Yinyin Liao

Functional characterizations of β-glucosidases involved in aroma compound formation in tea (Camellia sinensis)

Tea (Camellia sinensis) aroma is an important factor affecting tea quality. Many tea aroma compounds are present as glycosidically conjugated forms in tea leaves, and can be hydrolyzed by β-glucosidase (β-Glu) and β-primeverosidase to release free tea aromas. β-Primeverosidase has been identified and functionally characterized, while β-Glu has not been identified in tea leaves. In the present study, we established a yeast expression system to recombine CsGH1BG1, CsGH3BG1, and CsGH5BG1, which belonged to GH1, GH3, and GH5 families in plants, respectively. These three recombinant Csβ-Glus hydrolyzed the β-glucopyranosidically conjugated aromas to form free aromas, suggesting that there was no specific Csβ-Glus for the hydrolysis of β-glucopyranosidically conjugated aromas in vitro. Furthermore, subcellular localization of the Csβ-Glus indicated that CsGH1BG1 and CsGH3BG1 were located in the cytosol and vacuole, respectively, while CsGH5BG1 was located in the cell wall. This suggested that CsGH1BG1 and CsGH3BG1 might be responsible for the hydrolysis of β-glucopyranosidically conjugated aromas in tea leaves during the tea manufacturing process. This study provides the first evidence of Csβ-Glus in tea leaves, and will advance understanding of tea aroma formation.

Studies on the Biochemical Formation Pathway of the Amino Acid l-Theanine in Tea (Camellia sinensis) and Other Plants

Tea (Camellia sinensis) is the most widely consumed beverage aside from water. The flavor of tea is conferred by certain metabolites, especially l-theanine, in C. sinensis. To determine why more l-theanine accumulates in C. sinensis than in other plants, we compare l-theanine contents between C. sinensis and other plant species (Camellia nitidissima, Camellia japonica, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum) and use a stable isotope labeling approach to elucidate its biosynthetic route. We quantify relevant intermediates and metabolites by mass spectrometry. l-Glutamic acid, a precursor of l-theanine, is present in most plants, while ethylamine, another precursor of l-theanine, specifically accumulates in Camellia species, especially C. sinensis. Most plants contain the enzyme/gene catalyzing the conversion of ethylamine and l-glutamic acid to l-theanine. After supplementation with [2H5]ethylamine, all the plants produce [2H5]l-theanine, which suggests that ethylamine availability is the reason for the difference in l-theanine accumulation between C. sinensis and other plants.

An alternative pathway for the formation of aromatic aroma compounds derived from l-phenylalanine via phenylpyruvic acid in tea (Camellia sinensis (L.) O. Kuntze) leaves

Aromatic aroma compounds contribute to flavor of tea (Camellia sinensis (L.) O. Kuntze) and they are mostly derived from l-phenylalanine via trans-cinnamic acid or directly from l-phenylalanine. The objective of this study was to investigate whether an alternative pathway derived from l-phenylalanine via phenylpyruvic acid is involved in formation of aroma compounds in tea. Enzyme reaction with phenylpyruvic acid showed that benzaldehyde, benzyl alcohol, and methyl benzoate were derived from phenylpyruvic acid in tea leaves. Feeding experiments using [2H8]l-phenylalanine indicated that phenylpyruvic acid was derived from l-phenylalanine in a reaction catalyzed by aromatic amino acid aminotransferases (AAATs). CsAAAT1 showed higher catalytic efficiency towards l-phenylalanine (p ≤ 0.001) while CsAAAT2 showed higher catalytic efficiency towards l-tyrosine (p ≤ 0.001). Both CsAAATs were localized in the cytoplasm of leaf cells. In conclusion, an alternative pathway for the formation of aromatic aroma compounds derived from l-phenylalanine via phenylpyruvic acid occurred in tea leaves.

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.

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.

Analytical method for metabolites involved in biosynthesis of plant volatile compounds

Plants synthesize and emit a large variety of volatile organic compounds, which possess extremely important ecological functions. Rapid progress in the method and instruments available to separate and identify plant metabolites has led to intensive investigations of the biosynthetic pathways that form plant volatiles, which has resulted in insightful and useful discoveries. In this review, we summarize and present the successful techniques currently used for studying metabolites involved in the formation of the major classes of plant volatile compounds, including volatile fatty acid derivatives, volatile isoprenoids, volatile phenylpropanoids/benzenoids, carotenoid-derived aroma compounds, and glycosidically bound volatile compounds. We discuss the extraction, identification, and quantitative analysis of many of these metabolites, which are unstable and occur in plants at trace levels.

