A tau class glutathione S-transferase in tea plant, CsGSTU45, facilitates tea plant susceptibility to Colletotrichum camelliae infection mediated by jasmonate signaling pathway.

Tea plant [Camellia sinensis (L.) O. Kuntze], as one of the most important commercial crops, frequently suffers from anthracnose caused by Colletotrichum camelliae. The plant-specific tau (U) class of glutathione S-transferases (GSTU) participates in ROS homeostasis. Here, we identified a plant-specific GST tau class gene from tea plant, CsGSTU45, which is induced by various stresses, including C. camelliae infection, by analyzing multiple transcriptomes. CsGSTU45 plays a negative role in disease resistance against C. camelliae by accumulating H2 O2 . JA negatively regulates the resistance of tea plants against C. camelliae, which depends on CsGSTU45. CsMYC2.2, which is the key regulator in the JA signaling pathway, directly binds to and activates the promoter of CsGSTU45. Furthermore, silencing CsMYC2.2 increased disease resistance associated with reduced transcript and protein levels of CsGSTU45, and decreased contents of H2 O2 . Therefore, CsMYC2.2 suppresses disease resistance against C. camelliae by binding to the promoter of the CsGSTU45 gene and activating CsGSTU45. CsJAZ1 interacts with CsMYC2.2. Silencing CsJAZ1 attenuates disease resistance, upregulates the expression of CsMYC2.2 elevates the level of the CsGSTU45 protein, and promotes the accumulation of H2 O2 . As a result, CsJAZ1 interacts with CsMYC2.2 and acts as its repressor to suppress the level of CsGSTU45 protein, eventually enhancing disease resistance in tea plants. Taken together, the results show that the JA signaling pathway mediated by CsJAZ1-CsMYC2.2 modulates tea plant susceptibility to C. camelliae by regulating CsGSTU45 to accumulate H2 O2 .

HN-PPISP: a hybrid network based on MLP-Mixer for protein-protein interaction site prediction.

MOTIVATION: Biological experimental approaches to protein-protein interaction (PPI) site prediction are critical for understanding the mechanisms of biochemical processes but are time-consuming and laborious. With the development of Deep Learning (DL) techniques, the most popular Convolutional Neural Networks (CNN)-based methods have been proposed to address these problems. Although significant progress has been made, these methods still have limitations in encoding the characteristics of each amino acid in protein sequences. Current methods cannot efficiently explore the nature of Position Specific Scoring Matrix (PSSM), secondary structure and raw protein sequences by processing them all together. For PPI site prediction, how to effectively model the PPI context with attention to prediction remains an open problem. In addition, the long-distance dependencies of PPI features are important, which is very challenging for many CNN-based methods because the innate ability of CNN is difficult to outperform auto-regressive models like Transformers. RESULTS: To effectively mine the properties of PPI features, a novel hybrid neural network named HN-PPISP is proposed, which integrates a Multi-layer Perceptron Mixer (MLP-Mixer) module for local feature extraction and a two-stage multi-branch module for global feature capture. The model merits Transformer, TextCNN and Bi-LSTM as a powerful alternative for PPI site prediction. On the one hand, this is the first application of an advanced Transformer (i.e. MLP-Mixer) with a hybrid network for sequence-based PPI prediction. On the other hand, unlike existing methods that treat global features altogether, the proposed two-stage multi-branch hybrid module firstly assigns different attention scores to the input features and then encodes the feature through different branch modules. In the first stage, different improved attention modules are hybridized to extract features from the raw protein sequences, secondary structure and PSSM, respectively. In the second stage, a multi-branch network is designed to aggregate information from both branches in parallel. The two branches encode the features and extract dependencies through several operations such as TextCNN, Bi-LSTM and different activation functions. Experimental results on real-world public datasets show that our model consistently achieves state-of-the-art performance over seven remarkable baselines. AVAILABILITY: The source code of HN-PPISP model is available at https://github.com/ylxu05/HN-PPISP.

LUX ARRHYTHMO links CBF pathway and jasmonic acid metabolism to regulate cold tolerance of tea plants.

