臺灣檜木葉子揮發性有機化合物之釋出特性

Abstract

植物所釋放的生物源揮發性有機化合物（Biogenic volatile organic compounds，BVOCs）可作為植物抵抗環境逆境、外來侵襲及適應生長環境的保護機制，同時也是光化學反應的前驅化合物，對大氣空氣品質有重要的影響。迄今關於臺灣植物BVOCs釋放特性之了解仍十分有限。本研究以紅檜（Chamaecyparis formosensis）及臺灣扁柏（C. obtusa var. formosana）林木與幼木為題材，分別量測單萜類（Monoterpenoids，MTs）、倍半萜類（Sesquiterpenoids，STs）與二萜類（Diterpenoids，DTs）等化合物之實際釋出速率（Actual emission rates，ERs）、標準釋出速率（Basal emission rates，Es）與相對釋出速率（Relative emission rates，RERs）等，並探討不同化合物釋出之季節變化，以及不同環境因子分別對林木與幼木BVOCs釋出之影響。 首先，利用頂空萃取技術建立快速且準確鑑別三種扁柏屬（Chamaecyparis）植物的方法。本研究採集臺灣不同地區之紅檜、臺灣扁柏與日本扁柏（C. obtusa）葉子，分別以不同頂空萃取條件所獲得之樣本與其精油成分相互比較。試驗結果發現，150℃平衡50 min之頂空萃取法獲得之各樣本揮發成分與其精油成分之相對量呈現良好之相關（r = 0.555 - 0.985；p ＜ 0.01 - 0.001）。進一步以群團分析及主成分分析比較其親緣相似度，三樹種分別根據其主要特徵成分可明顯區分為3個族群。研究結果顯示頂空萃取法是一種高效率且可行的化學分類策略，可作為針對特殊成分在不同環境中成分變化之追蹤與監測工具。 此外，本研究以原位（in situ）採樣裝置搭配Tenax TA 60-80 mesh吸附劑監測林木與幼木活體葉子在不同季節釋出之BVOCs差異，由野外試驗結果顯示，紅檜共測得21種植株釋放之萜類化合物，其中包括13種MTs、4種STs與4種DTs化合物，無論林木或幼木，MTs皆為紅檜主要之釋出類型，佔總釋出比例80%以上；臺灣扁柏之主要釋出BVOCs也以MTs為主，尤以夏（69.82 ± 6.03% - 84.69 ± 4.36%）、秋（84.65 ± 6.72% - 86.22 ± 1.40%）二季高於冬（60.71 ± 6.12% - 65.25 ± 2.28%）、春（67.31 ± 9.09% - 71.82 ± 7.03%）二季。DTs之釋出比例於冬季（21.72 ± 7.41% - 23.08 ± 3.69%）與春季（17.06 ± 8.06 - 20.83 ± 9.99%）較高。 至於BVOCs之總釋出速率，林木與幼木在不同季節之釋出速率呈現相反之趨勢。紅檜與臺灣扁柏林木在高溫季節具有較高的釋出速率，紅檜在高溫與低溫季節的釋出速率範圍分別為101.49 ± 12.29 -181.35 ± 80.15 ng g-1 h-1與35.32 ± 6.74 -40.15 ± 4.69 ng g-1 h-1；臺灣扁柏林木同樣以高溫季節（176.60 ± 62.68 - 198.52 ± 46.94 ng g-1 h-1）之釋出速率高於低溫季節（104.55 ± 18.90 - 111.59 ± 6.86 ng g-1 h-1）；相反地，幼木於低溫季節之總釋出速率（TERs）（紅檜：64.44 ± 13.18 - 140.74 ± 18.90 ng g-1 h-1；臺灣扁柏：57.52 ± 13.50 - 68.41 ± 15.76 ng g-1 h-1）高於溫暖季節（紅檜：55.63 ± 15.84 - 63.48 ± 11.85 ng g-1 h-1；臺灣扁柏：32.29 ± 3.12 - 40.62 ± 4.29 ng g-1 h-1）。根據迴歸分析統計結果顯示，溫度為影響林木BVOCs釋出速率之主要因子，而幼木BVOCs之釋出速率則主要受到光量之調控。 此外，許多特定化合物之相對釋出速率也呈現出顯著的季節性變化，例如紅檜釋出之β-Myrcene、α-Terpinene、trans-β-Ocimene、Terpinen-4-ol、α-Cedrene及trans-β-Farnesene等化合物，以及臺灣扁柏釋出之δ-3-Carene、Terpinolene、β-Cedrene、(Z,Z)-α-Farnesene及Kaur-16-ene等化合物，上述成分之相對釋出速率在冬季皆呈現顯著較高的趨勢。 進一步探討光量與溫度分別對紅檜與臺灣扁柏幼木BVOCs釋出之影響，在人工氣候室控制環境下，紅檜與臺灣扁柏幼木之BVOCs釋出速率皆隨光量之提升而增加，臺灣扁柏變化之幅度略小於紅檜，但其倍半萜與二萜類化合物之釋出速率在高光下明顯提升。當生長溫度自20℃提高至30℃時，二樹種之BVOCs釋出速率皆明顯下降。由蛋白質體之分析結果顯示，當溫度或光量改變時，紅檜與臺灣扁柏分別有46與15個蛋白質之表現量具有顯著差異，這些蛋白質整體的表現量與釋出速率呈現相似的趨勢，光量增加時調升蛋白質之比例提高，而溫度提高時調降之蛋白質比例增加。他們主要參與光合作用、碳水化合物代謝、胺基酸和蛋白質代謝、信號傳導與防禦作用。紅檜幼木在環境條件改變時啟動一系列之蛋白質進行調控，而臺灣扁柏差異表現蛋白質數量較少，OEE2、RA A與TK等蛋白質在紅檜與臺灣扁柏幼木皆有共同的表現趨勢，可能是影響二者在不同環境條件下BVOCs釋出之主要調控因子；而影響臺灣扁柏在不同光量條件下萜類化合物組成的調控機制，則有待未來進一步探討。

Biogenic volatile organic compounds (BVOCs) produced by plants are involved in plant adaptation to the environment and have important roles in protecting plant against biotic and abiotic stresses. They are also important precursors of photochemical oxidants, and have significant influences on atmospheric chemistry and pose potential threat to air quality. However, reports on volatile emissions from living trees in Taiwan