Micro-scale chlorophyll analysis and developmental expression of a cytokinin oxidase/dehydrogenase gene during leaf development and senescence
2012
Evans, Thomas | Song, Jiancheng | Jameson, Paula E.
Chlorophyll content is a baseline measurement in many plant studies. With the miniaturization of many molecular and biochemical assays the sample size needed for these assays has been greatly reduced. Chlorophyll is commonly determined by spectrophotometry. Traditionally this analysis has been carried out in 10 mm cuvettes with volumes of 0.2–2.0 ml. The NanoDrop™ spectrophotometer requires a sample volume of 1–2 μl which provides the opportunity to analyze far smaller samples. Chlorophyll analyses are a critical part of any study of senescence and the cytokinins are considered to play a key role in regulating senescence. The tissue specific levels of cytokinins are thought to be regulated, at least in part, by expression of the cytokinin degradation enzyme cytokinin oxidase/dehydrogenase (CKX). Here an assay is described for the spectral determination of chlorophyll using the NanoDrop™ spectrophotometer that allows the determination of chlorophyll from part samples of clover leaves while leaving sufficient tissue for quantitative real-time PCR study of CKX expression. The expression of TrCKX2 increased during leaf development along the stolon of Trifolium repens but was stable during the onset and progression of senescence suggesting that TrCKX2 was not an initiator of leaf senescence but may have facilitated the progression.
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