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MEP, carotenoid and chlorophyll biosynthetic pathways in plants, and expression patterns of genes related to pigment metabolism in ‘Xiaoxueya’ leaves. (A) Genes involved in the MEP biosynthetic pathway. (B) Genes involved in the carotenoid biosynthetic pathway. (C) Genes involved in the chlorophyll biosynthetic pathway. Genes used for expression analysis are shown in red. The heat maps were generated using the mean of three replicates and MeV software. The blue-white-red schemes are labelled above the heat map. Red and blue represent relatively high and low expression levels, respectively, compared to those shown in white. Asterisks (*) indicate significant differences (p value ≤ 0.05) and |log2FoldChange| ≥ 1 between the values from the sample and third leaf.
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The tea cultivar ‘Xiaoxueya’, a temperature-sensitive albino mutant, is a rare tea germplasm because of its highly enriched amino acid content and brisk flavour. In comparison with green leaf tissues of ‘Xiaoxueya’, albino leaves show significant deficiency in chlorophylls and carotenoids and severely disrupted chloroplasts. Furthermore, the accumu...
Citations
... The chloroplast ultrastructure of the second leaves from ZH2-Y and ZH2-G was investigated via transmission electron microscopy (TEM; Li et al., 2019). Briefly, leaf discs (1-2 mm 2 ) were sequentially fixed with glutaraldehyde (Leagene, China) and OsO 4 (SPI-Chem, USA), dehydrated with ethanol and acetone (Sinopharm, China), infiltrated with a mixture of absolute acetone and epoxy resin (SPI-Chem, USA), sectioned in an EM UC7 ultratome (Leica, Germany), stained with uranyl acetate (SPI-Chem, USA) and alkaline lead citrate (Sinopharm, China), and observed by JEM-1230 TEM (JEOL, Japan). ...
... The rice faded green leaf (fgl) mutant, which is caused by a one bp deletion in the second exon of OsPORB and then a frameshift mutation and premature translational termination, displayed yellow/white leaf variegation and necrotic lesions during leaf elongation in field-grown plants (Sakuraba et al., 2013). Moreover, the expression of POR genes was downregulated in several chlorophyll-deficient tea cultivars, including 'Baijiguan', 'Xiaoxueya' and 'Huangjinya' (Wu et al., 2016;Li et al., 2019;Tian et al., 2022). Transgenic tomato plants overexpressing ShCHLP showed increased chlorophyll content in leaves; in contrast, the chlorophyll content was decreased in ShCHLP suppression plants, which exhibited a yellow leaf phenotype . ...
... Generally, chlorophyll-deficient tea plants show a certain sensitivity to the surrounding environmental conditions. For instance, 'Baijiguan' and 'Huangjinya' with yellow tender shoots are sensitive to light (Wu et al., 2016;Song et al., 2017), and 'White Leaf 1' and 'Xiaoxueya' with white tender shoots are sensitive to temperature (Xiong et al., 2013;Li et al., 2019). Likewise, ZH2 exhibited high sensitivity to sunlight intensity because its leaves were vulnerable to burning by natural sunlight . ...
... Baiye1 was due to chloroplast dysplasiaand the blocking synthesis of Pchlide a from Mg-proto IX 58 . Deficiency in grana stacking in chloroplasts and inhibition of gene expression related to chloroplast localization may also lead to the production of albino seedlings 59 . By assembling and comparing the chloroplast genomes of the G. microphylla mutant and G. microphylla, we found that their sequences were completely identical. ...
