Study of physiological changes induced by ectopic expression of AtAMY3 C-terminal domain in Arabidopsis leaves
Date Issued
2015
Date
2015
Author(s)
Huang, Hsin-Yao
Abstract
Forty to fifty percent of photosynthate is synthesized as transitory starch during daytime and then degraded during nighttime in chloroplasts. To achieve the diurnal transitory starch metabolism, enzymes for starch degradation should be properly regulated. In Arabidopsis chloroplast, AtAMY3 is an α-amylase which hydrolyzes α-1,4-glucosidic linkage of starch. It is known that AtAMY3 has an N-terminal domain which may negatively regulate AtAMY3 itself and a C-terminal domain which is responsible for amylolytic activity. Transformants overexpressing a fusion protein of AtAMY3 C-terminal domain and EYFP (AMY3C-EY) were generated, and the starch content in leaves of transformants was greatly reduced. In addition, AMY3C-EY showed distinct phenotypes such as chlorotic and reticulate leaves, dwarf, and late flowering. How altered starch metabolism in AMY3C-EY affects plant growth and produces these phenotypes remains unclear. It is possible that AMY3C-EY phenotypes are induced for two reasons. First, these phenotypes could be induced by accumulation of small glucans released from the AMY3C-EY amylolytic activity. Second, these phenotypes could be caused by energy overconsumption because of constitutive starch degradation during photosynthesis. To test these hypotheses, I isolated mutants from-irradiation mutagenized AMY3C-EY progenies. In these mutants, mutations which rescued AMY3C-EY phenotypes would provide information why AMY3C-EY phenotypes were produced. One of the revertants, 47, was shown to be an adg1 mutant without ADP-glucose pyrophosphorylase activity and starch synthesis. The study of 47 suggested that starch synthesis is needed for producing AMY3C-EY phenotypes, which indirectly supported both the hypotheses. To find out how AMY3C-EY alters plant physiology, I constructed an inducible expression system for AMY3C-EY with LexA-VP16-ER (XVE) chimeric transcription factor. Through controlling the activation of XVE by 17--estradiol, the AMY3C-EY expression could be induced and restricted at certain parts of a plant. 17--Estradiol was applied to shoot apical meristem or mature leaves of XVE AMY3C-EY double transformant. Only newly formed leaves with reduced starch content showed the chlorotic and reticulate phenotypes. This suggests that improper regulation of AtAMY3 affects chloroplast at early development. In summary, this study indicates that ectopic expression of AtAMY3 C-terminal domain affects early chloroplast development, revealing the importance of properly regulated AtAMY3 amylolytic activity for Arabidopsis leaves.
Subjects
chloroplast
transitory starch
alpha-amylase
gamma-irradiation mutagenesis
17-beta-estradiol
Type
thesis
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