Manipulation of the levels of pyruvate decarboxylase and alcohol dehydrogenase for submergence tolerance in rice

Abstract

Rice plants are partially or completely submerged when fields are flooded. During submergence, rice plants encounter anaerobic conditions, and suffer severe injury, often death, leading to major crop losses in countries affected by monsoonal flooding. Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) catalyse thanol fermentation (EF), the major energy-producing pathway under conditions of low oxygen and are among the anaerobic polypeptides induced under such conditions. The importance of EF is emphasised by the reduced survival and germination of the ADH null mutants of maize, barley, rice and Arabidopsis under anaerobic conditions. The research described in this thesis has taken a transgenic approach to manipulate the levels of PDC and ADH to determine whether altering the EF pathway can affect anaerobic tolerance in rice. It was found that one antisense ADHl (rice gene)line had decreased levels of both ADHl and ADH2 polypeptides, and greatly reduced ADH activity of 6% of that of wild type (WT- untransformed Taipei). This antisense ADHI line showed reduced ethanol production and coleoptile growth under anoxia, and mature plants exhibited reduced survival when submerged in anaerobic water, suggesting ADH plays a role in seed germination and plant survival under anoxia. One sense ADH2 (cotton gene) line had significantly increased levels of ADH activity compared to WT and a flooding tolerant rice variety FR13A in air and under hypoxia. No significant increase in ethanol production was observed in the line which overproduced ADH by 439% of WT. Similar levels of anoxia tolerance were found in mature plants of the line which over-produced ADH and WT whereas in the flooding tolerant variety, anoxia tolerance was much higher. This suggests that over-production of ADH increases neither ethanol production nor anaerobic survival. Three independently transformed lines of the rice PDCl driven by an anaerobically inducible promoter contained an increased level of PDC1 polypeptides. A moderate increase in PDC activity and ethanol production compared to WT was also observed in these lines under anaerobic conditions. Effects of anoxia on seed germination were assayed in these lines over-producing PDC and neither retardation nor acceleration of germination was observed. However, mature plants showed decreased survival under anaerobiosis. On the contrary, hybrid plants over-expressing both PDC and ADH were found to have better anaerobic tolerance than plants over-producing PDC alone. These results indicate that overproducing PDC plants suffered from some kind of toxicity which was counterbalanced and/or neutralised in plants over-producing ADH along with PDC. Acetaldehyde levels were appreciably higher in the plants over-producing PDC compared to WT and hybrid plants over-producing both PDC and ADH indicating that acetaldehyde might cause early senescence in plants over-producing PDC alone under anaerobic conditions. No transformed lines with either over-producing PDC, ADH or both PDC and ADH had increased submergence tolerance relative to the WT, however lines often had different metabolic rates and demonstrated the versatility of a molecular approach to evaluating metabolic controls affecting plant growth and survival. A second objective of this research was to study the expression of the rice PDCl and PDC3 promoters in various tissues of rice by GUS histochemical analysis. Translational fusion of the PDCl promoter-GUS gave positive blue staining in embryos, endosperm, shoots, and roots and showed strong anaerobic induction in shoots and roots. GUS staining was found in anthers but absent in pollen. In immunoblotting analysis using an antibody raised against the rice PDC1 polypeptide, the PDC band corresponding to PDCl was also absent in pollen of untransformed Taipei, suggesting that the rice PDCl gene is not expressed in pollen. Nine independently transformed lines of the rice PDC3 promoter-GUS fusion (translational) did not express GUS in any vegetative tissues even under anaerobic conditions. GUS staining was seen in the pollen of three independently transformed lines of PDC3-GUS. A novel PDC band with a MWt of approx. 62 kDa was found in immunoblots of pollen of untransformed Taipei using an antibody generated against the rice PDCl, indicating that the rice PDC3 has pollen specific expression. Expression of PDC3 was seen after the first mitosis of microspores and increased with maturation, implying that it may have a role during pollen germination. The final objective was to study the cis-acting regulatory elements required for anaerobic induction in the rice PDCl promoter. GUS histochemical analysis of transcriptional fusions of the various lengths of 5' truncated PDCl promoter-GUS revealed that the regulation of the GUS reporter gene did not mirror that of the endogenous PDCl and the PDCl promoter acted in a constitutive manner. Show more