Intercropping fruit trees in tea plantation improves soil properties and the formation of tea quality components.

Intercropping has been recommended as a beneficial cropping practice for improving soil characteristic and tea quality. However, there is limited research on the effects of intercropping fruit trees on soil chemical properties, soil aggregate structure, and tea quality components. In this study, intercropping fruit trees, specifically loquats and citrus, had a significant impact on the total available nutrients, AMN, and AP in soil. During spring and autumn seasons, the soil large-macroaggregates (>2 mm) proportion increased by 5.93% and 19.03%, as well as 29.23% and 19.14%, respectively, when intercropping loquats and citrus. Similarly, intercropping waxberry resulted in a highest small-macroaggregates (0.25 mm-2 mm) proportion at 54.89% and 77.32%. Soil aggregate stability parameters of the R0.25, MWD, and GMD were generally considered better soil aggregate stability indicators, and significantly improved in intercropping systems. Intercropping waxberry with higher values for those aggregate stability parameters and lower D values, showed a better soil aggregate distribution, while intercropping loquats and citrus at higher levels of AMN and AP in different soil aggregate sizes. As the soil aggregate sizes increased, the AMN and AP contents gradually decreased. Furthermore, the enhanced levels of amino acids were observed under loquat, waxberry, and citrus intercropping in spring, which increased by 27.98%, 27.35%, and 26.21%, respectively. The contents of tea polyphenol and caffeine were lower under loquat and citrus intercropping in spring. These findings indicated that intercropping fruit trees, specifically loquat and citrus, have immense potential in promoting the green and sustainable development of tea plantations.

Minigene Assay as an Effective Molecular Diagnostic Strategy in Determining the Pathogenicity of Noncanonical Splice-Site Variants in FLCN.

Primary spontaneous pneumothorax (PSP) or pulmonary cyst is one of the manifestations of Birt-Hogg-Dubé syndrome, which is caused by pathogenic variants in FLCN gene. Genetic testing in patients with PSP identifies a certain number of missense or intronic variants. These variants are usually considered as variants of uncertain significance, whose functional interpretations pose a challenge in clinical genetics. To improve recognition of pathogenic splice-altering variants in FLCN gene, computational tools are used to prioritize potential splice-altering variants and then a hybrid minigene assay is performed to verify the RNA splicing pattern. Herein, variants in FLCN exon 11 and its flanking sequence are focused. Eight variants detected in 11 patients with PSP are evaluated, and six variants are prioritized by in silico tools as potential splice-altering variants of uncertain significance. Four variants (c.1177-5_1177-3delCTC, c.1292_1300+4del, c.1300+4C>T, and c.1300+5G>A) are demonstrated by minigene assay to alter RNA splicing of FLCN, and the last three of them are novel. RT-PCR of patient-derived RNA gives consistent results. Genotype-phenotype correlation analysis in patients with PSP with these variants demonstrates good concordance. Our results underline the importance of RNA analysis, which could provide molecular evidence for pathogenicity of a variant, and provide essential information for the clinical interpretation of variants. Combining the clinical information, a definitive diagnosis could be made.

Genome-Wide Analysis of the TCP Gene Family and Their Expression Pattern Analysis in Tea Plant (Camellia sinensis).

TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors TEOSINTE BRANCHED1/CYCLOIDEA/PCF have been suggested to control the cell growth and proliferation in meristems and lateral organs. A total of 37 CsTCP genes were identified and divided into two classes, class I (PCF, group 1) and class II (CIN CYC/TB1, groups 2, and 3). The residues of TEOSINTE BRANCHED1/CYCLOIDEA/PCF of Camellia sinensis (Tea plant) (CsTCP) proteins between class I and class II were definitely different in the loop, helix I, and helix II regions; however, eighteen conserved tandem was found in bHLH. There are a large number of CsTCP homologous gene pairs in three groups. Additionally, most CsTCP proteins have obvious differences in motif composition. The results illuminated that CsTCP proteins in different groups are supposed to have complementary functions, whereas those in the same class seem to display function redundancies. There is no relationship between the number of CsTCP gene members and genome size, and the CsTCP gene family has only expanded since the divergence of monocots and eudicots. WGD/segmental duplication played a vital role in the expansion of the CsTCP gene family in tea plant, and the CsTCP gene family has expanded a lot. Most CsTCP genes of group 1 are more widely and non-specifically expressed, and the CsTCP genes of group 2 are mainly expressed in buds, flowers, and leaves. Most genes of group 1 and some genes of group 2 were up-/downregulated in varying degrees under different stress, CsTCP genes of group 3 basically do not respond to stress. TCP genes involved in abiotic stress response mostly belong to PCF group. Some CsTCP genes may have the same function as the homologous genes in Arabidopsis, but there is functional differentiation.

CsAAP7.2 is involved in the uptake of amino acids from soil and the long-distance transport of theanine in tea plants (Camellia sinensis L.).

