The fluoride exporter (CsFEX) regulates fluoride uptake/accumulation in Camellia sinensis under different pH.

Fluoride (F) can be absorbed from the environment and hyperaccumulate in leaves of Camellia sinensis without exhibiting any toxic symptoms. Fluoride exporter in C. sinensis (CsFEX) could transport F to extracellular environment to alleviate F accumulation and F toxicity, but its functional mechanism remains unclear. Here, combining with pH condition of C. sinensis growth, the characteristics of CsFEX and mechanism of F detoxification were further explored. The results showed that F accumulation was influenced by various pH, and pH 4.5 and 6.5 had a greater impact on the F accumulation of C. sinensis. Through Non-invasive Micro-test Technology (NMT) detection, it was found that F uptake/accumulation of C. sinensis and Arabidopsis thaliana might be affected by pH through changing the transmembrane electrochemical proton gradient of roots. Furthermore, diverse expression patterns of CsFEX were induced by F treatment under different pH, which was basically up-regulated in response to high F accumulation, indicating that CsFEX was likely to participate in the process of F accumulation in C. sinensis and this process might be regulated by pH. Additionally, CsFEX functioned in the mitigation of F sensitivity and accumulation strengthened by lower pH in Escherichia coli and A. thaliana. Moreover, the changes of H+ flux and potential gradient caused by F were relieved as well in transgenic lines, also suggesting that CsFEX might play an important role in the process of F accumulation. Above all, F uptake/accumulation were alleviated in E. coli and A. thaliana by CsFEX through exporting F-, especially at lower pH, implying that CsFEX might regulate F accumulation in C. sinensis.

Single-nucleus RNA sequencing and mRNA hybridization indicate key bud events and LcFT1 and LcTFL1-2 mRNA transportability during floral transition in litchi.

In flowering plants, floral induction signals intersect at the shoot apex to modulate meristem determinacy and growth form. Here, we report a single-nucleus RNA sequence analysis of litchi apical buds at different developmental stages. A total of 41 641 nuclei expressing 21 402 genes were analyzed, revealing 35 cell clusters corresponding to 12 broad populations. We identify genes associated with floral transition and propose a model that profiles the key events associated with litchi floral meristem identity by analyzing 567 identified floral meristem cells at single cell resolution. Interestingly, single-nucleus RNA-sequencing data indicated that all putative FT and TFL1 genes were not expressed in bud nuclei, but significant expression was detected in bud samples by RT-PCR. Based on the expression patterns and gene silencing results, we highlight the critical role of LcTFL1-2 in inhibiting flowering and propose that the LcFT1/LcTFL1-2 expression ratio may determine the success of floral transition. In addition, the transport of LcFT1 and LcTFL1-2 mRNA from the leaf to the shoot apical meristem is proposed based on in situ and dot-blot hybridization results. These findings allow a more comprehensive understanding of the molecular events during the litchi floral transition, as well as the identification of new regulators.

Integrated metabolome and transcriptome analysis reveals the cause of anthocyanin biosynthesis deficiency in litchi aril.

Anthocyanins are health-promoting compounds with strong antioxidant properties that play important roles in disease prevention. Litchi chinensis Sonn. is a well-known and economically significant fruit due to its appealing appearance and nutritional value. The mature pericarp of litchi is rich in anthocyanins, whereas the aril (flesh) has an extremely low anthocyanin content. However, the mechanism of anthocyanin differential accumulation in litchi pericarp and aril remained unknown. Here, metabolome and transcriptome analysis were performed to unveil the cause of the deficiency of anthocyanin biosynthesis in litchi aril. Numerous anthocyanin biosynthesis-related metabolites and their derivatives were found in the aril, and the levels of rutin and (-)-epicatechin in the aril were comparable to those found in the pericarp, while anthocyanin levels were negligible. This suggests that the biosynthetic pathway from phenylalanine to cyanidin was present but that a block in cyanidin glycosylation could result in extremely low anthocyanin accumulation in the aril. Furthermore, 54 candidate genes were screened using weighted gene co-expression network analysis (WGCNA), and 9 genes (LcUFGT1, LcGST1, LcMYB1, LcSGR, LcCYP75B1, LcMATE, LcTPP, LcSWEET10, and LcERF61) might play a significant role in regulating anthocyanin biosynthesis. The dual-luciferase reporter (DLR) assay revealed that LcMYB1 strongly activated the promoters of LcUFGT1, LcGST4, and LcSWEET10. The results imply that LcMYB1 is the primary qualitative gene responsible for the deficiency of anthocyanin biosynthesis in litchi aril, which was confirmed by a transient transformation assay. Our findings shed light on the molecular mechanisms underlying tissue-specific anthocyanin accumulation and will help developing new red-fleshed litchi germplasm.