Cold stress declines the quality and yield of tea, yet the molecular basis underlying cold tolerance of tea plants (Camellia sinensis) remains largely unknown. Here, we identified a circadian rhythm component LUX ARRHYTHMO (LUX) that potentially regulates cold tolerance of tea plants through a genome-wide association study and transcriptomic analysis. The expression of CsLUX phased with sunrise and sunset and was strongly induced by cold stress. Genetic assays indicated that CsLUX is a positive regulator of freezing tolerance in tea plants. CsLUX was directly activated by CsCBF1 and repressed the expression level of CsLOX2, which regulates the cold tolerance of tea plants through dynamically modulating jasmonic acid content. Furthermore, we showed that the CsLUX-CsJAZ1 complex attenuated the physical interaction of CsJAZ1 with CsICE1, liberating CsICE1 with transcriptional activities to withstand cold stress. Notably, a single-nucleotide variation of C-to-A in the coding region of CsLUX was functionally validated as the potential elite haplotype for cold response, which provided valuable molecular markers for future cold resistance breeding in tea plants.

Transcriptomic analysis of hub genes regulating albinism in light- and temperature-sensitive albino tea cultivars 'Zhonghuang 1' and 'Zhonghuang 2'.

Albino tea cultivars have high economic value because their young leaves contain enhanced free amino acids that improve the quality and properties of tea. Zhonghuang 1 (ZH1) and Zhonghuang 2 (ZH2) are two such cultivars widely planted in China; however, the environmental factors and molecular mechanisms regulating their yellow-leaf phenotype remain unclear. In this study, we demonstrated that both ZH1 and ZH2 are light- and temperature-sensitive. Under natural sunlight and low-temperature conditions, their young shoots were yellow with decreased chlorophyll and an abnormal chloroplast ultrastructure. Conversely, young shoots were green with increased chlorophyll and a normal chloroplast ultrastructure under shading and high-temperature conditions. RNA-seq analysis was performed for high light and low light conditions, and pairwise comparisons identified genes exhibiting different light responses between albino and green-leaf cultivars, including transcription factors, cytochrome P450 genes, and heat shock proteins. Weighted gene coexpression network analyses of RNA-seq data identified the modules related to chlorophyll differences between cultivars. Genes involved in chloroplast biogenesis and development, light signaling, and JA biosynthesis and signaling were typically downregulated in albino cultivars, accompanied by a decrease in JA-ILE content in ZH2 during the albino period. Furthermore, we identified the hub genes that may regulate the yellow-leaf phenotype of ZH1 and ZH2, including CsGDC1, CsALB4, CsGUN4, and a TPR gene (TEA010575.1), which were related to chloroplast biogenesis. This study provides new insights into the molecular mechanisms underlying leaf color formation in albino tea cultivars.

Thermoreflectance-based thermometry of silicon thin films with resonantly enhanced temperature sensitivity.

We demonstrate a thermoreflectance-based thermometry technique with an ultimate temperature resolution of 60 µK in a 2.6 mHz bandwidth. This temperature resolution was achieved using a 532 nm-wavelength probe laser and a ∼1 µm-thick silicon transducer film with a thermoreflectance coefficient of -4.7 × 10-3 K-1 at room temperature. The thermoreflectance sensitivity reported here is over an order-of-magnitude greater than that of metal transducers, and is comparable to the sensitivity of traditional resistance thermometers. Supporting calculations reveal that the enhancement in sensitivity is due to optical interference in the thin film.

MMDB: Multimodal dual-branch model for multi-functional bioactive peptide prediction.

