TLTC, a T5 exonuclease–mediated low-temperature DNA cloning method

Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease–mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp–25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120 fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5 U of T5 exonuclease at 0°C for 5 min. Then, the mixture was transformed into Escherichia coli to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments

Meta-analysis of the effect of expression of MYB transcription factor genes on abiotic stress

Background MYB proteins are a large group of transcription factors. The overexpression of MYB genes has been reported to improve abiotic stress tolerance in plant. However, due to the variety of plant species studied and the types of gene donors/recipients, along with different experimental conditions, it is difficult to interpret the roles of MYB in abiotic stress tolerance from published data. Methods Using meta-analysis approach, we investigated the plant characteristics involved in cold, drought, and salt stress in MYB-overexpressing plants and analyzed the degrees of influence on plant performance by experimental variables. Results The results show that two of the four measured plant parameters in cold-stressed plants, two of the six in drought-stressed, and four of the 13 in salt-stressed were significantly impacted by MYB overexpression by 22% or more, and the treatment medium, donor/recipient species, and donor type significantly influence the effects of MYB-overexpression on drought stress tolerance. Also, the donor/recipient species, donor type, and stress duration all significantly affected the extent of MYB-mediated salt stress tolerance. In summary, this study compiles and analyzes the data across studies to help us understand the complex interactions that dictate the efficacy of heterologous MYB expression designed for improved abiotic stress tolerance in plants

The tea plant CsLHT1 and CsLHT6 transporters take up amino acids, as a nitrogen source, from the soil of organic tea plantations

Organic tea is more popular than conventional tea that originates from fertilized plants. Amino acids inorganic soils constitute a substantial pool nitrogen (N) available for plants. However, the amino-acid contents in soils of tea plantations and how tea plants take up these amino acids remain largely unknown. In this study, we show that the amino-acid content in the soil of an organic tea plantation is significantly higher than that of a conventional tea plantation. Glutamate, alanine, valine, and leucine were the most abundant amino acids in the soil of this tea plantation. When 15N-glutamate was fed to tea plants, it was efficiently absorbed and significantly increased the contents of other amino acids in the roots. We cloned seven CsLHT genes encoding amino-acid transporters and found that the expression of CsLHT1, CsLHT2, and CsLHT6 in the roots significantly increased upon glutamate feeding. Moreover, the expression of CsLHT1 or CsLHT6 in a yeast amino-acid uptake-defective mutant, 22∆10α, enabled growth on media with amino acids constituting the sole N source. Amino-acid uptake assays indicated that CsLHT1 and CsLHT6 are H+-dependent high- and low-affinity amino-acid transporters, respectively. We further demonstrated that CsLHT1 and CsLHT6 are highly expressed in the roots and are localized to the plasma membrane. Moreover, overexpression of CsLHT1 and CsLHT6 in Arabidopsis significantly improved the uptake of exogenously supplied 15N-glutamate and 15N-glutamine. Taken together, our findings are consistent with the involvement of CsLHT1 and CsLHT6 in amino-acid uptake from the soil, which is particularly important for tea plants grown inorganic tea plantations

Immobilizing catalysts on porous materials

Significant advances in the immobilization of homogeneous and enzyme catalysts on ordered mesoporous materials (OMM), were described. Ordered mesoporous silicas provide excellent opportunities for the immobilization of both homogeneous and enzyme catalysts via covalent binding because of the availability of well-defined silanol groups. A homogeneous catalyst can be immobilized on the surface of OMMs during hydrothermal synthesis. Meanwhile, electrostatic interaction between the homogeneous catalyst and support has been shown to be sufficiently strong to minimize leaching. Enzymes immobilized on OMMs exhibit not only improved stability and reusability, but also enhanced activity

CsICE1 Functions in Cold Tolerance by Regulating Polyamine levels May through Interacting with Arginine Decarboxylase in the Tea Tree

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

Intercropping Cover Crops for a Vital Ecosystem Service: A Review of the Biocontrol of Insect Pests in Tea Agroecosystems

The intercropping of cover crops has been adopted in several agroecosystems, including tea agroecosystems, which promotes ecological intensification. Prior studies have shown that growing cover crops in tea plantations provided different ecological services, including the biocontrol of pests. Cover crops enrich soil nutrients, reduce soil erosion, suppress weeds and insect pests, and increase the abundance of natural enemies (predators and parasitoids). We have reviewed the potential cover crops that can be incorporated into the tea agroecosystem, particularly emphasizing the ecological services of cover crops in pest control. Cover crops were categorized into cereals (buckwheat, sorghum), legumes (guar, cowpea, tephrosia, hairy indigo, and sunn hemp), aromatic plants (lavender, marigold, basil, and semen cassiae), and others (maize, mountain pepper, white clover, round-leaf cassia, and creeping indigo). Legumes and aromatic plants are the most potent cover crop species that can be intercropped in monoculture tea plantations due to their exceptional benefits. These cover crop species improve crop diversity and help with atmospheric nitrogen fixation, including with the emission of functional plant volatiles, which enhances the diversity and abundance of natural enemies, thereby assisting in the biocontrol of tea insect pests. The vital ecological services rendered by cover crops to monoculture tea plantations, including regarding the prevalent natural enemies and their pivotal role in the biocontrol of insect pests in the tea plantation, have also been reviewed. Climate-resilient crops (sorghum, cowpea) and volatile blends emitting aromatic plants (semen cassiae, marigold, flemingia) are recommended as cover crops that can be intercropped in tea plantations. These recommended cover crop species attract diverse natural enemies and suppress major tea pests (tea green leaf hopper, white flies, tea aphids, and mirid bugs). It is presumed that the incorporation of cover crops within the rows of tea plantations will be a promising strategy for mitigating pest attacks via the conservation biological control, thereby increasing tea yield and conserving agrobiodiversity. Furthermore, a cropping system with intercropped cover crop species would be environmentally benign and offer the opportunity to increase natural enemy abundance, delaying pest colonization and/or preventing pest outbreaks for pest management sustainability