Tea plant roots can uptake both inorganic nitrogen (NH4+ and NO3-) and organic nitrogen (amino acids) from the soil. These amino acids are subsequently assimilated into theanine and transported to young shoots through the xylem. Our previous study showed that CsLHT1 and CsLHT6 transporters take up amino acids from the soil, and CsAAPs participate in the transport of theanine. However, whether other amino acid transporters are involved in this process remains unknown. In this study, we identified two new CsAAPs homologous to CsAAP7, named CsAAP7.1 and CsAAP7.2. Heterologous expression of CsAAP7.1 and CsAAP7.2 in the yeast mutant 22Δ10α showed that CsAAP7.2 had the capacity to transport theanine and other amino acids, whereas CsAAP7.1 had no transport activity. Transient expression of the CsAAP7.2-GFP fusion protein in tobacco leaf epidermal cells confirmed its localization to the endoplasmic reticulum. Tissue-specific analysis showed that CsAAP7.2 was highly expressed in roots and stems. In addition, CsAAP7.2 overexpression lines were more sensitive to high concentrations of theanine due to the high accumulation of theanine in seedlings. Taken together, these findings suggested that CsAAP7.2 plays an important role in the uptake of amino acids from soil and the long-distance transport of theanine. These results provide valuable tools for nitrogen nutrition studies and enrich our understanding of theanine transport in tea plants.

CsCuAOs and CsAMADH1 Are Required for Putrescine-Derived γ-Aminobutyric Acid Accumulation in Tea.

Polyamines are a potential source of γ-aminobutyric acid (GABA) in plants under abiotic stress. However, studies on GABA enrichment in tea mostly focus on the GABA shunt, while the correlation between polyamine degradation and GABA formation in tea is largely unknown. In this study, tea plants responded to exogenous putrescine, resulting in a significant increase in GABA content, while the glutamate level did not change. At the same time, five copper-containing amine oxidase (CuAO) and eight aminoaldehyde dehydrogenase (AMADH) genes involved in the putrescine-derived GABA pathway were identified from the Tea Plant Information Archive. Expression analysis indicated that CsCuAO1, CsCuAO3 as well as CsAMADH1 were induced to play an important function in response to exogenous putrescine. Thus, the three genes were cloned and the catalytic efficiency of soluble recombinant proteins was determined. CsCuAOs and CsAMADH1 exhibited indispensable functions in the GABA production from putrescine in vitro. Subcellular localization assays indicated that CsAMADH1 was localized in plastid, while both CsCuAO1 and CsCuAO3 were localized in peroxisome. In addition, the synergistic effects of CsCuAOs and CsAMADH1 were investigated by a transient co-expression system in Nicotiana benthamiana. Our data suggest that these three genes regulate the accumulation of GABA in tea by participating in the polyamine degradation pathway and improve the content of GABA in tea to a certain extent. The results will greatly contribute to the production of GABA tea.

Effects of GABA on the polyphenol accumulation and antioxidant activities in tea plants (Camellia sinensis L.) under heat-stress conditions.

Polyphenols are important active components in tea plants, which have strong biological activity and antioxidant activity. A certain degree of stress or exogenous substances can significantly increase the content of polyphenols in plants. γ-Aminobutyric acid (GABA), a natural functional amino acid, was used to study whether exogenous GABA can increase the content of polyphenols and enhance antioxidant activity in tea plants under heat-stress conditions. The results showed that the content of GABA was positively correlated with the content of polyphenols (r = 0.649), especially with the content of total catechins (r = 0.837). Most of the related genes encoding flavonoid metabolism (PAL, C4H, 4CL, CHS, CHI, F3H, F3'H, F3'5'H, DFR, LAR, ANS, ANR and FLS) as well as enzyme activities (PAL, C4H and 4CL) were upregulated. In addition, the activities of antioxidant enzymes were induced under heat-stress conditions. However, 3-mercaptopropionic acid (3-MPA), an inhibitor of GABA synthesis, exhibited opposite results under heat-stress conditions compared with GABA treatment. These results indicated that GABA plays a key role in the accumulation of polyphenols and the upregulation of the antioxidant system in tea plants under heat-stress conditions.

Aluminum relieves fluoride stress through stimulation of organic acid production in Camellia sinensis.

Tea plants (Camellia sinensis O. Kuntze) can hyperaccumulate fluoride (F) in leaves. Although, aluminum (Al) can alleviate F toxicity in C. sinensis, the mechanisms driving this process remain unclear. Here, we measured root length, root activity, soluble proteins content, and levels of peroxidase, superoxide dismutase, catalase, malondialdehyde (MDA), and chlorophyll in tea leaves after treatment with different F concentrations. In addition, we focused on the content of organic acids, the gene transcription of malate dehydrogenase (MDH), glycolate oxidase (GO) and citrate synthase (CS) and the relative enzyme activity involved in the tolerance to F in C. sinensis. We also examined the role of Al in this process by analyzing the content of these physiological indicators in tea leaves treated with F and Al. Our results demonstrate that increased MDA content, together with decreased chlorophyll content and soluble proteins are responsible for oxidative damage and metabolism inhibition at high F concentration. Moreover, increased antioxidant enzymes activity regulates ROS damage to protect tea leaves during F stress. Furthermore, exogenous Al alleviated F stress in tea leaves through the regulation of MDA content and antioxidant enzymes activity. In addition, organic acids in exudate stimulated root growth in tea plants exposed to low F concentrations are regulated by MDH, GO, and CS. In addition, Al can stimulate the exudation of organic acids, and may participate in regulating rhizosphere pH of the roots through the interaction with F, eventually leading to the response to F stress in C. sinensis.