Bioactive peptides can hinder oxidative processes and microbial spoilage in foodstuffs and play important roles in treating diverse diseases and disorders. While most of the methods focus on single-functional bioactive peptides and have obtained promising prediction performance, it is still a significant challenge to accurately detect complex and diverse functions simultaneously with the quick increase of multi-functional bioactive peptides. In contrast to previous research on multi-functional bioactive peptide prediction based solely on sequence, we propose a novel multimodal dual-branch (MMDB) lightweight deep learning model that designs two different branches to effectively capture the complementary information of peptide sequence and structural properties. Specifically, a multi-scale dilated convolution with Bi-LSTM branch is presented to effectively model the different scales sequence properties of peptides while a multi-layer convolution branch is proposed to capture structural information. To the best of our knowledge, this is the first effective extraction of peptide sequence features using multi-scale dilated convolution without parameter increase. Multimodal features from both branches are integrated via a fully connected layer for multi-label classification. Compared to state-of-the-art methods, our MMDB model exhibits competitive results across metrics, with a 9.1% Coverage increase and 5.3% and 3.5% improvements in Precision and Accuracy, respectively.

Helmet-Roled Molecules Carrying Double Metronidazole Frameworks and Phenyl Ring for Strengthening Adsorption and Anticorrosion on Mild Steel.

This study prepared new helmet-roled molecules (HMs) carrying metronidazole frameworks and a phenyl ring for strengthening adsorption and anticorrosion on mild steel. The adsorption of the HMs on the copper surface was understood by material simulation computation. Furthermore, the surface analysis experiments suggest that the studied molecules could be adsorbed to a mild steel surface through the chemical coordination bonding. The remarkable corrosion resistance of the HMs for mild steel in HCl was surveyed by potentiodynamic polarization and electrochemical impedance spectroscopy at 298 K. The HMs including two metronidazole skeletons displayed the stronger corrosion inhibition effect on mild steel than the HM1 bearing one single metronidazole part (the corrosion inhibition efficiency, HM3, 98.03%, HM2, 95.14%, HM1, 88.72%). The results presented in this study provided an efficient strategy to develop new clinical medicine-based corrosion inhibitors for metal in acid medium through molecular preconstruction.

Integrative transcriptome and whole-genome bisulfite sequencing analyses of a temperature-sensitive albino tea plant cultivar.

Green tea made from albino buds and leaves has a strong umami taste and aroma. The cultivar 'Zhonghuang 2' (ZH2, Camellia sinensis) is a natural mutant with young shoots that are yellow in spring and green or yellow-green in summer. However, the mechanism of leaf color change remains unclear. Here, we found that young shoots of ZH2 were yellow at low temperature (LT) and green at high temperature (HT), indicating that ZH2 is a temperature-sensitive cultivar. Transmission electron microscopy analysis showed that the grana in the chloroplasts of young shoots grown at LT were poorly stacked, which caused a lack of photoreactions and chlorophyll. RNA-seq results showed 1279 genes differentially expressed in the young shoots grown at LT compared with those at HT, including genes related to cytochrome synthesis, chloroplast development, photosynthesis, and DNA methylation. A whole-genome bisulfite sequencing assay revealed that the dynamics of DNA methylation levels in the CG, CHG, and CHH contexts decreased under LT, and the change was most obvious in the CHH context. Furthermore, 72 genes showed significant changes in both expression and DNA methylation levels, and most of them were related to cytochrome synthesis, chloroplast development, photosynthesis, transcription factors, and signaling pathways. These results demonstrate that DNA methylation is involved in the LT-regulated albino processes of ZH2. Changes in DNA methylation levels were associated with changes in gene expression levels, affecting the structure and function of chloroplasts, which may have a phenotypic impact on shoot and leaf color.

Developing a predictive model for clinically significant prostate cancer by combining age, PSA density, and mpMRI.

PURPOSE: The study aimed to construct a predictive model for clinically significant prostate cancer (csPCa) and investigate its clinical efficacy to reduce unnecessary prostate biopsies. METHODS: A total of 847 patients from institute 1 were included in cohort 1 for model development. Cohort 2 included a total of 208 patients from institute 2 for external validation of the model. The data obtained were used for retrospective analysis. The results of magnetic resonance imaging were obtained using Prostate Imaging Reporting and Data System version 2.1 (PI-RADS v2.1). Univariate and multivariate analyses were performed to determine significant predictors of csPCa. The diagnostic performances were compared using the receiver operating characteristic (ROC) curve and decision curve analyses. RESULTS: Age, prostate-specific antigen density (PSAD), and PI-RADS v2.1 scores were used as predictors of the model. In the development cohort, the areas under the ROC curve (AUC) for csPCa about age, PSAD, PI-RADS v2.1 scores, and the model were 0.675, 0.823, 0.875, and 0.938, respectively. In the external validation cohort, the AUC values predicted by the four were 0.619, 0.811, 0.863, and 0.914, respectively. Decision curve analysis revealed that the clear net benefit of the model was higher than PI-RADS v2.1 scores and PSAD. The model significantly reduced unnecessary prostate biopsies within the risk threshold of > 10%. CONCLUSIONS: In both internal and external validation, the model constructed by combining age, PSAD, and PI-RADS v2.1 scores exhibited excellent clinical efficacy and can be utilized to reduce unnecessary prostate biopsies.

