豆科植物綠豆、決明根際黃酮類化合物分泌及其與根瘤菌的關聯性

Abstract

固氮作用是豆科植物的主要特徵之一，然而經歷演化後，現今地球上並存不能固氮及能與根瘤菌共生固氮的豆科植物。當前已知豆科植物以黃酮類化合物作為吸引相容根瘤菌的共生信號分子，可藉由觀察特定黃酮類化合物的分泌，以瞭解豆科植物與根瘤菌及環境互動情形。因此，本研究目的有二，首先為改善根際黃酮類化合物萃取及分析方法，二為藉由種植能固氮與不能固氮的豆科植物，並為其接種不同根瘤菌，以探討豆科植物根際黃酮類化合物分泌與根瘤菌的關聯性。 本研究使用70%甲醇和超音波輔助對盆栽介質進行萃取，並在有無鹼水解的條件下，探討此方法對介質中黃酮類化合物及其糖苷的萃取成果。研究結果顯示，鹼水解有助於提高綠豆介質中vitexin和isovitexin相關化合物的萃取效果，但對daidzein、genistein和coumestrol等其他根際黃酮類化合物的影響不顯著。除此之外，本研究亦對總類黃酮測定普遍採用的氯化鋁比色法進行探討，過往研究中當量標準品多採用rutin、quercetin和catechin，但豆科植物根際分泌物含有其它與根瘤密切相關黃酮類化合物，故本研究增加coumestrol、genistein、naringenin和daidzein作為分析參考標準品，以分析不同黃酮類化合物在氯化鋁比色法中的差異。結果顯示，不同黃酮類化合物與氯化鋁反應後形成的複合物具有顯著不同的吸光波長和吸收強度，且發現daidzein不與鋁螯合呈色，此說明必須選擇樣品中所含的主要黃酮類化合物作為標準參考物質，才能使氯化鋁比色法定量樣品中總類黃酮含量具有意義。 在豆科植物根際黃酮類化合物的研究中，選用決明 (Senna tora) 與綠豆 (Vigna radiata) 為植物材料，兩者分別屬於無法固氮及能夠固氮的豆科植物，其根際分泌物分析結果顯示，綠豆能分泌genistein、coumestrol、isoliquiritigenin、naringenin以及daidzein等根瘤相關黃酮類化合物；然而，決明僅分泌少量與根瘤相關的黃酮類化合物apigenin。LC-MS/MS定性分析結果亦檢測到綠豆根際分泌物中含有genistein、daidzein和coumestrol的苷元及糖苷，且發現biochanin A或calycosin的苷元和糖苷以及luteolin或kaempferol的糖苷。將綠豆與決明間種後發現，高氮環境下綠豆會妨礙決明生長，低氮環境下綠豆的固氮作用並未促進決明生長。綠豆與決明間種相較於兩株綠豆比鄰種植時，根瘤數目更多。為了驗證不同根瘤菌與豆科植物的互動關係是否相同，試驗進一步以Ensifer fredii USDA 205、Bradyrhizobium diazoefficiens USDA 110、Bradyrhizobium arachidis CCBAU 051107、Bradyrhizobium elkanii USDA 76和Bradyrhizobium japonicum USDA 6五種根瘤菌接種綠豆，觀察根瘤及黃酮類化合物分泌差異。結果顯示，綠豆的根瘤型態有大而少、小而多兩種型態。B. arachidis和 B. japonicum形成的是小而多的根瘤，而B. diazoefficiens和B. elkanii 形成的則是大而少的根瘤。從栽培介質萃取之根際分泌物的分析結果顯示，根瘤小而多組別的genistein分泌量多於根瘤大而少組別，這可能是不同根瘤菌根瘤型態的關鍵。在高氮組中，用福林酚試劑測量的根際分泌物總還原能力明顯高於低氮組，相較之下，naringenin濃度卻隨氮濃度的增加而減少，這表明naringenin可能是綠豆調控不同氮營養條件下根瘤形成的決定因素。根瘤誘導因子isoliquiritigenin的量少且在各接種組間沒有顯著差異，可能與綠豆根瘤形成的關係較小。 綜上所述，本研究證實不形成根瘤的決明與會形成根瘤的綠豆，其根際分泌黃酮類化合物的差異，及間種下對彼此生長的影響，並觀察到綠豆與不同根瘤菌形成不同型態的根瘤，而根瘤菌的根瘤型態以及不同氮條件下的根瘤數目，分別與特定黃酮類化合物相關。 關鍵字: 綠豆 (Vigna radiata)、決明 (Senna tora)、根瘤、根際分泌物、黃酮類化合物、氯化鋁比色法