The genus Gleditsia has significant medicinal and economic value, but information about the chloroplast genomic characteristics of Gleditsia species has been limited. Using the Illumina sequencing, we assembled and annotated the whole chloroplast genomes of seven Gleditsia species (Gleditsia sinensis, Gleditsia japonica var. delavayi (G. delavayi), G. fera, G. japonica, G. microphylla, Fructus Gleditsiae Abnormalis (Zhū Yá Zào), G. microphylla mutant). The assembled genomes revealed that Gleditsia species have a typical circular tetrad structure, with genome sizes ranging from 162,746 to 170,907 bp. Comparative genomic analysis showed that most (65.8–75.8%) of the abundant simple sequence repeats in Gleditsia and Gymnocladus species were located in the large single copy region. The Gleditsia chloroplast genome prefer T/A-ending codons and avoid C/G-ending codons, positive selection was acting on the rpoA, rpl20, atpB, ndhA and ycf4 genes, most of the chloroplast genes of Gleditsia species underwent purifying selection. Expansion and contraction of the inverted repeat (IR)/single copy (SC) region showed similar patterns within the Gleditsia genus. Polymorphism analysis revealed that coding regions were more conserved than non-coding regions, and the IR region was more conserved than the SC region. Mutational hotspots were mostly found in intergenic regions such as “rps16-trnQ”, “trnT-trnL”, “ndhG-ndhI”, and "rpl32-trnL” in Gleditsia. Phylogenetic analysis showed that G. fera is most closely related to G. sinensis,G. japonica and G. delavayi are relatively closely related. Zhū Yá Zào can be considered a bud mutation of the G. sinensis. The albino phenotype of G. microphylla mutant is not caused by variations in the chloroplast genome, and that the occurrence of the albino phenotype may be due to mutations in chloroplast-related genes involved in splicing or localization functions. This study will help us enhance our exploration of the genetic evolution and geographical origins of the Gleditsia genus.
... The abnormal chloroplast structure directly destroyed the regular deposition of chlorophyll in cells and thus determined the leaf phenotype. This mechanism of leaf variation was widely confirmed in previous studies [19]. Interestingly, compared with the complete disruption of grana lamella in Y-G and Y, the granum structure stacked by thylakoid was partially reserved in Y-R. ...
Leaf color variations in tea plants were widely considered due to their attractive phenotypes and characteristic flavors. The molecular mechanism of color formation was extensively investigated. But few studies focused on the transformation process of leaf color change. In this study, four strains of ‘Baijiguan’ F1 half-sib generation with similar genetic backgrounds but different colors were used as materials, including Green (G), Yellow-Green (Y-G), Yellow (Y), and Yellow-Red (Y-R). The results of broadly targeted metabolomics showed that 47 metabolites were differentially accumulated in etiolated leaves (Y-G, Y, and Y-R) as compared with G. Among them, lipids were the main downregulated primary metabolites in etiolated leaves, which were closely linked with the thylakoid membrane and chloroplast structure. Flavones and flavonols were the dominant upregulated secondary metabolites in etiolated leaves, which might be a repair strategy for reducing the negative effects of dysfunctional chloroplasts. Further integrated analysis with the transcriptome indicated different variation mechanisms of leaf phenotype in Y-G, Y, and Y-R. The leaf color formation of Y-G and Y was largely determined by the increased content of eriodictyol-7-O-neohesperidoside and the enhanced activities of its modification process, while the color formation of Y-R depended on the increased contents of apigenin derivates and the vigorous processes of their transportation and transcription factor regulation. The key candidate genes, including UDPG, HCT, CsGSTF1, AN1/CsMYB75, and bHLH62, might play important roles in the flavonoid pathway.
... including 'Anji Baicha (Baiye 1)', 'Qiannianxue', 'Huabai 1', and 'Xiaoxueya' (Li et al., 2019;Ma et al., 2018;Zeng et al., 2019). ...
... As an albino cultivar, ZH2 has a higher content of theanine and free amino acids and a lower content of carotenoids, catechins, and anthocyanin than the evergreen tea cultivar 'Longjing43' whitish when the environmental temperature is below 20 C in early spring (Li et al., 2019;Zeng et al., 2019). Although the critical temperature of ZH2 was not determined in this study, its temperature range was narrow. ...