Operative management of trigeminal schwannomas: based on a modified classification in a study of 93 cases.

BACKGROUND: Advances in microscopic and endoscopic surgical techniques have outpaced traditional classification and transcranial surgical strategies, especially with reference to the treatment of trigeminal schwannomas (TSs). A modified TS classification is proposed and appropriate surgical strategies are discussed. METHODS: The cases of 93 patients who underwent surgical treatment in Beijing Tiantan Hospital in the previous 6 years were analyzed retrospectively, and a literature review was conducted. RESULTS: Classification is based on surgical direction. Tumors were classified as follows: type A, backward orientation, located in the orbit or orbit and middle cranial fossa (8 cases, 8.6%); type B, upward orientation, located in the pterygopalatine fossa, infratemporal fossa or pterygopalatine fossa, infratemporal fossa, and middle cranial fossa (23 cases, 24.7%); type C, forward and backward orientations, located in the middle cranial fossa, posterior cranial fossa or both (58 cases, 62.4%); and type D, located in multiple regions (4 cases, 4.3%). 91.40% of patients underwent gross total resection (GTR) with 29 cases receiving endoscopic resection of whom 93.10% (27/29) experienced GTR. CONCLUSION: The 93 cases were satisfactorily divided into four types, according to tumor location and surgical orientation, enabling safe and effective removal by appropriate surgery.

CsCIPK11-Regulated Metalloprotease CsFtsH5 Mediates the Cold Response of Tea Plants.

Photosystem II repair in chloroplasts is a critical process involved in maintaining a plant's photosynthetic activity under cold stress. FtsH (filamentation temperature-sensitive H) is an essential metalloprotease that is required for chloroplast photosystem II repair. However, the role of FtsH in tea plants and its regulatory mechanism under cold stress remains elusive. In this study, we cloned a FtsH homolog gene in tea plants, named CsFtsH5, and found that CsFtsH5 was located in the chloroplast and cytomembrane. RT-qPCR showed that the expression of CsFtsH5 was increased with leaf maturity and was significantly induced by light and cold stress. Transient knockdown CsFtsH5 expression in tea leaves using antisense oligonucleotides resulted in hypersensitivity to cold stress, along with higher relative electrolyte leakage and lower Fv/Fm values. To investigate the molecular mechanism underlying CsFtsH5 involvement in the cold stress, we focused on the calcineurin B-like-interacting protein kinase 11 (CsCIPK11), which had a tissue expression pattern similar to that of CsFtsH5 and was also upregulated by light and cold stress. Yeast two-hybrid and dual luciferase (Luc) complementation assays revealed that CsFtsH5 interacted with CsCIPK11. Furthermore, the Dual-Luc assay showed that CsCIPK11-CsFtsH5 interaction might enhance CsFtsH5 stability. Altogether, our study demonstrates that CsFtsH5 is associated with CsCIPK11 and plays a positive role in maintaining the photosynthetic activity of tea plants in response to low temperatures.

Transcriptome Analysis Reveals That Ascorbic Acid Treatment Enhances the Cold Tolerance of Tea Plants through Cell Wall Remodeling.