are limited. The present study measured actual emission rates (ERs), basal emissions rates (Es), and relative emission rates (RERs) of monoterpenoids (MTs), sesquiterpenoids (STs), and diterpenoids (DTs) in different ages of Chamaecyparis formosensis and C. obtusa var. formosana. The relative contributions of individual compounds to the total emission were compared, and the seasonal variations were investigated. Furthermore, the dominant environmental factors affecting the emission behavior in trees of different ages were identified. Firstly, a rapid and accurate analytical method was developed, to differentiate three Chamaecyparis species (C. formosensis, C. obtusa, and C. obtusa var. formosana) that could not be easily distinguished by morphologic characteristics. Leaf samples from three species were analyzed by static-headspace (static-HS) coupled with gas chromatography-mass spectrometry (GC-MS). The optimized static-HS procedure, 150℃ for 50 min, yielded a high Pearson correlation between the composition from essential oil and VOC (r = 0.555 - 0.985; p < 0.01 - 0.001). Using cluster analysis (CA) and principal component analysis (PCA), three different species was clearly differenicated. The static-HS-based procedure greatly enhanced the speed of precise analysis of chemical fingerprint in small sample amounts, and thus provided a fast and reliable technique for the prediction of constituent characteristics in volatile terpenoids. This offers good opportunities for studying the role of these feature compounds in chemotaxonomy or ecophysiology. In addition, the seasonal variations of emission data from sapling and adult trees were measured using in situ sampling technique coupled with Tenax TA absorbents. A total of 21 terpenoids were detected from leaves of C. formosensis, of which there were 13 monoterpenoids (MTs), 4 sesquiterpenoids (STs), and 4 diterpenoids (DTs). MTs dominated the emissions in both adult trees and saplings and produced more than 80% of terpene emissions in C. formosensis. MTs were also the most dominant BVOCs emitted from C. obtusa var. formosana in warm seasons, especially in summer (69.82 ± 6.03% - 84.69 ± 4.36%) and fall (84.65 ± 6.72% - 86.22 ± 1.40%), which are higher than winter (60.71 ± 6.12% - 65.25 ± 2.28%) and spring (67.31 ± 9.09% - 71.82 ± 7.03%)。The proportion of diterpenoids (DTs) were higher in winter (21.72 ± 7.41% - 23.08 ± 3.69%) and spring (17.06 ± 8.06 - 20.83 ± 9.99%). Contrasting seasonal pattern between adult trees and saplings was found. Total actual emissions from adult trees were higher in warm seasons (101.49 ± 12.29 - 181.35 ± 80.15 ng g-1 h-1 in C. formosensis and 176.60 ± 62.68 - 198.52 ± 46.94 ng g-1 h-1 in C. obtusa var. formosana) than in cold seasons (35.32 ± 6.74 - 40.15 ± 4.69 ng g-1 h-1 in C. formosensis and 104.55 ± 