... (G) DMR distribution in each context under different gene regions.content was dramatically reduced, and stacked grana were absent in its young shoots, which caused the albino phenotype(Figure 1), similar to that of some other temperature-sensitive albino tea trees, such as 'Huabai 1,' 'Xiaoxueya,' and 'Anji Baicha'(Li et al., 2019; Ma et al., 2018;Zeng et al., 2019). However, the critical temperature for leaf color change varies among temperature-sensitive albino tea cultivars. ...
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.
... The GC-MS parameters used for ethylamine determination were set according to a previous study [30] . The splitless mode was used and the MS scan was 30−400 m/z and MS were operated under selective ion monitoring (SIM) mode. ...
... It was reported that EA content positively correlates with the theanine biosynthesis activity [20,30] . Interestingly, we also noticed that accumulation patterns of Glu and EA were similar with that of theanine in these cultivars (Figs 2c, 4a & b). ...
Tea processed from albino/etiolated tea shoots is favored by consumers because of its high theanine accumulation. To explore why theanine accumulates highly in new shoots of albino/etiolated tea cultivars, we compared theanine content in shoots and roots of albino/etiolated and common green tea cultivars. Results suggested that high theanine accumulation in albino/etiolated tea shoots was likely not caused by higher theanine biosynthesis in roots. Further analyses suggested that CsAlaDC-catalyzed ethylamine biosynthesis and CsGOGAT1-catalyzed glutamate biosynthesis were more active, and CsGGT2-catalyzed theanine catabolism was weaker in new shoots of these albino/etiolated tea plant cultivars. Therefore, the high theanine accumulation in albino/etiolated shoots is probably contributed by the strong theanine biosynthesis and weak catabolism in new shoots. These findings provided more comprehensive insights into the high accumulation of theanine in new shoots of albino/etiolated tea cultivars, and the knowledge can be used in plant breeding for new cultivars with higher theanine accumulation. Citation: Zhu B, Qiao S, Li M, Cheng H, Ma Q, et al. 2023. Strong biosynthesis and weak catabolism of theanine in new shoots contribute to the high theanine accumulation in Albino/etiolated tea plant (Camellia sinensis). Beverage Plant Research 3:23 https://doi.
... After CsTCS1 was inhibited, the levels of expression of the key genes CsSAM and CsTIDH involved in caffeine synthesis were also reduced. Based on the results, the new leaves of the cuttings grew pale pink when CsTCS1 was silenced, and we hypothesized that the silencing of CsTCS1 in the leaves could have inhibited the genes related to the control chlorophyll synthesis [43] and upregulated the expression of genes that control anthocyanin synthesis [44], resulting in the changes in leaf color in the CsTCS1-silenced plants. In addition, the accumulation of caffeine in tea leaves was reduced. ...
Tea (Camellia sinensis [L.] O. Kuntze) is an important global economic crop and is considered to enhance health. However, the functions of many genes in tea plants are unknown. Virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) is an effective tool for the analysis of gene functions, although this method has rarely been reported in tea plants. In this study, we established an effective VIGS-mediated gene knockout technology to understand the functional identification of large-scale genomic sequences in tea plants. The results showed that the VIGS system was verified by detecting the virus and using a real-time quantitative reverse transcription PCR (qRT-PCR) analysis. The reporter gene CsPOR1 (protochlorophyllide oxidoreductase) was silenced using the vacuum infiltration method, and typical photobleaching and albino symptoms were observed in newly sprouted leaves at the whole plant level of tea after infection for 12 d and 25 d. After optimization, the VIGS system was successfully used to silence the tea plant CsTCS1 (caffeine synthase) gene. The results showed that the relative caffeine content was reduced 6.26-fold compared with the control, and the level of expression of CsPOR1 decreased by approximately 3.12-fold in plants in which CsPOR1 was silenced. These results demonstrate that VIGS can be quickly and efficiently used to analyze the function of genes in tea plants. The successful establishment of VIGS could eliminate the need for tissue culture by providing an effective method to study gene function in tea plants and accelerate the process of functional genome research in tea.
