Bellasio Chandra, Burgess Steven J, Griffiths Howard, Hibberd Julian M
Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK.
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK.
J Exp Bot. 2014 Jul;65(13):3769-79. doi: 10.1093/jxb/eru238. Epub 2014 Jun 13.
Large-scale research programmes seeking to characterize the C4 pathway have a requirement for a simple, high throughput screen that quantifies photorespiratory activity in C3 and C4 model systems. At present, approaches rely on model-fitting to assimilatory responses (A/C i curves, PSII quantum yield) or real-time carbon isotope discrimination, which are complicated and time-consuming. Here we present a method, and the associated theory, to determine the effectiveness of the C4 carboxylation, carbon concentration mechanism (CCM) by assessing the responsiveness of V O/V C, the ratio of RuBisCO oxygenase to carboxylase activity, upon transfer to low O2. This determination compares concurrent gas exchange and pulse-modulated chlorophyll fluorescence under ambient and low O2, using widely available equipment. Run time for the procedure can take as little as 6 minutes if plants are pre-adapted. The responsiveness of V O/V C is derived for typical C3 (tobacco, rice, wheat) and C4 (maize, Miscanthus, cleome) plants, and compared with full C3 and C4 model systems. We also undertake sensitivity analyses to determine the impact of R LIGHT (respiration in the light) and the effectiveness of the light saturating pulse used by fluorescence systems. The results show that the method can readily resolve variations in photorespiratory activity between C3 and C4 plants and could be used to rapidly screen large numbers of mutants or transformants in high throughput studies.
旨在表征C4途径的大规模研究项目需要一种简单的高通量筛选方法，用于量化C3和C4模式系统中的光呼吸活性。目前，相关方法依赖于对同化响应（A/Ci曲线、PSII量子产率）进行模型拟合或实时碳同位素判别，这些方法既复杂又耗时。在此，我们提出一种方法及相关理论，通过评估转移至低氧环境时RuBisCO加氧酶与羧化酶活性之比VO/VC的响应性，来确定C4羧化、碳浓缩机制（CCM）的有效性。该测定使用广泛可用的设备，比较在环境氧浓度和低氧条件下同步进行的气体交换和脉冲调制叶绿素荧光。如果植物预先适应，该程序的运行时间最短可达6分钟。推导了典型C3（烟草、水稻、小麦）和C4（玉米、芒草、醉蝶花）植物的VO/VC响应性，并与完整的C3和C4模式系统进行比较。我们还进行敏感性分析，以确定光呼吸（R LIGHT）的影响以及荧光系统所使用的光饱和脉冲的有效性。结果表明，该方法能够轻松分辨C3和C4植物之间光呼吸活性的差异，可用于在高通量研究中快速筛选大量突变体或转化体。