Transcriptome analysis reveals the roles of phytohormone signaling in tea plant (Camellia sinensis L.) flower development.

BACKGROUND: Tea plant (Camellia sinensis (L.) O. Kuntze) is an important economic tea crop, but flowering will consume a lot of nutrients of C. sinensis, which will seriously affect the nutritional growth of C. sinensis. However, there are few studies on the development mechanism of C. sinensis flower, and most studies focus on a single C. sinensis cultivar. RESULTS: Here, we identified a 92-genes' C. sinensis flower development core transcriptome from the transcriptome of three C. sinensis cultivars ('BaiYe1', 'HuangJinYa' and 'SuChaZao') in three developmental stages (bud stage, white bud stage and blooming stage). In addition, we also reveal the changes in endogenous hormone contents and the expression of genes related to synthesis and signal transduction during the development of C. sinensis flower. The results showed that most genes of the core transcriptome were involved in circadian rhythm and autonomous pathways. Moreover, there were only a few flowering time integrators, only 1 HD3A, 1 SOC1 and 1 LFY, and SOC1 played a dominant role in the development of C. sinensis flower. Furthermore, we screened out 217 differentially expressed genes related to plant hormone synthesis and 199 differentially expressed genes related to plant hormone signal transduction in C. sinensis flower development stage. CONCLUSIONS: By constructing a complex hormone regulation network of C. sinensis flowering, we speculate that MYC, FT, SOC1 and LFY play key roles in the process of endogenous hormones regulating C. sinensis flowering development. The results of this study can a provide reference for the further study of C. sinensis flowering mechanism.

Multiomic Analyses Reveal the Effects of Supplementing Phytosterols on the Metabolic Function of the Rumen Microbiota in Perinatal Cows.

Phytosterols are natural steroids in plants, possessing bioactivities that could modify gut microbes. This experiment aimed to evaluate the effects of feeding phytosterols on the community structures and metabolic functions of the rumen microbiota in perinatal cows. Perinatal cows were supplied with 0 mg (control) or 200 mg (treatment) phytosterols per day. Multiomic analyses were used to analyze the community structures and metabolic functions of rumen microbiota. Results showed that dietary phytosterols increased the copy number of total ruminal bacteria, the concentration of microbial crude protein, and the molar percentage of propionate in the rumen of perinatal cows but had no effects on the alpha diversity of ruminal bacteria. However, they enriched three genera (i.e., Fibrobacter) and seven species (i.e., Fibrobacter succinogenes) within active ruminal bacteria. Metatranscriptomic and metabolomic analyses revealed that dietary phytosterols enhanced the pathway of glycolysis and the family of glycoside hydrolase 13 but depressed the citrate cycle and pyruvate metabolism and several pathways of amino acid biosynthesis. In conclusion, dietary addition of phytosterols improved the growth of ruminal bacteria and changed rumen fermentation by modifying the rumen microbiome and the energy metabolism pathways, which would be beneficial for the energy utilization of perinatal cows. IMPORTANCE Perinatal cows suffer serious physiological stress and energy deficiency. Phytosterols have bioactive functions for gut microbes. However, little knowledge is available on their effects on rumen microbiota and rumen fermentation. Results of the present experiment revealed that dietary supplementation of phytosterols could improve the growth of ruminal bacteria and changed the rumen fermentation to provide more glycogenetic precursors for the perinatal cows by modifying the ruminal bacteria community and altering the energy metabolism pathways of the rumen microbiota. These findings suggest that dietary supplementation of phytosterols would be beneficial for perinatal cows suffering from a negative energy balance.

The performance of water-soluble fluoride transformation in soil-tea-tea infusion chain system and the potential health risk assessment.

