Strategies for studying in vivo biochemical formation pathways and multilevel distributions of quality or function-related specialized metabolites in tea (Camellia sinensis).

Tea (Camellia sinensis) contains bioactive metabolites such as catechins, amino acids, caffeine, and aroma compounds that contribute to characteristic tea function and flavor. Therefore, studies on biochemical formation pathways and occurrences of these characteristic specialized metabolites in tea plants are important, providing essential information for the regulation and improvement of tea quality and function. Owing to the lack of a stable genetic transformation system, obtaining direct in vivo evidence of the formation of characteristic tea specialized metabolites is difficult. Herein, we review potential strategies for studying in vivo biochemical formation pathways and multilevel distributions of specialized metabolites in tea. At the individual plant level, stable isotope-labeled precursor tracing is an approach to discovering the pathways of some specialized metabolites specifically occurring in tea and elucidating the formation of tea specialized metabolites in response to stresses. At the within-tissue level, imaging mass spectrometry can be used to investigate the in situ localization of characteristic specialized metabolites within tea tissue without sample destruction. At the cellular or subcellular level, nonaqueous fractionation is a feasible method for characterizing the distributions of nonvolatile metabolites in subcellular organs. These approaches will help explain the characteristic scientific problems in tea secondary metabolism and provide more precise information to improve tea quality or function. HighlightsMultilevel distributions of metabolites in tea are important for tea quality improvement.Stable isotope-labeled precursor tracing method can be used to study formations of tea metabolites at individual plant level.Imaging mass spectrometry can be used to investigate the in situ localization of metabolites within tea tissue.Nonaqueous fractionation is a feasible method for characterizing the distributions of metabolites in subcellular organs.

Nonaqueous fractionation and overexpression of fluorescent-tagged enzymes reveals the subcellular sites of L-theanine biosynthesis in tea.

l-Theanine is a specialized metabolite in the tea (Camellia sinensis) plant which can constitute over 50% of the total amino acids. This makes an important contribution to tea functionality and quality, but the subcellular location and mechanism of biosynthesis of l-theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizing l-theanine in tea. Using a nonaqueous fractionation method, we determined the subcellular distribution of l-theanine in tea shoots and roots and used transient expression in Nicotiana or Arabidopsis to investigate in vivo functions of l-theanine synthetase and also to determine the subcellular localization of fluorescent-tagged proteins by confocal laser scanning microscopy. In tea root tissue, the cytosol was the main site of l-theanine biosynthesis, and cytosol-located CsTSI was the key l-theanine synthase. In tea shoot tissue, l-theanine biosynthesis occurred mainly in the cytosol and chloroplasts and CsGS1.1 and CsGS2 were most likely the key l-theanine synthases. In addition, l-theanine content and distribution were affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds.

Metabolism of Gallic Acid and Its Distributions in Tea (Camellia sinensis) Plants at the Tissue and Subcellular Levels.

In tea (Camellia sinensis) plants, polyphenols are the representative metabolites and play important roles during their growth. Among tea polyphenols, catechins are extensively studied, while very little attention has been paid to other polyphenols such as gallic acid (GA) that occur in tea leaves with relatively high content. In this study, GA was able to be transformed into methyl gallate (MG), suggesting that GA is not only a precursor of catechins, but also can be transformed into other metabolites in tea plants. GA content in tea leaves was higher than MG content-regardless of the cultivar, plucking month or leaf position. These two metabolites occurred with higher amounts in tender leaves. Using nonaqueous fractionation techniques, it was found that GA and MG were abundantly accumulated in peroxisome. In addition, GA and MG were found to have strong antifungal activity against two main tea plant diseases, Colletotrichum camelliae and Pseudopestalotiopsis camelliae-sinensis. The information will advance our understanding on formation and biologic functions of polyphenols in tea plants and also provide a good reference for studying in vivo occurrence of specialized metabolites in economic plants.

Microscopic Analyses of Fruit Cell Plastid Development in Loquat (Eriobotrya japonica) during Fruit Ripening.

