levels in tea plants. The results showed that exogenous GABA spraying effectively alleviated the drought-induced physical damage. Arabidopsis lines overexpressing CsCuAO1 and CsAMADH1 exhibited enhanced resistance to drought, which promoted the synthesis of GABA and putrescine by stimulating reactive oxygen species’ scavenging capacity and stomatal movement. However, the suppression of CsCuAO1 or CsAMADH1 in tea plants resulted in increased sensitivity to drought treatment. Moreover, co-overexpressing plants increased GABA accumulation both in an Agrobacterium-mediated Nicotiana benthamiana transient assay and transgenic Arabidopsis plants. In addition, a GABA transporter gene, CsGAT1, was identified, whose expression was strongly correlated with GABA accumulation levels in different tissues under drought stress. Taken together, CsCuAO1 and CsAMADH1 were involved in the response to drought stress through a dynamic GABA-putrescine balance. Our data will contribute to the characterization of GABA’s biological functions in response to environmental stresses in plants. Full article

Nitrate nitrogen is an important nitrogen source for tea plants’ growth and development. LBD transcription factors play important roles in response to the presence of nitrate in plants. The functional study of LBD transcription factors in tea plants remains limited. In this study, the LBD family gene CsLBD39 was isolated and characterized from tea plants. Sequence analysis indicated that CsLBD39 contained a highly conserved CX₂CX₆CX₃CX domain. The phylogenetic tree assay showed that CsLBD39 belonged to class II subfamily of the LBD family. CsLBD39 was highly expressed in flowers and root; we determined that its expression could be induced by nitrate treatment. The CsLBD39 protein was located in the nucleus and has transcriptional activation activity in yeast. Compared with the wild type, overexpression of CsLBD39 gene in Arabidopsis resulted in smaller rosettes, shorter main roots, reduced lateral roots and lower plant weights. The nitrate content and the expression levels of genes related to nitrate transport and regulation were decreased in transgenic Arabidopsis hosting CsLBD39 gene. Compared with the wild type, CsLBD39 overexpression in transgenic Arabidopsis had smaller cell structure of leaves, shorter diameter of stem cross section, and slender and compact cell of stem longitudinal section. Under KNO₃ treatment, the contents of nitrate, anthocyanins, and chlorophyll in leaves, and the content of nitrate in roots of Arabidopsis overexpressing CsLBD39 were reduced, the expression levels of nitrate transport and regulation related genes were decreased. The results revealed that CsLBD39 may be involved in nitrate signal transduction in tea plants as a negative regulator and laid the groundwork for future studies into the mechanism of nitrate response. Full article

Inherited retinal dystrophies and optic neuropathies cause chronic disabling loss of visual function. The development of recombinant adeno-associated viral vectors (rAAV) gene therapies in all disease fields have been promising, but the translation to the clinic has been slow. The safety and efficacy profiles of rAAV are linked to the dose of applied vectors. DNA changes in the rAAV gene cassette affect potency, the expression pattern (cell-specificity), and the production yield. Here, we present a library of rAAV vectors and elements that provide a workflow to design novel vectors. We first performed a meta-analysis on recombinant rAAV elements in clinical trials (2007–2020) for ocular gene therapies. We analyzed 33 unique rAAV gene cassettes used in 57 ocular clinical trials. The rAAV gene therapy vectors used six unique capsid variants, 16 different promoters, and six unique polyadenylation sequences. Further, we compiled a list of promoters, enhancers, and other sequences used in current rAAV gene cassettes in preclinical studies. Then, we give an update on pro-viral plasmid backbones used to produce the gene therapy vectors, inverted terminal repeats, production yield, and rAAV safety considerations. Finally, we assess rAAV transgene and bioactivity assays applied to cells or organoids in vitro, explants ex vivo, and clinical studies. Full article

Background: The identification of C-repeat binding factor (CBF), and the characterization as an inducer of CBF Expression 1 (ICE1), and a major activator for C-repeat binding factor, were important breakthroughs in the cold signaling network. Methods: In the present study, the full length cDNA of ICE1 was isolated from the tea tree (Camellia sinensis). CsICE1 protein was located in the cell nucleus as revealed by subcellular localization analysis. To investigate the biological functions of CsICE1, a transgenic line fused with the CsICE1 gene in Arabidopsis thaliana (arabidopsis) was generated by the floral dip method. Results: The CsICE1 was expressed differentially in various tea tree tissues, mostly in buds and leaves, and the transcript level of CsICE1 was increased after 1 h and peaked at 2 h under cold treatment. Transcription activity assay indicated that the spermine synthase (SPMS) and arginine decarboxylase (ADC) genes were possible targets of CsICE1. In addition, the values of net photosynthetic rate, transpiration rate, stomatal conductance in transgenic lines declined by less extent than wild-type plants under low temperatures. Furthermore, transcript levels of ADC genes in the transgenic lines had no apparent alteration under normal growth conditions but substantially increased under cold conditions, consistent of changes in free polyamine levels. Taken together, these results demonstrated that CsICE1 plays a positive role in cold tolerance, which may be due to the modulation of polyamine levels through interacting with CsADC. Full article