BACKGROUND: Water-soluble fluoride (WS-F) can be absorbed directly by tea plants from soil and comprises a major source of dietary F in tea consumers. To reveal the WS-F accumulation in tea leaves and assess WS-F health risks, 70 sets of samples including tea leaves at three maturity stages and corresponding topsoil were collected from Xinyang, China. The WS-F contents in tea samples and pH values in soil samples were determined. RESULTS: The contents of WS-F in tea leaves exhibited a positive correlation with leaf maturity. The contents of WS-F in tea leaves showed a positive correlation with WS-F contents in the soil as the soil pH value exceeds 5. All the bud with two leaves samples, 84.29% of the third to sixth leaves samples, and 78.57% mature leaves samples in 5-min infusion tend to be no health threat. The leaching characteristics of WS-F from tea leaves were influenced by the leaf maturity and soaking time. CONCLUSION: Taking measures to control pH and WS-F concentration of plantations soil, as well as drinking tea infusion made from young leaves or reducing soaking time could decrease the WS-F health risk. © 2021 Society of Chemical Industry.

Effect of archwire plane and archwire size on anterior teeth movement in sliding mechanics in customized labial orthodontics: a 3D finite element study.

BACKGROUND: The aim of this study was to evaluate anterior teeth movement with different archwire planes and archwire sizes during space closure with and without miniscrew in sliding mechanics. METHODS: A 3D finite element method was applied to simulate anterior teeth retraction with and without miniscrew and power arm. Initial displacements and pressure stresses of periodontal tissue in anterior teeth were calculated after the teeth were applied with retraction forces with different archwire planes and archwire sizes. RESULTS: High archwire plane showed better torque control of anterior teeth in both sliding mechanics. With intramaxillary retraction, anterior teeth showed lingual tipping and extrusion movement, whereas larger-size archwires did not reduce it. In miniscrew sliding mechanics, anterior teeth showed labial tipping and intrusion movement. Compared with intramaxillary retraction, the retraction force produced less pressure stress on periodontal tissue in miniscrew sliding mechanics with long power arm. CONCLUSIONS: Higher archwire plane is conducive to anterior teeth torque control. In order to achieve the bodily movement of the anterior teeth during space closure, it is more important to choose the appropriate method (miniscrew sliding mechanics with long power arm), instead of increasing the size of the archwire.

Identification and response analysis of xyloglucan endotransglycosylase/hydrolases (XTH) family to fluoride and aluminum treatment in Camellia sinensis.

BACKGROUND: Xyloglucan endotransglycosylase/hydrolases (XTH) can disrupt and reconnect the xyloglucan chains, modify the cellulose-xyloglucan complex structure in the cell wall to reconstruct the cell wall. Previous studies have reported that XTH plays a key role in the aluminum (Al) tolerance of tea plants (Camellia sinensis), which is a typical plant that accumulates Al and fluoride (F), but its role in F resistance has not been reported. RESULTS: Here, 14 CsXTH genes were identified from C. sinensis and named as CsXTH1-14. The phylogenetic analysis revealed that CsXTH members were divided into 3 subclasses, and conserved motif analysis showed that all these members included catalytic active region. Furthermore, the expressions of all CsXTH genes showed tissue-specific and were regulated by Al3+ and F- treatments. CsXTH1, CsXTH4, CsXTH6-8 and CsXTH11-14 were up-regulated under Al3+ treatments; CsXTH1-10 and CsXTH12-14 responded to different concentrations of F- treatments. The content of xyloglucan oligosaccharide determined by immunofluorescence labeling increased to the highest level at low concentrations of Al3+ or F- treatments (0.4 mM Al3+ or 8 mg/L F-), accompanying by the activity of XET (Xyloglucan endotransglucosylase) peaked. CONCLUSION: In conclusion, CsXTH activities were regulated by Al or F via controlling the expressions of CsXTH genes and the content of xyloglucan oligosaccharide in C. sinensis roots was affected by Al or F, which might finally influence the elongation of roots and the growth of plants.

Genome-wide identification and expression analysis of the CLC superfamily genes in tea plants (Camellia sinensis).