Cold stress is a major environmental factor that adversely affects the growth and productivity of tea plants. Upon cold stress, tea plants accumulate multiple metabolites, including ascorbic acid. However, the role of ascorbic acid in the cold stress response of tea plants is not well understood. Here, we report that exogenous ascorbic acid treatment improves the cold tolerance of tea plants. We show that ascorbic acid treatment reduces lipid peroxidation and increases the Fv/Fm of tea plants under cold stress. Transcriptome analysis indicates that ascorbic acid treatment down-regulates the expression of ascorbic acid biosynthesis genes and ROS-scavenging-related genes, while modulating the expression of cell wall remodeling-related genes. Our findings suggest that ascorbic acid treatment negatively regulates the ROS-scavenging system to maintain ROS homeostasis in the cold stress response of tea plants and that ascorbic acid's protective role in minimizing the harmful effects of cold stress on tea plants may occur through cell wall remodeling. Ascorbic acid can be used as a potential agent to increase the cold tolerance of tea plants with no pesticide residual concerns in tea.

A Fluoride-Ion-Conducting Solid Electrolyte with Both High Conductivity and Excellent Electrochemical Stability.

Solid-state fluoride-ion batteries (FIBs) circumvent multiple formidable bottlenecks of lithium-ion batteries, but their overall performance remains inferior due to the absence of appropriate solid electrolytes. Presently the conductivity of most solid electrolytes for FIBs is too low to enable room-temperature cycling, while the few sufficiently conductive ones only allow for very low discharge voltages because of the narrow electrochemical stability window (ESW). Here, high room-temperature conductivity and a decent ESW are simultaneously achieved by designing a solid electrolyte CsPb0.9 K0.1 F2.9 . Its room-temperature conductivity is 1.23 × 10-3  S cm-1 , comparable to the most conductive system reported so far (PbSnF4 , 5.44 × 10-4 -1.6 × 10-3  S cm-1 ), but the ESW is several times broader. With these appealing characteristics simultaneously achieved in the solid electrolyte, a cell with much higher voltages than other room-temperature-operable solid-state FIBs in literature is successfully constructed, and stably cycled at 25 °C for 4581 h without considerable capacity fade.

Diverse phosphate and auxin transport loci distinguish phosphate tolerant from sensitive Arabidopsis accessions.

Phosphorus (P) is an essential element for plant growth often limiting agroecosystems. To identify genetic determinants of performance under variable phosphate (Pi) supply, we conducted genome-wide association studies on five highly predictive Pi starvation response traits in 200 Arabidopsis (Arabidopsis thaliana) accessions. Pi concentration in Pi-limited organs had the strongest, and primary root length had the weakest genetic component. Of 70 trait-associated candidate genes, 17 responded to Pi withdrawal. The PHOSPHATE TRANSPORTER1 gene cluster on chromosome 5 comprises PHT1;1, PHT1;2, and PHT1;3 with known impact on P status. A second locus featured uncharacterized endomembrane-associated auxin efflux carrier encoding PIN-LIKES7 (PILS7) which was more strongly suppressed in Pi-limited roots of Pi-starvation sensitive accessions. In the Col-0 background, Pi uptake and organ growth were impaired in both Pi-limited pht1;1 and two pils7 T-DNA insertion mutants, while Pi -limited pht1;2 had higher biomass and pht1;3 was indistinguishable from wild-type. Copy number variation at the PHT1 locus with loss of the PHT1;3 gene and smaller scale deletions in PHT1;1 and PHT1;2 predicted to alter both protein structure and function suggest diversification of PHT1 is a key driver for adaptation to P limitation. Haplogroup analysis revealed a phosphorylation site in the protein encoded by the PILS7 allele from stress-sensitive accessions as well as additional auxin-responsive elements in the promoter of the "stress tolerant" allele. The former allele's inability to complement the pils7-1 mutant in the Col-0 background implies the presence of a kinase signaling loop controlling PILS7 activity in accessions from P-rich environments, while survival in P-poor environments requires fine-tuning of stress-responsive root auxin signaling.

A Tea Plant (Camellia sinensis) FLOWERING LOCUS C-like Gene, CsFLC1, Is Correlated to Bud Dormancy and Triggers Early Flowering in Arabidopsis.