α-Farnesene and ocimene induce metabolite changes by volatile signaling in neighboring tea (Camellia sinensis) plants

Herbivore-induced plant volatiles (HIPVs) act as direct defenses against herbivores and as indirect defenses by attracting herbivore enemies. However, the involvement of HIPVs in within-plant or plant-to-plant signaling is not fully clarified. Furthermore, in contrast to model plants, HIPV signaling roles in crops have hardly been reported. Here, we investigated HIPVs emitted from tea (Camellia sinensis) plants, an important crop used for beverages, and their involvement in tea plant-to-plant signaling. To ensure uniform and sufficient exposure to HIPVs, jasmonic acid combined with mechanical damage (JAMD) was used to simulate herbivore attacks. Metabonomics techniques based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and gas chromatography-mass spectrometry were employed to determine metabolite changes in undamaged tea plants exposed to JAMD-stimulated volatiles. JAMD-stimulated volatiles mainly enhanced the amounts of 1-O-galloyl-6-O-luteoyl-α-d-glucose, assamicain C, 2,3,4,5-tetrahydroxy-6-oxohexyl gallate, quercetagitrin, 2-(2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-8-yl)-4,5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-3-yl, 3,4-dimethoxybenzoate, 1,3,4,5,6,7-hexahydroxyheptan-2-one, and methyl gallate in neighboring undamaged tea leaves. Furthermore, α-farnesene and β-ocimene, which were produced after JAMD treatments, were identified as two main JAMD-stimulated volatiles altering metabolite profiles of the neighboring undamaged tea leaves. This research advances our understanding of the ecological functions of HIPVs and can be used to develop crop biological control agents against pest insects in the future.

Influence of Plant Growth Retardants on Quality of Codonopsis Radix

Plant growth retardant (PGR) refers to organics that can inhibit the cell division of plant stem tip sub-apical meristem cells or primordial meristem cell. They are widely used in the cultivation of rhizomatous functional plants; such as Codonopsis Radix, that is a famous Chinese traditional herb. However, it is still unclear whether PGR affects the medicinal quality of C. Radix. In the present study, amino acid analyses, targeted and non-targeted analyses by ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC-TOF-MS) and gas chromatography-MS were used to analyze and compare the composition of untreated C. Radix and C. Radix treated with PGR. The contents of two key bioactive compounds, lobetyolin and atractylenolide III, were not affected by PGR treatment. The amounts of polysaccharides and some internal volatiles were significantly decreased by PGR treatment; while the free amino acids content was generally increased. Fifteen metabolites whose abundance were affected by PGR treatment were identified by UPLC-TOF-MS. Five of the up-regulated compounds have been reported to show immune activity, which might contribute to the healing efficacy (“buqi”) of C. Radix. The results of this study showed that treatment of C. Radix with PGR during cultivation has economic benefits and affected some main bioactive compounds in C. Radix.

The sphingolipid biosynthetic enzyme Sphingolipid delta8 desaturase is important for chilling resistance of tomato.

The physiological functions of sphingolipids in animals have been intensively studied, while less attention has been paid to their roles in plants. Here, we reveal the involvement of sphingolipid delta8 desaturase (SlSLD) in the chilling resistance of tomato (Solanum lycopersicum cv. Micro-Tom). We used the virus-induced gene silencing (VIGS) approach to knock-down SlSLD expression in tomato leaves, and then evaluated chilling resistance. Changes in leaf cell structure under a chilling treatment were observed by transmission electron microscopy. In control plants, SlSLD was highly expressed in the fruit and leaves in response to a chilling treatment. The degree of chilling damage was greater in SlSLD-silenced plants than in control plants, indicating that SlSLD knock-down significantly reduced the chilling resistance of tomato. Compared with control plants, SlSLD-silenced plants showed higher relative electrolytic leakage and malondialdehyde content, and lower superoxide dismutase and peroxidase activities after a chilling treatment. Chilling severely damaged the chloroplasts in SlSLD-silenced plants, resulting in the disruption of chloroplast membranes, swelling of thylakoids, and reduced granal stacking. Together, these results show that SlSLD is crucial for chilling resistance in tomato.