Impacts of Ecological Shading by Roadside Trees on Tea Foliar Nutritional and Bioactive Components, Community Diversity of Insects and Soil Microbes in Tea Plantation

Roadside trees not only add aesthetic appeal to tea plantations, but also serve important ecological purposes for the shaded tea plants. In this study, we selected tea orchards with two access roads, from east to west (EW-road) and from south to north (SN-road), and the roadside trees formed three types of ecological shading of the adjoining tea plants; i.e., south shading (SS) by the roadside trees on the EW-road, and east shading and west shading (ES and WS) by the roadside trees on the SN-road. We studied the impacts of ecological shading by roadside trees on the tea plants, insects, and soil microbes in the tea plantation, by measuring the contents of soluble nutrients, bioactive compounds in the tea, and tea quality indices; and by investigating the population occurrence of key species of insects and calculating insect community indexes, while simultaneously assaying the soil microbiome. The results vividly demonstrated that the shading formed by roadside tree lines on the surrounding tea plantation (SS, ES, and WS) had adverse effects on the concentration of tea soluble sugars but enhanced the foliar contents of bioactive components and improved the overall tea quality, in contrast to the no-shading control tea plants. In addition, the roadside tree lines seemed to be beneficial for the tea plantation, as they reduced pest occurrence, and ES shading enhanced the microbial soil diversity in the rhizosphere of the tea plants

Metabolic analyses reveal different mechanisms of leaf color change in two purple-leaf tea plant (Camellia sinensis L.) cultivars

Plant breeding: Coloring tea leaves Researchers in China have uncovered how the leaves of purple-leaf tea varieties acquire their color. Wanping Fang and Xujun Zhu of Nanjing Agricultural University analyzed the biochemical profile and gene expression patterns in the young purple leaves and older green leaves of two purple tea varieties, one new and one previously studied. The purple leaves of both varieties had higher levels of red/purple pigments known as flavonoids, as well as increased expression of genes related to their synthesis. However, only the new variety also had increased chlorophyll levels in green leaves. Both varieties are purple because flavonoids mask the color of chlorophyll, but the process is driven by different mechanisms in each. These findings clarify how tea varieties develop different levels of flavonoids, which affect tea quality due to their antioxidant and antimicrobial activity

Identification of the varietal origin of processed loose-leaf tea based on analysis of a single leaf by SNP nanofluidic array

Tea is an important cash crop, representing a $40 billion-a-year global market. Differentiation of the tea market has resulted in increasing demand for tea products that are sustainably and responsibly produced. Tea authentication is important because of growing concerns about fraud involving premium tea products. Analytical technologies are needed for protection and value enhancement of high-quality brands. For loose-leaf teas, the challenge is that the authentication needs to be established on the basis of a single leaf, so that the products can be traced back to the original varieties. A new generation of molecular markers offers an ideal solution for authentication of processed agricultural products. Using a nanofluidic array to identify variant SNP sequences, we tested genetic identities using DNA extracted from single leaves of 14 processed commercial tea products. Based on the profiles of 60 SNP markers, the genetic identity of each tea sample was unambiguously identified by multilocus matching and ordination analysis. Results for repeated samples of multiple tea leaves from the same products (using three independent DNA extractions) showed 100% concordance, showing that the nanofluidic system is a reliable platform for generating tea DNA fingerprints with high accuracy. The method worked well on green, oolong, and black teas, and can handle a large number of samples in a short period of time. It is robust and cost-effective, thus showing high potential for practical application in the value chain of the tea industry

Impacts of Intercropped Maize Ecological Shading on Tea Foliar and Functional Components, Insect Pest Diversity and Soil Microbes

Ecological shading fueled by maize intercropping in tea plantations can improve tea quality and flavor, and efficiently control the population occurrence of main insect pests. In this study, tea plants were intercropped with maize in two planting directions from east to west (i.e., south shading (SS)) and from north to south (i.e., east shading (ES) and west shading (WS)) to form ecological shading, and the effects on tea quality, and the population occurrence and community diversity of insect pests and soil microbes were studied. When compared with the non-shading control, the tea foliar nutrition contents of free fatty acids have been significantly affected by the ecological shading. SS, ES, and WS all significantly increased the foliar content of theanine and caffeine and the catechin quality index in the leaves of tea plants, simultaneously significantly reducing the foliar content of total polyphenols and the phenol/ammonia ratio. Moreover, ES and WS both significantly reduced the population occurrences of Empoasca onukii and Trialeurodes vaporariorum. Ecological shading significantly affected the composition of soil microbial communities in tea plantations, in which WS significantly reduced the diversity of soil microorganisms