Transcriptome Analysis Reveals the Mechanism of Fluoride Treatment Affecting Biochemical Components in Camellia sinensis.

Tea (Camellia sinensis (L.) O. Kuntze), one of the main crops in China, is high in various bioactive compounds including flavonoids, catechins, caffeine, theanine, and other amino acids. C. sinensis is also known as an accumulator of fluoride (F), and the bioactive compounds are affected by F, however, the mechanism remains unclear. Here, the effects of F treatment on the accumulation of F and major bioactive compounds and gene expression were investigated, revealing the molecular mechanisms affecting the accumulation of bioactive compounds by F treatment. The results showed that F accumulation in tea leaves gradually increased under exogenous F treatments. Similarly, the flavonoid content also increased in the F treatment. In contrast, the polyphenol content, free amino acids, and the total catechins decreased significantly. Special amino acids, such as sulfur-containing amino acids and proline, had the opposite trend of free amino acids. Caffeine was obviously induced by exogenous F, while the theanine content peaked after two day-treatment. These results suggest that the F accumulation and content of bioactive compounds were dramatically affected by F treatment. Furthermore, differentially expressed genes (DEGs) related to the metabolism of main bioactive compounds and amino acids, especially the pivotal regulatory genes of catechins, caffeine, and theanine biosynthesis pathways, were identified and analyzed using high-throughput Illumina RNA-Seq technology and qRT-PCR. The expression of pivotal regulatory genes is consistent with the changes of the main bioactive compounds in C. sinensis leaves, indicating a complicated molecular mechanism for the above findings. Overall, these data provide a reference for exploring the possible molecular mechanism of the accumulation of major bioactive components such as flavonoid, catechins, caffeine, theanine and other amino acids in tea leaves in response to fluoride treatment.

Transcriptome-wide analysis of MADS-box family genes involved in aluminum and fluoride assimilation in Camellia sinensis.

MADS-box transcription factors (TFs) are involved in a variety of processes in flowering plants ranging from root growth to flower and fruit development. However, studies of the tolerance-related functions of MADS-box genes are very limited, and to date no such studies have been conducted on Camellia sinensis. To gain insight into the functions of genes of this family and to elucidate the role they may play in tissue development and Al and F response, we identified 45 MADS-box genes through transcriptomic analysis of C. sinensis. Phylogenetic analysis of these CsMADS-box genes, along with their homologues in Arabidopsis thaliana, enabled us to classify them into distinct groups, including: M-type (Mα), MIKC* and MIKCc (which contains the SOC1, AGL12, AGL32, SEP, ANR1, SVP, and FLC subgroups). Conserved motif analysis of the CsMADS-box proteins revealed diverse motif compositions indicating a complex evolutionary relationship. Finally, we examined the expression patterns of CsMADS-box genes in various tissues and under different Al and F concentration treatments. Our qPCR results showed that these CsMADS-box genes were involved in Al and F accumulation and root growth in C. sinensis. These findings lay the foundation for future research on the function of CsMADS-box genes and their role in response to Al and F accumulation in root tissues.

Poly(glycidyl methacrylate) nanoparticle-coated capillary with oriented antibody immobilization for immunoaffinity in-tube solid phase microextraction: Preparation and characterization.

A combination between modification with nanoparticles (NP) and oriented antibody immobilization (OAI) on the inner face of capillary was for the first time developed for immunoaffinity in-tube solid-phase microextraction (SPME) to promise high antigen extraction capacity. ß2-microglobin (ß2MG) and cystatin C (Cys-C) were selected as model antigens. Poly(glycidyl methacrylate) (PGMA) NPs were chemically immobilized onto the capillary by a ring-opening reaction. Antibodies for ß2MG and Cys-C were immobilized on the NPs through OAI. Scanning electron micrograph of the OAI capillary clearly showed that the PGMA NPs were coated onto the inner surface of capillary in a dense monolayer. In addition, random antibody immobilized (RAI) capillaries and OAI capillaries without NP were also prepared as controls. The extraction capacities of OAI capillaries were 2.02 and 2.18mgm-1 for ß2MG and Cys-C, and were about 5 and 6 times as many as RAI capillaries and OAI capillaries without NP, respectively. The resultant capillaries were used as in-tube SPME materials to enrich ß2MG and Cys-C for particle-enhanced turbidimetric immunoassay. When using 1.0mgL-1 standard solutions, the recoveries of OAI capillaries, RAI capillaries and OAI capillaries without NP were 103.6% and 96.8%, 48.5% and 31.5%, and 24.2% and 25.7% for ß2MG and Cys-C, respectively. Furthermore, the method quantitation limit by OAI capillaries was 5 and 10 times lower than that by RAI capillaries and OAI capillaries without NP, respectively. This result indicated that the NP-coated capillaries with OAI are more suitable for using as immunoaffinity in-tube SPME materials than that with RAI.