The voltage-gated chloride channel (CLC) superfamily is one of the most important anion channels that is widely distributed in bacteria and plants. CLC is involved in transporting various anions such as chloride (Cl-) and fluoride (F-) in and out of cells. Although Camellia sinensis is a hyper-accumulated F plant, there is no studies on the CLC gene superfamily in the tea plant. Here, 8 CLC genes were identified from C. sinensis and they were named CsCLC1-8. The structure of CsCLC genes and the proteins were not conserved; the number of exons varied from 3 to 24, and the number of transmembrane domains contained 2 to 10. Furthermore, phylogenetic analysis revealed that CsCLC4-8 in subclass I contained the typical conserved domains GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III), and CsCLC1-3 in subclass II did not contain any of the three conserved residues. We measured the expression levels of CsCLCs in roots, stems and leaves to assess the responses to different concentrations of Cl- and F-. The result indicated that CsCLCs participated in subfunctionalization in response to Cl- and F-, and CsCLC1-3 was more sensitive to F- treatments than CsCLC4-8, CsCLC6 and CsCLC7 may participate in absorption and long-distance transport of Cl-.

Biosynthesis of quebrachitol, a transportable photosynthate, in Litchi chinensis.

Although methylated cyclitols constitute a major proportion of the carbohydrates in many plant species, their physiological roles and biosynthetic pathway are largely unknown. Quebrachitol (2-O-methyl-chiro-inositol) is one of the major methylated cyclitols in some plant species. In litchi, quebrachitol represents approximately 50% of soluble sugars in mature leaves and 40% of the total sugars in phloem exudate. In the present study, we identified bornesitol as a transient methylated intermediate of quebrachitol and measured the concentrations of methyl-inositols in different tissues and in tissues subjected to different treatments. 14CO2 feeding and phloem exudate experiments demonstrated that quebrachitol is one of the transportable photosynthates. In contrast to other plant species, the biosynthesis of quebrachitol in litchi is not associated with osmotic stress. High quebrachitol concentrations in tissues of the woody plant litchi might represent a unique carbon metabolic strategy that maintains osmolality under reduced-sucrose conditions. The presence of bornesitol but not ononitol in the leaves indicates a different biosynthetic pathway with pinitol. The biosynthesis of quebrachitol involves the methylation of myo-inositol and the subsequent epimerization of bornesitol. An inositol methyltransferase gene (LcIMT1) responsible for bornesitol biosynthesis was isolated and characterized for the first time, and the biosynthesis pathways of methyl-inositols are discussed.

Functional characterization of a glucosyltransferase gene, LcUFGT1, involved in the formation of cyanidin glucoside in the pericarp of Litchi chinensis.

Anthocyanins generate the red color in the pericarp of Litchi chinensis. UDP-glucose: flavonoid 3-O-glycosyltransferase (UFGT, EC. 2.4.1.91) stabilizes anthocyanidin by attaching sugar moieties to the anthocyanin aglycone. In this study, the function of an UFGT gene involved in the biosynthesis of anthocyanin was verified through heterologous expression and virus-induced gene silencing assays. A strong positive correlation between UFGT activity and anthocyanin accumulation capacity was observed in the pericarp of 15 cultivars. Four putative flavonoid 3-O-glycosyltransferase-like genes, designated as LcUFGT1 to LcUFGT4, were identified in the pericarp of litchi. Among the four UFGT gene members, only LcUFGT1 can use cyanidin as its substrate. The expression of LcUFGT1 was parallel with developmental anthocyanin accumulation, and the heterologously expressed protein of LcUFGT1 displayed catalytic activities in the formation of anthocyanin. The LcUFGT1 over-expression tobacco had darker petals and pigmented filaments and calyxes resulting from higher anthocyanin accumulations compared with non-transformed tobacco. In the pericarp with LcUFGT1 suppressed by virus-induced gene silencing, pigmentation was retarded, which was well correlated with the reduced-LcUFGT1 transcriptional activity. These results suggested that the glycosylation-related gene LcUFGT1 plays a critical role in red color formation in the pericarp of litchi.

Methyl-inositol, γ-aminobutyric acid and other health benefit compounds in the aril of litchi.