Nitrogen fixation is a fundamental trait of legumes. However, through evolutionary processes, some legumes can engage in nitrogen fixation via rhizobia symbiosis, while others cannot. Legumes employ flavonoids as signaling molecules to attract compatible rhizobia. Observing the secretion of specific flavonoid compounds enables a better understanding of interactions among legumes, rhizobia, and the environment. Therefore, this study has two main objectives: firstly, to improve the extraction and analysis of root-secreted flavonoid compounds; and secondly, to investigate the correlation between the secretion of flavonoids by leguminous plants and their interaction with rhizobia. This was achieved by planting both nitrogen-fixing and non-nitrogen-fixing leguminous plants and inoculating them with various rhizobia strains. In this study, 70% methanol with ultrasonic assistance was employed for the extraction of compounds from the potting medium. The investigation focused on the effects of alkaline hydrolysis on the extraction of flavonoids and their glycosides within the medium. The findings indicated that alkaline hydrolysis extraction enhanced the yield of specific compounds, particularly those associated with vitexin and isovitexin. However, this method showed no significant impact on flavonoids such as daidzein, genistein, and coumestrol. In addition, we explored the widely-used aluminum chloride colorimetric method for determining the total flavonoid content. Traditionally, compounds like rutin, quercetin, and catechin have been commonly used as equivalent standards for quantification of total flavonoid content. However, our investigation revealed that leguminous plant root exudates contain additional flavonoids closely associated with rhizobia. Consequently, we introduced coumestrol, genistein, naringenin, and daidzein as reference standards for the quantification analysis. The results demonstrated that different flavonoid standards formed distinct complexes with aluminum, significantly influencing the absorption wavelength and peak intensity of the analysis; furthermore, it was noted that daidzein failed to form a complex with aluminum. This observation highlights the critical importance of selecting appropriate primary flavonoids in the sample as equivalent standards to ensure the meaningful application of the aluminum chloride colorimetric method. For the study of legume root-exuded flavonoids, Senna tora and Vigna radiata (mung bean) were selected as plant materials. These two plants belong to the categories of non-nitrogen-fixing and nitrogen-fixing legumes, respectively. The results showed that V. radiata can secrete nodule-related flavonoids such as genistein, coumestrol, isoliquiritigenin, naringenin, and daidzein. However, S. tora secreted only small amounts of apigenin, a flavonoid compound related to root nodules. LC-MS/MS qualitative analysis further identified aglycone and glycoside forms of genistein, daidzein, and coumestrol in the root exudate of V. radiata, along with the presence of biochanin A or calycosin aglycone and glycosides, and luteolin or kaempferol glycosides. Intercropping V. radiata and S. tora revealed that V. radiata hindered the growth of S. tora under high nitrogen conditions, and the nitrogen-fixing ability of V. radiata did not promote the growth of S. tora under low nitrogen conditions. Additionally, compared to planting each species separately, more nodules were observed when V. radiata and S. tora were interplanted. To verify whether the interaction between different rhizobia and leguminous plants was similar, experiments were conducted with five rhizobia strains (Ensifer fredii USDA 205, Bradyrhizobium diazoefficiens USDA 110, Bradyrhizobium arachidis CCBAU 051107, Bradyrhizobium elkanii USDA 76, and Bradyrhizobium japonicum USDA 6) inoculated into V. radiata. Differences in nodule formation and flavonoids secretion were observed. The findings indicated that V. radiata exhibited two distinct nodule patterns. Specifically, inoculation with B. arachidis and B. japonicum led to the formation of numerous small nodules, while inoculation with B. diazoefficiens and B. elkanii resulted in larger and fewer nodules. Analysis of root exudates indicated that the group with small and numerous nodules secreted a significantly higher amount of genistein than the group with large and few nodules, suggesting that genistein might play a key role in the formation of distinct nodule patterns induced by different rhizobia. Additionally, the antioxidant capacity of root exudates, measured using the Folin-Ciocalteu reagent, was significantly higher in the high nitrogen group than in the low nitrogen group. While flavonoid compounds exhibited antioxidant capabilities, the concentration of naringenin decreased with increasing nitrogen levels. This implied that naringenin may be a determining factor in regulating nodule formation in V. radiata under varying nitrogen conditions. In contrast, a low concentration of the nodule-inducing factor isoliquiritigenin was observed, with no significant difference among the inoculation groups, indicating that it had a limited association with V. radiata root nodule formation. In summary, this study demonstrated the differences in the secretion of flavonoid compounds by S. tora, which does not form nodules, and V. radiata, which does form nodules, and the effects of intercropping on their respective growth. It was also observed that V. radiata formed different nodule patterns with different rhizobia strains, and these nodule patterns and numbers under different nitrogen conditions were associated with specific flavonoid compounds. Key words: mung bean (Vigna radiata), cassia seed (Senna tora), nodulation, flavonoids, root exudate, aluminum chloride assay