I am working on a rice mutant which showed leaf yellowing phenotype. Initially, very tiny lesion mimic spots appeared randomly on the old leaves, then the spots expand and finally, the leaves completely turned yellow, dried and died. However, young leaf seemed normal. At the mature stage, mutant plants died earlier than wild type. I conducted dark treatment assay, which is usually used to discover the senescence status, there showed no difference between mutant and wild plants. I also checked the expression level of several pathogen-related genes (PRs), such as pr1a, pr4 and pbz1. However, no significant difference was identified as well. Based on the phenotype, I suspected either senescence or immunity induced PCD was affected the mutant performance. However, the simple experiments I did seemed rule out my hypothesis. How can I determine is the senescence or PCD triggered the phenotype of my mutant? Or can anyone give some marker genes to distinguish these two physiological processes in rice? Many thanks in advance.
You can go for TUNEL assay. For this you have to make leaf section followed by TUNEL labeling. The higher number of nicks in your mutant plant will indicate PCD phenomenon. If you will need the protocol then do write me, i will send you the protocol i used.
Alternatively, you can check the photosynthetic efficiency of plants through chlorophyll fluorescence using PAM or IRGA. A plant under going senescence has a particular pattern of ETR which. If your plant will show that pattern it will confirm senescence. But I have no idea if anyone has done the photosynthetic efficiency assay on plants under going PCD, so I can say if a plant under going PCD will show a pattern change in chlorophyll fluorescence.
You can also check for senescence associated genes (SAG) that are well studied in rice.
SAG genes are usually used as Senescence markers. In Arabidopsis plants, a common assay to determine senescence-related phenotypes is transform plants with the reporter construccion SAG12promoter::GUS. that allows you to see the parts of the plant undergoing senescence. Probably there is an equivalent promoter in rice.
You could try some cell staining assays, trypan blue stains dead cells, decolorized aniline blue stains callose (often deposited in lesion mimics), and do a time course to compare your mutant to the controls. That way you can see how early cells begin to die in the mutant and what the pattern is.
You could check the expression of the rice orthologue of the arabidopsis SAG12 and SAG13. Both should be induced if it is senescence but only SAG13 would be if it is PCD.
See Brodersen et al. 2002 for details
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