Flowering and bud dormancy are crucial stages in the life cycle of perennial angiosperms in temperate climates. MADS-box family genes are involved in many plant growth and development processes. Here, we identified three MADS-box genes in tea plant belonging to the FLOWERING LOCUS C (CsFLC) family. We monitored CsFLC1 transcription throughout the year and found that CsFLC1 was expressed at a higher level during the winter bud dormancy and flowering phases. To clarify the function of CsFLC1, we developed transgenic Arabidopsis thaliana plants heterologously expressing 35S::CsFLC1. These lines bolted and bloomed earlier than the WT (Col-0), and the seed germination rate was inversely proportional to the increased CsFLC1 expression level. The RNA-seq of 35S::CsFLC1 transgenic Arabidopsis showed that many genes responding to ageing, flower development and leaf senescence were affected, and phytohormone-related pathways were especially enriched. According to the results of hormone content detection and RNA transcript level analysis, CsFLC1 controls flowering time possibly by regulating SOC1, AGL42, SEP3 and AP3 and hormone signaling, accumulation and metabolism. This is the first time a study has identified FLC-like genes and characterized CsFLC1 in tea plant. Our results suggest that CsFLC1 might play dual roles in flowering and winter bud dormancy and provide new insight into the molecular mechanisms of FLC in tea plants as well as other plant species.

Genome-wide identification, characterization, and expression analysis of tea plant autophagy-related genes (CsARGs) demonstrates that they play diverse roles during development and under abiotic stress.

BACKGROUND: Autophagy, meaning 'self-eating', is required for the degradation and recycling of cytoplasmic constituents under stressful and non-stressful conditions, which helps to maintain cellular homeostasis and delay aging and longevity in eukaryotes. To date, the functions of autophagy have been heavily studied in yeast, mammals and model plants, but few studies have focused on economically important crops, especially tea plants (Camellia sinensis). The roles played by autophagy in coping with various environmental stimuli have not been fully elucidated to date. Therefore, investigating the functions of autophagy-related genes in tea plants may help to elucidate the mechanism governing autophagy in response to stresses in woody plants. RESULTS: In this study, we identified 35 C. sinensis autophagy-related genes (CsARGs). Each CsARG is highly conserved with its homologues from other plant species, except for CsATG14. Tissue-specific expression analysis demonstrated that the abundances of CsARGs varied across different tissues, but CsATG8c/i showed a degree of tissue specificity. Under hormone and abiotic stress conditions, most CsARGs were upregulated at different time points during the treatment. In addition, the expression levels of 10 CsARGs were higher in the cold-resistant cultivar 'Longjing43' than in the cold-susceptible cultivar 'Damianbai' during the CA period; however, the expression of CsATG101 showed the opposite tendency. CONCLUSIONS: We performed a comprehensive bioinformatic and physiological analysis of CsARGs in tea plants, and these results may help to establish a foundation for further research investigating the molecular mechanisms governing autophagy in tea plant growth, development and response to stress. Meanwhile, some CsARGs could serve as putative molecular markers for the breeding of cold-resistant tea plants in future research.

Colorimetric analysis of extracellular vesicle surface proteins based on controlled growth of Au aptasensors.

Protein profiling of extracellular vesicles (EVs) provides important information in both clinical cancer diagnosis and relevant biological research studies. Although a variety of bioanalytical techniques have been investigated for EV characterization, limitations such as time-consuming operations, the requirement of large sample volume and demand for specialized instruments hinder their practical applications. Here, we report a simple and wash-free homogeneous colorimetric assay for sensitive detection of surface proteins on EVs. Au nanoparticles were modified with thiolated aptamers to fabricate aptasensors and incubated with EVs. Upon addition of a Au growth reagent, the solution color changed from light red to blue in the presence of target proteins and became deep red when the targets were absent. Expression of CD63, epithelial cell adhesion molecules (EpCAM), and mucin1 in EVs derived from two breast cancer cell lines (MCF-7 and MDA-MB-231) were compared, showing results consistent with western blotting results. The colorimetric assay achieves a limit of detection (LOD) down to 0.7 ng µL-1 against MCF-7 EVs based on the assessment of EpCAM expression, suggesting its potential to be applied in clinical breast cancer diagnosis.