18.90 - 111.59 ± 6.86 ng g-1 h-1 in C. obtusa var. formosana), and temperature was found to be the most important factor affecting the terpene emissions of adult trees. On the contrary, higher emissions were found in cold seasons for saplings (64.44 ± 13.18 - 140.74 ± 18.90 ng g-1 h-1 in C. formosensis and 52.47 ± 15.16 - 68.72 ± 22.87 ng g-1 h-1 in C. obtusa var. formosana) than in warm seasons (55.63 ± 15.84 - 63.48 ± 11.85 ng g-1 h-1 in C. formosensis and 32.29 ± 3.12 - 40.62 ± 4.29 ng g-1 h-1 in C. obtusa var. formosana), and the emissions of saplings were mainly regulated by the photosynthetically active radiation (PAR). Some compounds in both age trees showed comparably higher relative emission rates in cold seasons, such as β-myrcene, α-terpinene, trans-β-ocimene, terpinen-4-ol, α-cedrene and trans-β-farnesene from C. formosensis; δ-3-carene, terpinolene, β-cedrene, (Z,Z)-α-farnesene and kaur-16-ene from C. obtusa var. formosana. We further investigated the effects of light intensity and temperature on the emission of BVOCs from C. formosensis and C. obtusa var. formosana saplings. Under artificially controlled environments, the emission rates of BVOCs from C. formosensis and C. obtusa var. formosana saplings raised with the increase in light intensity. The variations of TERs in C. obtusa var. formosana was smaller than that in C. formosensis, while the emission rates of STs and DTs were obviously increased with light intensity. When the growth temperature was increased from 20°C to 30°C, the emission rates of BVOCs in both species decreased apparently. According to the proteomic analysis, it showed that when the temperature or the light intensity changed, there were significant differences in the abundance of 46 and 15 proteins of C. formosensis and C. obtusa var. formosana, respectively. The overall expression of these proteins showed similar trends with the emission rates. The proportion of up-regulated proteins raises as the light intensity increases, whereas the proportion of down-regulated proteins enhances when the temperature increases. These proteins were mainly involved in photosynthesis, carbohydrate metabolism, amino acid and protein metabolism, and signaling and defense. When the environmental conditions changed, a series of proteins were activated and regulated in C. formosensis saplings, while there were only a few proteins in C. obtusa var. formosana saplings showed significantly differential expression. In addition, OEE2, RA A and TK proteins had the same performance trend in both species, and these might be the main regulatory factors that affect the emission of BVOCs under different environmental conditions. As regard to the regulatory mechanism of the composition changes in terpenoids under different light intensities were required further investigation.