The available components in the flesh of litchi seem insufficient to interpret its wide and significant physiological effects. Some unusual compounds, including myo-inositol, inositol methyl derivatives and γ-aminobutyric acid (GABA) were identified as main constituents in the flesh of litchi. Their concentrations varied among cultivars but remain relatively constant during development. Litchi flesh was shown to contain moderate myo-inositol (0.28-0.78 mg g(-1) FW), ascorbic acid (0.08-0.39 mg g(-1) FW) and phenolics (0.47-1.60 mg g(-1) FW), but abundant l-quebrachitol (1.6-6.4 mg g(-1) FW) and GABA (1.7-3.5 mg g(-1) FW). The concentration of GABA in the flesh of litchi was about 100 times higher than in other fruits. And l-quebrachitol is not a common component in fruits. The biological and physiological activities of inositols, inositol derivatives and GABA have been extensively documented. These compounds are probably important compositional characteristic contributing to the widely shown health benefits of litchi.

Sugar uptake in the Aril of litchi fruit depends on the apoplasmic post-phloem transport and the activity of proton pumps and the putative transporter LcSUT4.

The post-phloem unloading pathway and the mechanism of sugar accumulation remain unclear in litchi fruit. A combination of electron microscopy, transport of phloem-mobile symplasmic tracer (carboxyfluorescein, CF) and biochemical and molecular assays was used to explore the post-phloem transport pathway and the mechanism of aril sugar accumulation in litchi. In the funicle, where the aril originates, abundant plasmodesmata were observed, and CF introduced from the peduncle diffused to the parenchyma cells. In addition, abundant starch and pentasaccharide were detected and the sugar concentration was positively correlated with activities of sucrose hydrolysis enzymes. These results clearly showed that the phloem unloading and post-phloem transport in the funicle were symplastic. On the other hand, imaging of CF showed that it remained confined to the parenchyma cells in funicle tissues connecting the aril. Infiltration of both an ATPase inhibitor [eosin B (EB)] and a sucrose transporter inhibitor [p-chloromercuribenzene sulfonate (PCMBS)] inhibited sugar accumulation in the aril. These results indicated an apoplasmic post-phloem sugar transport from the funicle to the aril. Although facilitated diffusion might help sucrose uptake from the cytosol to the vacuole in cultivars with high soluble invertase, membrane ATPases in the aril, especially tonoplast ATPase, are crucial for aril sugar accumulation. The expression of a putative aril vacuolar membrane sucrose transporter gene (LcSUT4) was highly correlated with the sugar accumulation in the aril of litchi. These data suggest that apoplasmic transport is critical for sugar accumulation in litchi aril and that LcSUT4 is involved in this step.

Utilization of Ningxiang pig milk oligosaccharides by Akkermansia muciniphila in vitro fermentation: enhancing neonatal piglet survival.

Akkermansia muciniphila (A. muciniphila), an intestinal symbiont residing in the mucosal layer, shows promise as a probiotic. Our previous study found that the abundance of A. muciniphila was significantly higher in Ningxiang suckling piglets compared to other breeds, suggesting that early breast milk may play a crucial role. This study examines A. muciniphila's ability to utilize Ningxiang pig milk oligosaccharides. We discovered that A. muciniphila can thrive on both Ningxiang pig colostrum and purified pig milk oligosaccharides. Genetic analysis has shown that A. muciniphila harbors essential glycan-degrading enzymes, enabling it to effectively break down a broad spectrum of oligosaccharides. Our findings demonstrate that A. muciniphila can degrade pig milk oligosaccharides structures such as 3'-FL, 3'-SL, LNT, and LNnT, producing short-chain fatty acids in the process. The hydrolysis of these host-derived glycan structures enhances A. muciniphila's symbiotic interactions with other beneficial gut bacteria, contributing to a dynamic microbial ecological network. The capability of A. muciniphila to utilize pig milk oligosaccharides allows it to establish itself in the intestines of newborn piglets, effectively colonizing the mucosal layer early in life. This early colonization is key in supporting both mucosal and metabolic health, which is critical for enhancing piglet survival during lactation.

Dietary supplementing phytosterols improves the metabolic status of perinatal cows revealed by plasma metabolomics and faecal microbial metabolism.