Plastids are sites for carotenoid biosynthesis and accumulation, but detailed information on fruit plastid development and its relation to carotenoid accumulation remains largely unclear. Here, using Baisha (BS; white-fleshed) and Luoyangqing (LYQ; red-fleshed) loquat (Eriobotrya japonica), a detailed microscopic analysis of plastid development during fruit ripening was carried out. In peel cells, chloroplasts turned into smaller chromoplasts in both cultivars, and the quantity of plastids in LYQ increased by one-half during fruit ripening. The average number of chromoplasts per peel cell in fully ripe fruit was similar between the two cultivars, but LYQ peel cell plastids were 20% larger and had a higher colour density, associated with the presence of larger plastoglobules. In flesh cells, chromoplasts could be observed only in LYQ during the middle and late stages of ripening, and the quantity on a per-cell basis was higher than that in peel cells, but the size of chromoplasts was smaller. It was concluded that chromoplasts are derived from the direct conversion of chloroplasts to chromoplasts in the peel, and from de novo differentiation of proplastids into chromoplasts in flesh. The relationship between plastid development and carotenoid accumulation is discussed.

Recent Advances in the Emission and Functions of Plant Vegetative Volatiles.

Plants synthesize and emit a large variety of volatile organic compounds, which possess extremely important ecological functions. In most case, most plant volatiles are liquids, rather than gases, at room temperature. Some volatiles are emitted "on demand" when plants, especially vegetative parts, are exposed to abiotic or biotic stress. In this review, we summarize some of the highlights of plant vegetative volatile emission and functions research published during the past few years.

Involvement of multiple phytoene synthase genes in tissue- and cultivar-specific accumulation of carotenoids in loquat.

Differences in carotenoid accumulation between tissues and cultivars is common in plants. White-fleshed loquat cultivars had low levels of carotenoids in the flesh, but accumulated carotenoids in peel when ripe, and the leaves accumulated similar carotenoids to those in the red-fleshed loquat cultivars. The catalytic activity and expression patterns of four phytoene synthase (PSY) genes, EjPSY1, EjPSY2A, EjPSY2B, and EjPSY3, were analysed to understand their roles in different loquat (Eriobotrya japonica Lindl.) types. EjPSY1 was responsible for carotenoid synthesis in the fruit peel but not the flesh, whereas EjPSY2A was responsible for carotenoid accumulation in flesh of ripening fruit. A mutant EjPSY2A (d) , with the same tissue specificity and expression level as EjPSY2A, but lacking the C-terminal region and corresponding catalytic activity, was discovered in white-fleshed varieties, explaining the lack of carotenoids in the white flesh. The catalytic role of EjPSY2B was most significant in leaves. The tissue-specific expression of EjPSY1 and EjPSY2B explained well how peel and leaf tissues can still accumulate carotenoids in white-fleshed cultivars, which have lost the functional EjPSY2A. EjPSY3 mRNA abundance was ~1000-fold less than that of other PSY mRNAs in all tissues examined. In addition, neither the normal sized transcript nor two alternatively spliced forms, EjPSY3α in LYQ and EjPSY3ß in BS cultivars, encoded functional enzymes, and it is concluded that EjPSY3 plays no role in carotenoid accumulation. In addition, it was noted that recruitment of PSY genes for expression in specific tissues of different plants has occurred independently of gene structure and evolutionary origin.

Biochemical Pathways of Salicylic Acid Derived from l-Phenylalanine in Plants with Different Basal SA Levels.

As a plant hormone, salicylic acid (SA) has diverse regulatory roles in plant growth and stress resistance. Although SA is widely found in plants, there is substantial variation in basal SA among species. Tea plant is an economically important crop containing high contents of SA whose synthesis pathway remains unidentified. The phenylalanine ammonia-lyase (PAL) pathway is responsible for basal SA synthesis in plants. In this study, isotopic tracing and enzymatic assay experiments were used to verify the SA synthesis pathway in tea plants and evaluate the variation in phenylalanine-derived SA formation among 11 plant species with different levels of SA. The results indicated that SA could be synthesized via PAL in tea plants and conversion efficiency from benzoic acid to SA might account for variation in basal SA among plant species. This research lays the foundation for an improved understanding of the molecular regulatory mechanism for SA biosynthesis.

Biosynthetic Pathway and Bioactivity of Vanillin, a Highly Abundant Metabolite Distributed in the Root Cortex of Tea Plants (Camellia sinensis).