Genome-wide identification and expression analysis of flowering-related genes reveal putative floral induction and differentiation mechanisms in tea plant (Camellia sinensis).

The tea leaf is economically important, while reproductive growth reduce tea output. However, little is known about flowering mechanisms in tea plants. Here, we determined the approximate times of floral induction, floral transition and floral organ differentiation by morphological observation. We identified 401 and 356 flowering-related genes from the genomes of Camellia sinensis var. sinensis and Camellia sinensis var. assamica, respectively. Then, we compared the expression profiles of flowering-related genes in floriferous and oliganthous cultivars, the result showed that PRR7, GI, GID1B and GID1C expression is correlated with the floral induction; LFY, PNF and PNY expression was correlated with floral bud formation. Transcriptome analysis also showed that GI, PRR7 and GID1 were correlated with stress-induced flowering. Thus, we proposed putative mechanisms of flowering in tea plants. This study provides new insights into flowering and a theoretical basis for balancing vegetative and reproductive growth in tea plants and other economical plants.

Genome-wide identification, characterization, and expression analysis of nucleotide-binding leucine-rich repeats gene family under environmental stresses in tea (Camellia sinensis).

Plants often use nucleotide-binding leucine-rich repeats (NLRs) to recognize specific virulence proteins and activate the hypersensitive response thereby defending against invaders. However, data on NLRs and the resistance mechanism of NLR protein mediation in tea plant are extremely limited. In this study, 400 and 303 CsNLRs were identified from the genomes of C. sinensis var. sinensis (CSS) and C. sinensis var. assamica (CSA), respectively. Phylogenetic analysis revealed that the numbers in CNL groups are predominant in both CSS and CSA. RNA-Seq revealed that the expression of CsNLRs is induced by Colletotrichum fructicola, cold, drought, salt stress and exogenous methyl jasmonate. The 21 CsCNLs that are highly expressed in tea plant under biotic and abiotic stresses as well as during bud dormancy and in different tissues are identified. Gene structure analysis revealed several cis-regulatory elements associated with phytohormones and light responsiveness in the promoter regions of these 21 CsCNLs.

CsSWEET1a and CsSWEET17 Mediate Growth and Freezing Tolerance by Promoting Sugar Transport across the Plasma Membrane.

Sugars Will Eventually be Exported Transporters (SWEETs) are important in plant biological processes. Expression levels of CsSWEET1a and CsSWEET17 are induced by cold acclimation (CA) and cold stress in Camellia sinensis. Here, we found that CsSWEET17 was alternatively spliced, and its exclusion (Ex) transcript was associated with the CA process. Both plasma membrane-localized CsSWEET1a and CsSWEET17 transport hexoses, but cytoplasm-localized CsSWEET17-Ex does not. These results indicate that alternative splicing may be involved in regulating the function of SWEET transporters in response to low temperature in plants. The extra C-terminal of CsSWEET17, which is not found in the tonoplast fructose transporter AtSWEET17, did not affect its plasma membrane localization but promoted its sugar transport activities. The overexpression (OE) of CsSWEET1a and CsSWEET17 genes resulted in an increased sugar uptake in Arabidopsis, affecting plant germination and growth. The leaf and seed sizes of the CsSWEET17-OE lines were significantly larger than those of the wild type. Moreover, the OE of CsSWEET1a and CsSWEET17 significantly reduced the relative electrolyte leakage levels under freezing stress. Compared with the wild type, the expression of AtCWINV genes was suppressed in both CsSWEET1a-OE and CsSWEET17-OE lines, indicating the alteration in sugar contents in the cell walls of the OE lines. Furthermore, the interaction between CsSWEET1a and CsSWEET17 was confirmed using yeast two-hybrid and bimolecular fluorescence complementation assays. We showed that CsSWEET1a and CsSWEET17 form homo-/heterodimers in the plasma membrane and mediate the partitioning of sugars between the cytoplasm and the apoplast, thereby regulating plant growth and freezing tolerance.