Objective: Previous research reported that dietary addition with phytosterols improved the energy utilisation of the rumen microbiome, suggesting its potential to alleviate the negative energy balance of perinatal cows. This experiment aimed to explore the effects of feeding phytosterols on the metabolic status of perinatal cows through plasma metabolomics and faecal bacteria metabolism. Methods: Ten perinatal Holstein cows (multiparous, 2 parities) with a similar calving date were selected four weeks before calving. After 7 days for adaptation, cows were allocated to two groups (n=5), which respectively received the basal rations supplementing commercial phytosterols at 0 and 200 mg/d during a 42-day experiment. The milk yield of each cow was recorded daily after calving. On days 1 and 42, blood and faeces samples were all collected from perinatal cows before morning feeding for analysing plasma biochemicals and metabolome, and faecal bacteria metabolism. Results: Dietary addition with phytosterols at 200 mg/d had no effects on plasma cholesterol and numerically increased milk yield by 1.82 kg/d (p>0.10) but attenuated their negative energy balance in perinatal cows as observed from the significantly decreased plasma level of ß-hydroxybutyric acid (p=0.002). Dietary addition with phytosterols significantly altered 12 and 15 metabolites (p<0.05) within the plasma and faeces of perinatal cows, respectively. Of these metabolites, 5 upregulated plasma fatty acids indicated an improved energy status (i.e., C18:1T, C14:0, C17:0, C18:0, and C16:0). Milk yield negatively correlated with plasma concentrations of ketone bodies (p=0.035) and 5-methoxytryptamine (p=0.039). Furthermore, dietary addition with phytosterols at 200 mg/d had no effects on fermentation characteristics and bacterial diversity of cow faeces (p>0.10) but improved potentially beneficial bacteria such as Christensenellaceae family (p<0.05) that positively correlated with feed efficiency. Conclusion: Dietary addition with phytosterols at 200 mg/d could effectively improve the energy status in perinatal cows by attenuating their negative energy balance.

A homogeneous dopamine-silver nanocomposite coating: striking a balance between the antibacterial ability and cytocompatibility of dental implants.

Silver has been widely used for surface modification to prevent implant-associated infections. However, the inherent cytotoxicity of silver greatly limited the scope of its clinical applications. The construction of surfaces with both good antibacterial properties and favorable cytocompatibility still remains a challenge. In this study, a structurally homogeneous dopamine-silver (DA/Ag) nanocomposite was fabricated on the implant surface to balance the antibacterial activity and cytocompatibility of the implant. The results show that the DA/Ag nanocomposites prepared under the acidic conditions (pH = 4) on the titanium surface are homogeneous with higher Ag+ content, while an obvious core (AgNPs)-shell (PDA) structure is formed under neutral (pH = 7) and alkaline conditions (pH = 10), and the subsequent heat treatment enhanced the stability of PDA-AgNPs nanocomposite coatings on porous titanium. The antibacterial test, cytotoxicity test, hypodermic implantation and osteogenesis test revealed that the homogeneous PDA-AgNPs nanocomposite coating achieved the balance between the antibacterial ability and cytocompatibility, and had the best outcomes for soft tissue healing and bone formation around the implants. This study provides a facile strategy for preparing silver-loaded surfaces with both good antibacterial effect and favorable cytocompatibility, which is expected to further improve the therapeutic efficacy of silver composite-coated dental implants.

Disease resistance conferred by components of essential chrysanthemum oil and the epigenetic regulation of OsTPS1.

The sesquiterpene alpha-bisabolol is the predominant active ingredient in essential oils that are highly valued in the cosmetics industry due to its wound healing, anti-inflammatory, and skin-soothing properties. Alpha-bisabolol was thought to be restricted to Compositae plants. Here we reveal that alpha-bisabolol is also synthesized in rice, a non-Compositae plant, where it acts as a novel sesquiterpene phytoalexin. Overexpressing the gene responsible for the biosynthesis of alpha-bisabolol, OsTPS1, conferred bacterial blight resistance in rice. Phylogenomic analyses revealed that alpha-bisabolol-synthesizing enzymes in rice and Compositae evolved independently. Further experiments demonstrated that the natural variation in the disease resistance level was associated with differential transcription of OsTPS1 due to polymorphisms in its promoter. We demonstrated that OsTPS1 was regulated at the epigenetic level by JMJ705 through the methyl jasmonate pathway. These data reveal the cross-family accumulation and regulatory mechanisms of alpha-bisabolol production.

Camellia sinensis small GTPase gene (CsRAC1) involves in response to salt stress, drought stress and ABA signaling pathway.