... While, for light-sensitive tea mutants, strong sunlight up-regulated the expression of genes involved in the carotenoid pathway, resulting in the accumulation of carotenoids, and strong sunlight induced hypoplasia of chloroplasts by suppressing the development of grana stacking and thylakoids, so the lack of chlorophylls and the accumulation of carotenoids were considered to cause the yellow color formation in etiolated shoots [10]. Similar research results have been reported in some etiolated mutants, for example, 'Anji baicha' [26], 'Huabai 1' [13], 'Xiaoxueya' [27], 'Yujinxiang' [28], 'Huangjinya' [20], and 'Menghaihuangya' [29]. The current study results showed that the total chlorophyll content of normal green leaves of 'Yinghong 9' was significantly higher than that of yellow leaves of 'Huangyu' ( Figure 1B). ...
Leaf color is one of the key factors involved in determining the processing suitability of tea. It relates to differential accumulation of flavor compounds due to the different metabolic mechanisms. In recent years, photosensitive etiolation or albefaction is an interesting direction in tea research field. However, the molecular mechanism of color formation remains unclear since albino or etiolated mutants have different genetic backgrounds. In this study, wide-target metabolomic and transcriptomic analyses were used to reveal the biological mechanism of leaf etiolation for ‘Huangyu’, a bud mutant of ‘Yinghong 9’. The results indicated that the reduction in the content of chlorophyll and the ratio of chlorophyll to carotenoids might be the biochemical reasons for the etiolation of ‘Huangyu’ tea leaves, while the content of zeaxanthin was significantly higher. The differentially expressed genes (DEGs) involved in chlorophyll and chloroplast biogenesis were the biomolecular reasons for the formation of green or yellow color in tea leaves. In addition, our results also revealed that the changes of DEGs involved in light-induced proteins and circadian rhythm promoted the adaptation of etiolated tea leaves to light stress. Variant colors of tea leaves indicated different directions in metabolic flux and accumulation of flavor compounds.
... To overcome these barriers, two different strategies have been widely applied. The first one relies on another green tea cultivar as control [17,25,29,[34][35][36]. The drawback of this method is that the selected control green tea cultivar is genetically unrelated to the albino tea under study. ...
The non-volatile and volatile metabolites in tea confer the taste and odor characteristics of tea fusion, as well as shape the chemical base for tea quality. To date, it remains largely elusive whether there are metabolic crosstalks among non-volatile metabolites and volatile metabolites in the tea tree. Here, we generated an F1 half-sib population by using an albino cultivar of Camellia sinensis cv Baijiguan as the maternal parent, and then we quantified the non-volatile metabolites and volatile metabolites from individual half-sibs. We found that the EGC and EGCG contents of the albino half-sibs were significantly lower than those of the green half-sibs, while no significant differences were observed in total amino acids, caffeine, and other catechin types between these two groups. The phenylpropanoid pathway and the MEP pathway are the dominant routes for volatile synthesis in fresh tea leaves, followed by the MVA pathway and the fatty acid-derivative pathway. The total volatile contents derived from individual pathways showed large variations among half-sibs, there were no significant differences between the albino half-sibs and the green half-sibs. We performed a comprehensive correlation analysis, including correlations among non-volatile metabolites, between volatile synthesis pathways and non-volatile metabolites, and among the volatiles derived from same synthesis pathway, and we identified several significant positive or negative correlations. Our data suggest that the synthesis of non-volatile and volatile metabolites is potentially connected through shared intermediates; feedback inhibition, activation, or competition for common intermediates among branched pathways may co-exist; and cross-pathway activation or inhibition, as well as metabolome channeling, were also implicated. These multiple metabolic regulation modes could provide metabolic plasticity to direct carbon flux and lead to diverse metabolome among Baijiguan half-sibs. This study provides an essential knowledge base for rational tea germplasm improvements.
