Targeted Inactivation of the Plastid ndhB Gene in Tobacco Results in an Enhanced Sensitivity of Photosynthesis to Moderate Stomatal Closure

The ndh genes encoding for the subunits of NAD(P)H dehydrogenase complex represent the largest family of plastid genes without a clearly defined function. Tobacco (Nicotiana tabacum) plastid transformants were produced in which the ndhB gene was inactivated by replacing it with a mutant version possessing translational stops in the coding region. Western-blot analysis indicated that no functional NAD(P)H dehydrogenase complex can be assembled in the plastid transformants. Chlorophyll fluorescence measurements showed that dark reduction of the plastoquinone pool by stromal reductants was impaired in ndhB-inactivated plants. Both the phenotype and photosynthetic performance of the plastid transformants was completely normal under favorable conditions. However, an enhanced growth retardation of ndhB-inactivated plants was revealed under humidity stress conditions causing a moderate decline in photosynthesis via stomatal closure. This distinctive phenotype was mimicked under normal humidity by spraying plants with abscisic acid. Measurements of CO(2) fixation demonstrated an enhanced decline in photosynthesis in the mutant plants under humidity stress, which could be restored to wild-type levels by elevating the external CO(2) concentration. These results suggest that the plastid NAD(P)H:plastoquinone oxidoreductase in tobacco performs a significant physiological role by facilitating photosynthesis at moderate CO(2) limitation.

编码NAD(P)H脱氢酶复合体（NAD(P)H dehydrogenase complex）亚基的ndh基因，是目前功能尚未明确的最大质体基因家族。本研究以烟草（Nicotiana tabacum）为实验材料，构建了ndhB基因失活的质体转化植株：通过将内源ndhB基因替换为编码区携带翻译终止密码子的突变版本，实现基因功能失活。蛋白质印迹（Western-blot）分析结果显示，该质体转化植株无法组装具备正常功能的NAD(P)H脱氢酶复合体。叶绿素荧光测定表明，在ndhB失活植株中，基质还原剂介导的质体醌（plastoquinone）库暗还原过程受到显著损伤。在适宜生长条件下，该质体转化植株的表型与光合性能均完全正常；但在湿度胁迫环境中——此类胁迫可通过诱导气孔关闭引发光合速率适度下降——ndhB失活植株的生长迟滞现象显著加剧。在正常湿度条件下，向植株喷施脱落酸（abscisic acid）可模拟这一独特表型。二氧化碳固定测定结果显示，湿度胁迫下突变植株的光合速率下降幅度更大，而通过提高外界二氧化碳浓度可将其恢复至野生型水平。上述实验结果表明，烟草中的质体NAD(P)H：质体醌氧化还原酶（plastid NAD(P)H:plastoquinone oxidoreductase），可通过在适度二氧化碳限制条件下促进光合作用，发挥重要的生理功能。