Xiumin Fu

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.

Regulation of formation of volatile compounds of tea ( Camellia sinensis ) leaves by single light wavelength

Regulation of plant growth and development by light wavelength has been extensively studied. Less attention has been paid to effect of light wavelength on formation of plant metabolites. The objective of this study was to investigate whether formation of volatiles in preharvest and postharvest tea (Camellia sinensis) leaves can be regulated by light wavelength. In the present study, in contrast to the natural light or dark treatment, blue light (470 nm) and red light (660 nm) significantly increased most endogenous volatiles including volatile fatty acid derivatives (VFADs), volatile phenylpropanoids/benzenoids (VPBs), and volatile terpenes (VTs) in the preharvest tea leaves. Furthermore, blue and red lights significantly up-regulated the expression levels of 9/13-lipoxygenases involved in VFADs formation, phenylalanine ammonialyase involved in VPBs formation, and terpene synthases involved in VTs formation. Single light wavelength had less remarkable influences on formation of volatiles in the postharvest leaves compared with the preharvest leaves. These results suggest that blue and red lights can be promising technology for remodeling the aroma of preharvest tea leaves. Furthermore, our study provided evidence that light wavelength can activate the expression of key genes involved in formation of plant volatiles for the first time.

Does Enzymatic Hydrolysis of Glycosidically Bound Volatile Compounds Really Contribute to the Formation of Volatile Compounds During the Oolong Tea Manufacturing Process

It was generally thought that aroma of oolong tea resulted from hydrolysis of glycosidically bound volatiles (GBVs). In this study, most GBVs showed no reduction during the oolong tea manufacturing process. β-Glycosidases either at protein or gene level were not activated during the manufacturing process. Subcellular localization of β-primeverosidase provided evidence that β-primeverosidase was located in the leaf cell wall. The cell wall remained intact during the enzyme-active manufacturing process. After the leaf cell disruption, GBV content was reduced. These findings reveal that, during the enzyme-active process of oolong tea, nondisruption of the leaf cell walls resulted in impossibility of interaction of GBVs and β-glycosidases. Indole, jasmine lactone, and trans-nerolidol were characteristic volatiles produced from the manufacturing process. Interestingly, the contents of the three volatiles was reduced after the leaf cell disruption, suggesting that mechanical damage with the cell disruption, which is similar to black tea manufacturing, did not induce accumulation of the three volatiles. In addition, 11 volatiles with flavor dilution factor ≥4(4) were identified as relatively potent odorants in the oolong tea. These results suggest that enzymatic hydrolysis of GBVs was not involved in the formation of volatiles of oolong tea, and some characteristic volatiles with potent odorants were produced from the manufacturing process.

Proteolysis of chloroplast proteins is responsible for accumulation of free amino acids in dark-treated tea (Camellia sinensis) leaves.

Shade management (dark treatment) on tea (Camellia sinensis) plants is a common approach to improve free amino acids in raw materials of tea leaves. However, the reason for amino acid accumulation in dark-treated tea leaves is still unknown. In the present study, dark treatment significantly increased content of free amino acids and reduced content of soluble proteins in tea leaves. Quantitative proteomics analysis showed that most enzymes involved in biosyntheses of amino acids were down-accumulated by dark treatment. Chloroplast numbers reduced in dark-treated leaves and the content of soluble proteins reduced in the chloroplasts isolated from dark-treated leaves compared to control. These suggest that proteolysis of chloroplast proteins contributed to amino acid accumulation in dark-treated leaves. Two chloroplasts proteases, ATP-dependent Clp protease proteolytic subunit 3 and protease Do-like 2, were up-accumulated in dark-treated leaves. This study firstly elucidated the mechanism of accumulation of amino acids in dark-treated tea leaves. BIOLOGICAL SIGNIFICANCE Effect of dark on crop growth has been widely studied, while less attention has been paid to effect of dark on quality-related metabolites in crops. Shade management (dark treatment) on tea plants is a common approach to improve free amino acids in tea leaves. However, the reason for accumulation of free amino acids in dark-treated tea leaves is still unknown. In the present study, an iTRAQ-based quantitative proteomic analysis was performed and the results revealed the accumulation of free amino acids in dark-treated tea leaves was not due to activation of biosyntheses of amino acids, but resulted from proteolysis of chloroplast proteins. The information will advance our understanding of formation of quality or function-related metabolites in agricultural crops exposed to dark stress/shade management.