... To analyse the reasons for the yellow leaf phenotype of MHHY, we measured the pigment contents of MHHY_May, MHHY_July, and YK10_May and found that the chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids in MHHY were significantly lower than those of the control (Fig. 1B). Studies on various albino tea plants, such as 'Anji Baicha' (Li et al. 2016), 'Huabai 1' (Ma et al. 2018), 'Xiaoxueya' (Li et al. 2019), and 'Yujinxiang' (Xu et al. 2020), have also found that the chlorophyll content of albino leaves is significantly reduced. In addition, compared to carotenoids, MHHY_May and YK10_May had greater differences in chlorophyll a, chlorophyll b, and total chlorophyll, and chlorophyll b was the most different component (the difference was more than 6 times). ...
... Among the differentially expressed genes, we also obtained 22 differentially expressed genes related to photosynthesis, including 10 genes related to photosystem II, photosystem I, cytochrome b6/f complex, and photosynthetic election transport, and except for CSS0038460 (PetH), other genes were significantly downregulated in MHHY_May (Fig. 3B). Several previous studies (Li et al. 2019;Jiang et al. 2020) have found that photosynthesis complex-related proteins are significantly inhibited in albino tea plants, as well as a lack of chlorophylls and carotenoids in the leaves. As a result, inhibiting these light response complex-related genes might affect the development of thylakoids and the structure of chloroplasts and might reduce the chlorophyll content of MHHY. ...
Albino tea plants generally have higher theanine, which causes their tea leaves to taste fresher, and they are an important mutant for the breeding of tea plant varieties. Earlier, we reported an albino germplasm, ‘Menghai Huangye’ (MHHY), from Yunnan Province and found that it has a lower chlorophyll content during the yellowing stage, but the mechanism underlying low chlorophyll and the yellowing phenotype is still unclear. In this study, the pigment contents of MHHY_May (yellowing, low chlorophyll), MHHY_July (regreening, normal chlorophyll), and YK10_May (green leaves, normal chlorophyll) were determined, and the results showed that the lower chlorophyll content might be an important reason for the formation of the yellowing phenotype of MHHY. Through transcriptome sequencing, we obtained 654 candidates for differentially expressed genes (DEGs), among which 4 genes were related to chlorophyll synthesis, 10 were photosynthesis-related, 34 were HSP family genes, and 19 were transcription factor genes. In addition, we analysed the transcription levels of the key candidate genes in MHHY_May and MHHY_July and found that they are consistent with the expression trends in MHHY_May and YK10_May, which further indicates that the candidate differential genes we identified are likely to be key candidate factors involved in the low chlorophyll content and yellowing of MHHY. In summary, our findings will assist in revealing the low chlorophyll content of MHHY and the formation mechanism of yellowing tea plants and will be applied to the selection and breeding of albino tea cultivars in the future.
... However, the concentration of squalene in tea leaves varied with the development of tea shoots (23). Chemical compositions of tea leaves varied with tea cultivars (23)(24)(25)(26). However, the information on the squalene variation between tea cultivars has not been available yet. ...
Squalene is a precursor of steroids with diverse bioactivities. Tea was previously found to contain squalene, but its variation between tea cultivars remains unknown. In this study, tea leaf squalene sample preparation was optimized and the squalene variation among 30 tea cultivars was investigated. It shows that squalene in the unsaponified tea leaf extracts was well separated on gas chromatography profile. Saponification led to a partial loss of squalene in tea leaf extract and so it is not an essential step for preparing squalene samples from tea leaves. The tea leaf squalene content increased with the maturity of tea leaf and the old leaves grown in the previous year had the highest level of squalene among the tested samples. The squalene levels in the old leaves of the 30 tested cultivars differentiated greatly, ranging from 0.289 to 3.682 mg/g, in which cultivar “Pingyun” had the highest level of squalene. The old tea leaves and pruned littering, which are not used in tea production, are an alternative source for natural squalene extraction.