Volatiles are important for plant root stress resistance. The diseases in tea root are serious, causing major losses. The volatile composition in tea root and whether it can resist diseases remain unclear. In this study, the volatile composition in different tea tissues was revealed. The vanillin content was higher in the root (mainly in root cortex) than in aerial parts. The antifungal effects of vanillin on pathogenic fungi in tea root were equal to or greater than those of other metabolites. O-methyltransferase (CsOMT), a key enzyme in one of two biosynthetic pathways of vanillin, converted protocatechualdehyde to vanillin in vitro. Furthermore, its characteristics and kinetic parameters were studied. In Arabidopsis thaliana protoplasts, the transiently expressed CsOMT was localized in the cytoplasm and nucleus. These findings have clarified the formation and bioactivities of volatiles in tea roots and provided a theoretical basis for understanding how tea plants resist root diseases.

Effect of the biosynthesis of the volatile compound phenylacetaldehyde on chloroplast modifications in tea (Camellia sinensis) plants.

Plant volatile compounds have important physiological and ecological functions. Phenylacetaldehyde (PAld), a volatile phenylpropanoid/benzenoid, accumulates in the leaves of tea (Camellia sinensis) plants grown under continuous shading. This study was conducted to determine whether PAld production is correlated with light and to elucidate the physiological functions of PAld in tea plants. Specifically, the upstream mechanism modulating PAld biosynthesis in tea plants under different light conditions as well as the effects of PAld on chloroplast/chlorophyll were investigated. The biosynthesis of PAld was inhibited under light, whereas it was induced in darkness. The structural gene encoding aromatic amino acid aminotransferase 1 (CsAAAT1) was expressed at a high level in darkness, consistent with its importance for PAld accumulation. Additionally, the results of a transcriptional activation assay and an electrophoretic mobility shift assay indicated CsAAAT1 expression was slightly activated by phytochrome-interacting factor 3-2 (CsPIF3-2), which is a light-responsive transcription factor. Furthermore, PAld might promote the excitation of chlorophyll in dark-treated chloroplasts and mediate electron energy transfer in cells. However, the accumulated PAld can degrade chloroplasts and chlorophyll, with potentially detrimental effects on photosynthesis. Moreover, PAld biosynthesis is inhibited in tea leaves by red and blue light, thereby decreasing the adverse effects of PAld on chloroplasts during daytime. In conclusion, the regulated biosynthesis of PAld in tea plants under light and in darkness leads to chloroplast modifications. The results of this study have expanded our understanding of the biosynthesis and functions of volatile phenylpropanoids/benzenoids in tea leaves.

Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits.

Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 µg g(-1) and 13 µg g(-1) fresh weight (FW), respectively, and for BS 19 µg g(-1) and 0.27 µg g(-1) FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene ß-cyclase (CYCB) were higher in the peel, and CYCB and ß-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit.

Mechanism underlying the carotenoid accumulation in shaded tea leaves.

Carotenoids contribute to tea leaf coloration and are the precursors of important aromatic compounds. Shading can promote the accumulation of carotenoids in tea leaves, but the underlying mechanism remains unknown. In the study, we analyzed the content and composition of carotenoids, and transcript levels and functions of related genes in carotenoid biosynthesis using HPLC, qRT-PCR, and heterologous expression system. It was found that long-term shading (14 days, 90% shading) significantly increased the total carotenoid content in tea leaves, and increased the expression of non-mevalonate pathway (MEP) genes (CsDXS1 and CsDXS3) and key genes in carotenoid synthesis pathway (CsPSY, CsLCYB, and CsLCYE). Long-term exposure to darkness (14 days, 0 lx) decreased the transcription of most carotenoid biosynthetic genes and adversely affected carotenoid accumulation. Furthermore, CsDXS1, CsDXS3, CsPSY, CsLCYB, and CsLCYE were functionally identified and contributed to the enhanced accumulation of carotenoids in tea leaves in response to long-term shading.

Stable Isotope-Labeled Precursor Tracing Reveals that l-Alanine is Converted to l-Theanine via l-Glutamate not Ethylamine in Tea Plants In Vivo.