RAC/ROP gene (RACs) is a plant-specific small GTPases. RACs play an irreplaceable role in the tissue dynamics of cytoskeleton, vesicle transport and hormone signal transmission in plants. In the present study, a novel gene from RACs family, CsRAC1, was identified from tea [Camellia sinensis (L.) O. Kuntze]. CsRAC1 contained a 591-bp open reading frame and encoded a putative protein of 197 amino acids. Subcellular localization analysis in leaves of transgenic tobacco and root tips of Arabidopsis thaliana showed that CsRAC1 targeted the nucleus and cell membrane. The expression of CsRAC1 induced by abiotic stresses such as cold, heat, drought, salt and abscisic acid has also been verified by RT-qPCR. Further verification of biological function of CsRAC1 showed that overexpression of CsRAC1 increased the sensitivity of A. thaliana to salt stress, improved the tolerance of mature A. thaliana to drought stress, and enhanced the inhibition of ABA on seed germination of A. thaliana. In addition, the antioxidant system regulated by CsRAC1 mainly worked in mature A. thaliana. The results indicate that CsRAC1 is involved in the response of C. sinensis to salt, drought stress and ABA signaling pathway.

Systematic Methods for Isolating High Purity Nuclei from Ten Important Plants for Omics Interrogation.

Recent advances in developmental biology have been made possible by using multi-omic studies at single cell resolution. However, progress in plants has been slowed, owing to the tremendous difficulty in protoplast isolation from most plant tissues and/or oversize protoplasts during flow cytometry purification. Surprisingly, rapid innovations in nucleus research have shed light on plant studies in single cell resolution, which necessitates high quality and efficient nucleus isolation. Herein, we present efficient nuclei isolation protocols from the leaves of ten important plants including Arabidopsis, rice, maize, tomato, soybean, banana, grape, citrus, apple, and litchi. We provide a detailed procedure for nucleus isolation, flow cytometry purification, and absolute nucleus number quantification. The nucleus isolation buffer formula of the ten plants tested was optimized, and the results indicated a high nuclei yield. Microscope observations revealed high purity after flow cytometry sorting, and the DNA and RNA quality extract from isolated nuclei were monitored by using the nuclei in cell division cycle and single nucleus RNA sequencing (snRNA-seq) studies, with detailed procedures provided. The findings indicated that nucleus yield and quality meet the requirements of snRNA-seq, cell division cycle, and likely other omic studies. The protocol outlined here makes it feasible to perform plant omic studies at single cell resolution.

Nitric Oxide Participates in Aluminum-Stress-Induced Pollen Tube Growth Inhibition in Tea (Camelliasinensis) by Regulating CsALMTs.

Nitric oxide (NO), as a signal molecule, is involved in the mediation of heavy-metal-stress-induced physiological responses in plants. In this study, we investigated the effect of NO on Camellia sinensis pollen tubes exposed to aluminum (Al) stress. Exogenous application of the NO donor decreased the pollen germination rate and pollen tube length and increased the malondialdehyde (MDA) content and antioxidant enzyme activities under Al stress. Simultaneously, the NO donor effectively increased NO content in pollen tube of C. sinensis under Al stress and could aggravate the damage of Al3+ to C. sinensis pollen tubes by promoting the uptake of Al3+. In addition, application of the NO-specific scavenger significantly alleviated stress damage in C. sinensis pollen tube under Al stress. Moreover, 18 CsALMT members from a key Al-transporting gene family were identified, which could be divided into four subclasses. Pearson correlation analysis showed the expression level of CsALMT8 showed significant positive correlation with the Al3+ concentration gradient and NO levels, but a significant negative correlation with pollen germination rate and pollen tube length. The expression level of CsALMT5 was negatively correlated with the Al3+ concentration gradient and NO level, and positively correlated with pollen germination rate and pollen tube length. The expression level of CsALMT17 showed a significant negative correlation with Al3+ concentration and NO content in pollen tubes, but significant positive correlation with pollen germination rate and pollen tube length. In conclusion, a complex signal network regulated by NO-mediated CsALMTs revealed that CsALMT8 was regulated by environmental Al3+ and NO to assist Al3+ entry into pollen tubes; CsALMT5 might be influenced by the Al3+ signal, stimulate malate efflux in vacuoles and chelate with Al3+ to detoxify Al in C. sinensis pollen tube.