葡萄叶绿素荧光参数测量方法的优化与日变化特征分析

Optimization of measurement methods for chlorophyll fluorescence parameters and analysis of diurnal variation characteristics in grape leaves

【目的】确定葡萄叶绿素荧光参数测量最适的暗适应时间、测量光强度,分析葡萄叶绿素荧光参数日变化特征并确定最适测量时间,提出优化测量方法的建议,为进一步研究葡萄光合特性提供理论依据,以提高研究数据的准确性和可靠性。【方法】选择阳光玫瑰葡萄和巨峰葡萄叶龄为40~50 d的健康成熟叶片,利用JIP测定(JIP-test)和主成分分析(PCA)研究葡萄叶片在不同暗适应时间(5、10、15、20、25、30、35和40 min)、不同测量光强度[500、1000、1500、2000、2500、3000和3500μmol/(m^(2)·s)]和一天中不同时间点(8:00、10:00、12:00、14:00、16:00、18:00和20:00)的快速叶绿素荧光诱导动力学(OJIP)曲线特征,分析不同测量条件对葡萄叶片叶绿素荧光参数的影响。【结果】暗适应时间和测量光强度均明显影响葡萄叶片叶绿素荧光参数,较长的暗适应时间和较高的测量光强度能获得更稳定和可靠的测量结果。OJIP曲线特征显示,在较短的暗适应时间(5~15 min)内,各暗适应时间与暗适应5 min间的特征区间(OJ相、JI相和IP相)相对可变荧光差值(ΔWOJ、ΔWJI和ΔWIP)曲线变化幅度较大,初始荧光(F_(o))、最大荧光(F_(m))和最大光化学效率(F_(v)/F_(m))等参数波动较大,当暗适应时间达30 min以上时,各参数再无显著变化(P>0.05,下同),趋于稳定。随着测量光强度的增加,F_(m)、F_(v)/F_(m)、OJIP曲线的初始斜率(M_o)和单位面积反应中心数量(RC/CSm)等参数呈上升趋势,各测量光强度与500μmol/(m^(2)·s)测量光强度间的ΔWOJ、ΔWJI和ΔWIP曲线呈正K峰、负H峰和正G峰,当光强度达3000μmol/(m^(2)·s)以上时,各参数均无显著变化。葡萄叶片的多数叶绿素荧光参数随着一天中时间点的推移而变化,光系统II(PSII)光化学效率在12:00之前较高,14:00时光化学效率下降,16:00之后PSII逐渐恢复至最佳状态。【结论】针对葡萄叶片进行OJIP曲线测定时,暗适应时间应为30 min以上,最小测量光强度为3000μmol/(m^(2)·s),测量时间宜选择在12:00之前和16:00之后,以确保结果的准确性和可靠性。

【Objective】To determine the optimal dark adaptation duration and measurement light intensity for assessing chlorophyll fluorescence parameters in grape leaves,and to analyze the diurnal variation characteristicss of these parameters.This study aimed to provide recommendations for optimizing measurement protocols,thereby offering theoretical reference for further research into grape photosynthetic characteristics and enhancing the accuracy and reliability of experimental data.【Method】Healthy,mature leaves of Shine Muscat and Kyoho grape varieties aged 40-50 d were selected.The JIP-test and principal component analysis(PCA)were utilized to investigate the rapid chlorophyll fluorescence induction kinetics(OJIP)curves under different dark adaptation durations(5,10,15,20,25,30,35 and 40 min),various measurement light intensities[500,1000,1500,2000,2500,3000 and 3500μmol/(m^(2)·s)],and at different times throughout the day(8:00,10:00,12:00,14:00,16:00,18:00 and 20:00).The influence of these experimental conditions on the chlorophyll fluorescence parameters was evaluated.【Result】Dark adaptation duration and measurement light intensity greatly influenced the chlorophyll fluorescence parameters of grape leaves.Extended dark adaptation durations and increased measurement light intensities resulted in more stable and reliable fluorescence parameter measurements.OJIP curve characteristics indicated that under short dark adaptations(5-15 min),the characteristic interval(OJ phase,JI phase and IP phase)relatively variable fluorescence difference(ΔWOJ,ΔWJI andΔWIP)curves between each dark adaptation duration and dark adaptation 5 min had a large variation,parameters such as initial fluorescence(F_(o)),maximum fluorescence(F_(m))and maximum photochemical efficiency(F_(v)/F_(m))fluctuated greatly.When dark adaptation exceeded 30 min,the parameters had no significant difference(P>0.05,the same below),and tended to be stablized.With increasing measurement light intensity,F_(m),F_(v)/F_(m) the initial slope of OJIP curves(Mo),and number of reaction centers per unit area(RC/CSm)showed an upward trend,and theΔWOJ,ΔWJI andΔWIP curves between each measured light intensity and the measured light intensity at 500μmol/(m^(2)·s)showed positive K peaks,negative H peaks and positive G peaks.Once the light intensity exceeded 3000μmol/(m^(2)·s),no further significant changes in fluorescence parameters were observed.Most chlorophyll fluorescence parameters of grape leaves changed with diurnal variations of times,with higher photosystem II(PSII)photochemical efficiency before 12:00,a decline at 14:00,and a gradual recovery of PSII to optimal levels after 16:00.【Conclusion】F_(o)r accurate OJIP curve measurements of grape leaves,a dark adaptation duration of at least 30 min and a minimum measurement light intensity of 3000μmol/(m^(2)·s)are recommended.Optimal measurement times are before 12:00 and after 16:00 to ensure the accuracy and reliability of the results.