Tea plants (Camellia sinensis) specifically produce l-theanine, which contributes to tea function and taste. Ethylamine is a limiting factor differentiating l-theanine accumulation between tea and other plants. Ethylamine has long been assumed to be derived from l-alanine in tea. In this study, the l-alanine content in tea root cells was mainly located in vacuoles and mitochondria using a nonaqueous fractionation technique, while alanine decarboxylase in tea (CsADC) was located in the cytoplasm. Although CsADC was able to catalyze l-alanine decarboxylation to produce ethylamine in vitro, it may not provide the same enzyme activity in tea plants. Stable isotope-labeled precursor tracing in tea plants discovered that l-alanine is not a direct precursor of ethylamine but a precursor of l-glutamate, which is involved in l-theanine biosynthesis in tea. Cortex with epidermis from root tissue was the main location of ethylamine. In summary, l-alanine is converted to l-theanine via l-glutamate not ethylamine in tea plants in vivo.

Transformation of Salicylic Acid and Its Distribution in Tea Plants (Camellia sinensis) at the Tissue and Subcellular Levels.

Salicylic acid (SA) is a well-known immune-related hormone that has been well studied in model plants. However, less attention has been paid to the presence of SA and its derivatives in economic plants, such as tea plants (Camellia sinensis). This study showed that tea plants were rich in SA and responded differently to different pathogens. Feeding experiments in tea tissues further confirmed the transformation of SA into salicylic acid 2-O-ß-glucoside (SAG) and methyl salicylate. Nonaqueous fractionation techniques confirmed that SA and SAG were mostly distributed in the cytosol of tea leaves, consistent with distributions in other plant species. Furthermore, the stem epidermis contained more SA than the stem core both in C. sinensis cv. "Jinxuan" (small-leaf species) and "Yinghong No. 9" (large-leaf species). Compared with cv. "Yinghong No. 9", cv. "Jinxuan" contained more SAG in the stem epidermis, which might explain its lower incidence rate of wilt disease. This information will improve understanding of SA occurrence in tea plants and provide a basis for investigating the relationship between SA and disease resistance in tea plants.

Characterization of l-Theanine Hydrolase in Vitro and Subcellular Distribution of Its Specific Product Ethylamine in Tea (Camellia sinensis).

l-Theanine has a significant role in the taste of tea (Camellia sinensis) infusions. Our previous research indicated that the lower l-theanine metabolism in ethylamine and l-glutamate is a key factor that explains the higher content of l-theanine in albino tea with yellow or white leaves, compared with that of normal tea with green leaves. However, the specific genes encoding l-theanine hydrolase in tea remains unknown. In this study, CsPDX2.1 was cloned together with the homologous Arabidopsis PDX2 gene and the recombinant protein was shown to catalyze l-theanine hydrolysis into ethylamine and l-glutamate in vitro. There were higher CsPDX2.1 transcript levels in leaf tissue and lower transcripts in the types of albino (yellow leaf) teas compared with green controls. The subcellular location of ethylamine in tea leaves was shown to be in the mitochondria and peroxisome using a nonaqueous fractionation method. This study identified the l-theanine hydrolase gene and subcellular distribution of ethylamine in tea leaves, which improves our understanding of the l-theanine metabolism and the mechanism of differential accumulation of l-theanine among tea varieties.

Visualized analysis of within-tissue spatial distribution of specialized metabolites in tea (Camellia sinensis) using desorption electrospray ionization imaging mass spectrometry.

Although specialized metabolite distributions in different tea (Camellia sinensis) tissues has been studied extensively, little is known about their within-tissue distribution owing to the lack of nondestructive methodology. In this study, desorption electrospray ionization imaging mass spectrometry was used to investigate the within-tissue spatial distributions of specialized metabolites in tea. To overcome the negative effects of the large amount of wax on tea leaves, several sample preparation methods were compared, with a Teflon-imprint method established for tea leaves. Polyphenols are characteristic metabolites in tea leaves. Epicatechin gallate/catechin gallate, epigallocatechin gallate/gallocatechin gallate, and gallic acid were evenly distributed on both sides of the leaves, while epicatechin/catechin, epigallocatechin/gallocatechin, and assamicain A were distributed near the leaf vein. L-Theanine was mainly accumulated in tea roots. L-Theanine and valinol were distributed around the outer root cross-section. The results will advance our understanding of the precise localizations and in-vivo biosyntheses of specialized metabolites in tea.