Formation of Volatile Tea Constituent Indole During the Oolong Tea Manufacturing Process

Indole is a characteristic volatile constituent in oolong tea. Our previous study indicated that indole was mostly accumulated at the turn over stage of oolong tea manufacturing process. However, formation of indole in tea leaves remains unknown. In this study, one tryptophan synthase α-subunit (TSA) and three tryptophan synthase β-subunits (TSBs) from tea leaves were isolated, cloned, sequenced, and functionally characterized. Combination of CsTSA and CsTSB2 recombinant protein produced in Escherichia coli exhibited the ability of transformation from indole-3-glycerol phosphate to indole. CsTSB2 was highly expressed during the turn over process of oolong tea. Continuous mechanical damage, simulating the turn over process, significantly enhanced the expression level of CsTSB2 and amount of indole. These suggested that accumulation of indole in oolong tea was due to the activation of CsTSB2 by continuous wounding stress from the turn over process. Black teas contain much less indole, although wounding stress is also involved in the manufacturing process. Stable isotope labeling indicated that tea leaf cell disruption from the rolling process of black tea did not lead to the conversion of indole, but terminated the synthesis of indole. Our study provided evidence concerning formation of indole in tea leaves for the first time.

Formation of (E)-nerolidol in tea (Camellia sinensis) leaves exposed to multiple stresses during tea manufacturing

(E)-Nerolidol is a volatile sesquiterpene that contributes to the floral aroma of teas (Camellia sinensis). The unique manufacturing process for oolong tea involves multiple stresses, resulting in a high content of (E)-nerolidol, which is not known to form in tea leaves. This study aimed to determine the formation mechanism of (E)-nerolidol in tea exposed to multiple stresses during tea manufacture. C. sinensis (E)-nerolidol synthase (CsNES) recombinant protein, found in the cytosol, was found to transform farnesyl diphosphate into (E)-nerolidol. CsNES was highly expressed during the oolong tea turn over process, resulting in (E)-nerolidol accumulation. Continuous mechanical damage, simulating the turn over process, significantly enhanced CsNES expression level and (E)-nerolidol content. The combination of low temperature stress and mechanical damage had a synergistic effect on (E)-nerolidol formation. This is the first evidence of (E)-nerolidol formation mechanism in tea leaves and a characteristic example of plant volatile formation in response to dual stresses.

Does oolong tea (Camellia sinensis) made from a combination of leaf and stem smell more aromatic than leaf-only tea? Contribution of the stem to oolong tea aroma

The raw materials used to make oolong tea (Camellia sinensis) are a combination of leaf and stem. Oolong tea made from leaf and stem is thought to have a more aromatic smell than leaf-only tea. However, there is no available evidence to support the viewpoint. In this study, sensory evaluation and detailed characterization of emitted and internal volatiles (not readily emitted, but stored in samples) of dry oolong teas and infusions indicated that the presence of stem did not significantly improve the total aroma characteristics. During the enzyme-active processes, volatile monoterpenes and theanine were accumulated more abundantly in stem than in leaf, while jasmine lactone, indole, and trans-nerolidol were lower in stem than in leaf. Tissue-specific aroma-related gene expression and availability of precursors of aroma compounds resulted in different aroma distributions in leaf and stem. This study presents the first determination of the contribution of stem to oolong tea aroma.

Differential responses of four biosynthetic pathways of aroma compounds in postharvest strawberry (Fragaria × ananassa Duch.) under interaction of light and temperature

Light and temperature are two of the most important factors regulating postharvest strawberry aroma. To date the majority of research has been concentrated on the contribution of either light or temperature factors in isolation. In the present study, we investigated integrated effects of light and temperature on the formation of characteristic aromas during postharvest strawberry ripening process. Most volatiles including volatile esters, volatile furanones, and volatile terpenes showed increasing trends, whereas volatile benzenoids showed decreasing trends during postharvest ripening. Biosyntheses of volatile esters and volatile benzenoids were mainly affected by interaction of temperature and dark, whereas formation of volatile furanones and volatile terpenes were mostly influenced by temperature and dark, respectively. This study provided evidence of regulation of strawberry aroma by dual factors for the first time, and characterized a comprehensive profile of formations of strawberry aromas in response to light and temperature during postharvest ripening.

Formation and emission of linalool in tea (Camellia sinensis) leaves infested by tea green leafhopper (Empoasca (Matsumurasca) onukii Matsuda)

Famous oolong tea (Oriental Beauty), which is manufactured by tea leaves (Camellia sinensis) infected with tea green leafhoppers, contains characteristic volatile monoterpenes derived from linalool. This study aimed to determine the formation mechanism of linalool in tea exposed to tea green leafhopper attack. The tea green leafhopper responsible for inducing the production of characteristic volatiles was identified as Empoasca (Matsumurasca) onukii Matsuda. E. (M.) onukii attack significantly induced the emission of linalool from tea leaves (p<0.05) as a result of the up-regulation of the linalool synthases (CsLIS1 and CsLIS2) (p<0.05). Continuous mechanical damage significantly enhanced CsLIS1 and CsLIS2 expression levels and linalool emission (p<0.05). Therefore, continuous wounding was a key factor causing the formation and emission of linalool from tea leaves exposed to E. (M.) onukii attack. This information should prove helpful for the future use of stress responses of plant secondary metabolism to improve quality components of agricultural products.

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.