WRKY transcription factors (TFs) containing one or two WRKY domains are a class of plant TFs that respond to diverse abiotic stresses and are associated with developmental processes. However, little has been known about the function of WRKY gene in tea plant. In this study, a subgroup IId WRKY gene CsWRKY7 was isolated from Camellia sinensis, which displayed amino acid sequence homology with Arabidopsis AtWRKY7 and AtWRKY15. Subcellular localization prediction indicated that CsWRKY7 localized to nucleus. Cis-acting elements detected in the promotor region of CsWRKY7 are mainly involved in plant response to environmental stress and growth. Consistently, expression analysis showed that CsWRKY7 transcripts responded to NaCl, mannitol, PEG, and diverse hormones treatments. Additionally, CsWRKY7 exhibited a higher accumulation both in old leaves and roots compared to bud. Seed germination and root growth assay indicated that overexpressed CsWRKY7 in transgenic Arabidopsis was not sensitive to NaCl, mannitol, PEG, and low concentration of ABA treatments. CsWRKY7 overexpressing Arabidopsis showed a late-flowering phenotype under normal conditions compared to wild type. Furthermore, gene expression analysis showed that the transcription levels of the flowering time integrator gene FLOWERING LOCUS T (FT) and the floral meristem identity genes APETALA1 (AP1) and LEAFY (LFY) were lower in WRKY7-OE than in the WT. Taken together, these findings indicate that CsWRKY7 TF may participate in plant growth. This study provides a potential strategy to breed late-blooming tea cultivar. Full article

Flavan-3-ols and oligomeric proanthocyanidins (PAs) are the main nutritional polyphenols in green tea (Camellia sinensis), which provide numerous benefits to human health. To date, the regulatory mechanism of flavan-3-ol biosynthesis in green tea remains open to study. Herein, we report the characterization of a C. sinensis tryptophan-aspartic acid repeat protein (CsWD40) that interacts with myeloblastosis (MYB) and basic helix-loop-helix (bHLH) transcription factors (TFs) to regulate the biosynthesis of flavan-3-ols. Full length CsWD40 cDNA was cloned from leaves and was deduced to encode 342 amino acids. An in vitro yeast two-hybrid assay demonstrated that CsWD40 interacted with two bHLH TFs (CsGL3 and CsTT8) and two MYB TFs (CsAN2 and CsMYB5e). The overexpression of CsWD40 in Arabidopsis thaliana transparent testa glabra 1 (ttg1) restored normal trichome and seed coat development. Ectopic expression of CsWD40 alone in tobacco resulted in a significant increase in the anthocyanins of transgenic petals. CsWD40 was then coexpressed with CsMYB5e in tobacco plants to increase levels of both anthocyanins and PAs. Furthermore, gene expression analysis revealed that CsWD40 expression in tea plants could be induced by several abiotic stresses. Taken together, these data provide solid evidence that CsWD40 partners with bHLH and MYB TFs to form ternary WBM complexes to regulate anthocyanin, PA biosynthesis, and trichome development. Full article

Epidemiological studies on the effects of green tea consumption (Camellia sinensis) have demonstrated a reduction for the risk of age-related diseases. The investigation of the in vivo and in vitro antioxidant properties of an aqueous extract of green tea (GTE) was the aim of the current study. 2,2-Diphenyl-1-picrylhydrazyl (DPPH^•) and superoxide anion radical (O₂^•−) assays were used to estimate the GTE antioxidant activity. To investigate the protective effects of GTE against oxidative stress, wild-type N2 and transgenic strains (TJ374, hsp-16.2/GFP) of the model organism, Caenorhabditis elegans (C. elegans), were chosen. In the current study, the following catechins were identified by LC/ESI-MS: catechin, epicatechin, epicatechin gallate, gallocatechin, epigallocatechin and epigallocatechin gallate. GTE exhibited a free radical scavenging activity of DPPH^• and O₂^•− with IC₅₀ 8.37 and 91.34 µg/mL, respectively. In the C. elegans strain (TJ374, hsp-16.2/GFP), the expression of hsp-16.2/GFP was induced by a nonlethal dose of juglone, and the fluorescence density of hsp-16.2/GFP was measured. The hsp-16.2/GFP was reduced by 68.43% in the worms pretreated with 100 µg/mL GTE. N2 worms pretreated with 100 µg/mL GTE exhibited an increased survival rate of 48.31% after a lethal dose application of juglone. The results suggest that some green tea constituents are absorbed by the worms and play a substantial role to enhance oxidative stress resistance in C. elegans. Full article