Lycopene cyclases determine high α-/ß-carotene ratio and increased carotenoids in bananas ripening at high temperatures.

Bananas are a recommended food source to alleviate vitamin A deficiency because they contain a high ratio of provitamin A precursors. The objective of this study was to investigate carotenoid accumulation pattern in banana fruits during postharvest ripening and the mechanisms regulating this process. Ripe banana pulp had an unusually high α-/ß-carotene ratio (1.05), and the carotenoid contents increased (p ≤ 0.05) under light and high temperature. We analyzed the sequences, transcript levels, and functions of genes involved in carotenoid synthesis. The high ratio of α-/ß-carotene in ripe banana fruit was explained by the high flux to the α-carotene biosynthetic pathway, as reflected by high transcript levels of LCYE, and the weak flux to the ß-carotene branch of the biosynthetic pathway due to inactive MaLCYB1.2. High temperature during ripening up-regulated the transcript levels of genes involved in the α- and ß-carotene biosynthesis pathways and the activities of their encoded enzymes.

Differential accumulation of specialized metabolite l-theanine in green and albino-induced yellow tea (Camellia sinensis) leaves.

l-Theanine is a specialized metabolite in tea (Camellia sinensis) leaves that contributes to tea function and quality. Yellow tea leaves (albino) generally have higher l-theanine contents than green tea leaves (normal), but the reason is unknown. The objective of this study was to investigate why l-theanine is accumulated in yellow tea leaves. We compared original normal leaves (green) and light-sensitive albino leaves (yellow) of cv. Yinghong No. 9. The l-theanine content was significantly higher in yellow leaves than in green leaves (p ≤ 0.01). After supplementation with [2H5]-l-theanine, yellow leaves catabolized less [2H5]-l-theanine than green leaves (p ≤ 0.05). Furthermore, most plants contained the enzyme catalyzing l-theanine conversion to ethylamine and l-glutamic acid. In conclusion, l-theanine accumulation in albino-induced yellow tea leaves was due to weak l-theanine catabolism. The differential accumulation mechanism differed from the l-theanine accumulation mechanism in tea and other plants.

Comparative analysis of pigments in red and yellow banana fruit.

Color is an important characteristic determining the fruit value. Although ripe bananas usually have yellow peels, several banana cultivars have red peels. As details of the pigments in banana fruits are unknown, we investigated these pigments contents and compositions in the peel and pulp of red cultivar 'Hongjiaowang' and yellow cultivar 'Baxijiao' by UPLC-PDA-QTOF-MS and HPLC-PDA techniques. The 'Hongjiaowang' peel color was mainly determined by the presence of anthocyanin-containing epidermal cells. Rutinoside derivatives of cyanidin, peonidin, petunidin, and malvidin were unique to the red peel, and possibly responsible for the red color. 'Hongjiaowang' contained higher total content of carotenoids than 'Baxijiao' in both pulp and peel. Lutein, α-carotene, and ß-carotene were main carotenoids, which might play a more important role than flavonoids in producing the yellow banana color owing to the properties and distribution in the fruit. The information will help us understand a complete profile of pigments in banana.

Biosynthesis of Jasmine Lactone in Tea ( Camellia sinensis) Leaves and Its Formation in Response to Multiple Stresses.

Jasmine lactone has a potent odor that contributes to the fruity, sweet floral aroma of tea ( Camellia sinensis). Our previous study demonstrated that jasmine lactone was mostly accumulated at the turnover stage of the oolong tea manufacturing process. This study investigates the previously unknown mechanism of formation of jasmine lactone in tea leaves exposed to multiple stresses occurring during the growth and manufacturing processes. Both continuous mechanical damage and the dual stress of low temperature and mechanical damage enhanced jasmine lactone accumulation in tea leaves. In addition, only one pathway, via hydroperoxy fatty acids from unsaturated fatty acid, including linoleic acid and α-linolenic acid, under the action of lipoxygenases (LOXs), especially CsLOX1, was significantly affected by these stresses. This is the first evidence of the mechanism of jasmine lactone formation in tea leaves and is a characteristic example of plant volatile formation in response to dual stress.