The role of autophagy in Arabidopsis plants exposed to ultraviolet-B or high temperature stress
概要
Autophagy is one of the catabolic processes important for the survival of the cells by ensuring homeostasis under various conditions. Its functions are known both in a bulk degradation process where cytoplasmic components are unselectively degraded for nutrient recycling and in a selective degradation for the elimination of damaged organelles by vacuolar hydrolyses enzymes. Autophagy plays a central role in programmed cell death (PCD), which is an essential feature of normal plant development, and also in some of the environmental stresses. Recently, there are few reports related to autophagy role in the mechanisms of ultraviolet-B (UV-B) or high temperature (HT) tolerance. However, the knowledge about its role in UV- B responses is not known in plants and even controversy in the case of HT stress.
Chapter I: In addition to the substantial damage to plant macromolecules,1 UV-B induces lesions in nuclear, mitochondrial, and chloroplast genome by specifically inducing photoproducts between adjacent pyrimidines on the same DNA strand in plants.2 The cyclobutane pyrimidine dimers (CPDs), which make up the largest component (~75%) of photoproducts,4 can retard growth and development in Arabidopsis.3,4 The CPDs, are the principal cause of UV-B-induced growth inhibition in plants, thus often used as a direct indicator of UV-B damage.5, Photoreactivation by CPD photolyase is the primary mechanism of CPD repair in rice plants5 and has previously demonstrated that CPD photolyase is crucial for determining the resistance of rice to UV-B, and that elevated CPD photolyase activity can significantly alleviate UV-B-induced growth inhibition.5,7 In Arabidopsis, deficiency of CPD photolyase (PHR1/ UV RESISTANCE 2 [UVR2]; AT1G12370) induces UV-B sensitivity,3 indicating that UV-B-induced CPD accumulation is a principal cause of UV-B-induced cell death and growth inhibition in plants. Additionally, previous study that mutants of the autophagy-related genes (ATG2, 5, 7) exhibited UV-B-sensitive phenotypes like that of PHR1- deficient Arabidopsis thaliana mutants (uvr2).8 These results indicated that autophagy also plays an important role in the plant response to UV-B-induced damage.
In yeast and mammals, damaged organelles, especially mitochondria are removed by a selective autophagic process known as mitophagy.9 Mitophagy is linked to several physiological functions and human pathologies, such as neurodegenerative disease.10 However, little is known about the relationships between UV-B-induced CPD accumulation and mitochondrial dynamics or between mitochondrial dynamics and autophagy in UV-B damaged plants. Here, we analysed the mitochondrial dynamics and UV-B-induced CPD accumulation in WT, atg5, phr, atg5 phr, and AtPHRox plants in order to understand the role of mitochondria removal by autophagy in UV-B protection in plant cells. The severe UV-B-sensitive phenotype of the atg5 phr double mutant compared with the atg5 and phr single mutants, which were already more sensitive than WT, strongly suggest that observed phenotype is resulted from deficiency in two independent machineries in response to UV-B, i.e., autophagy and CPD photorepair. Furthermore, UV- B induced CPD level was similar between WT and atg5 plants indicating UV-B hypersensitivity of atg5 is not due to high CPDs accumulation. Here, I found that mitochondria, one of the reproductive organelle having its own genome, were fragmented and the number was significantly increased at 10 h after UV-B irradiation in all genotypes, but decreased in WT and phr1 plants at 24 h after UV-B irradiation while massively accumulated in atg mutants. In order to analyse activity of these accumulated mitochondria, tetramethylrhodamine, ethyl ester (TMRE) staining method was preformed and revealed that higher portion of the remaining mitochondria in UV-B treated plants were dysfunctional. Southern blot results for checking CPD levels on mtDNA were no different between WT and atg5 plants indicating that UV-B-induced mitochondrial fragmentation occurred in regardless of CPD accumulation on mtDNA. Taken together here we show that UV-B damages mitochondrial function and causes mitochondrial fragmentation in Arabidopsis, and the damaged dysfunctional mitochondria are removed by autophagy.
Chapter II: Microsporogenesis is one of the most sensitive process affected by HT.11 Anther development follows well-regulated programs and amidst them PCD is crucial to break down anther wall cells, such as tapetum and middle layer cells for maturing pollen grains and anther dehiscence. The proper timing of tapetal degradation by PCD is necessary for the production of viable pollen.11 MYB80 is a key transcription factor that controls tapetal PCD by regulating several genes, including directly upregulating UNDEAD.12 Autophagy is thought dispensable for regular reproductive development in Arabidopsis,13 intriguingly, a transgenic tobacco line overexpressing Arabidopsis AtATG6/BECLIN1 in the tapetum cells showed male sterility14 due to accelerated tapetum cell death therefore microsporogenesis abortion. Nevertheless, the rice autophagy null mutant OsATG7 showed a defect in tapetum cell degradation and sporophytic male sterility under normal culture conditions.15 These results indicate autophagy plays an important role in the tapetal degeneration, but the biological significance and its specific function in development are still controversial. Here, to check whether elevated temperatures directly induce autophagy, and if so, how autophagy is involved in upon HT stress, the contribution of autophagy to pollen and anther development under moderate HT stress is analysed in Arabidopsis plants.
Firstly, the physiologic parameters of plants grown at 30 °C were measured at different developmental stages in WT and atg mutant plants and found that most of the atg plants showed no difference than WT plants at the vegetative stage but in the reproductive stage. Furthermore, Arabidopsis atg mutants became almost completely male-sterile at 30 ℃ whilst WT plants still set some seeds indicating that autophagy is essential to for mitigating HT injury during pollen development. Meanwhile, ovules developed normal in both WT and atg5 under either temperature conditions. In the histologic observation of the anthers cross- sections of stage 13 anthers at 30 °C, the pollen size and number decreased in WT anthers, but there was still septa and stomium breakage. Whilst, septum and stomium breakage did not occur in atg5 anthers. These results indicate that autophagy is essential for septum and stomium breakage at 30 °C, that is necessary for the maturation and release of the pollens through degeneration of anther wall cells. Gene expression results of MYB80 and UNDEAD indicated that HT stress alters the timing of tapetal PCD through repression of MYB80 transcriptional regulation, which then sequentially activates autophagy to compensate reduction of tapetal PCD by HT. In addition to this hypothesis, here I also found that HT induces oxidative damage in developing anthers, much more prominently in the atg5 mutant. Altogether, in addition to HT- induced ROS, over-accumulation of ROS in autophagy deficiency may strongly affect the spatiotemporal ROS signalling for tapetal PCD. In either hypothesis, the proper timing of pollen development including tapetal PCD is strictly controlled, and autophagy seems to be important to minimize the effects of HT and to maintain this pollen specific development.
Conclusion: In this study, I showed that autophagy plays an important role in both UV-B and HT responses thorough involving in the cell death process, and therefore it can be used as a modulator mechanism to alleviate the decrease in plant growth and yield. atg mutants of Arabidopsis exhibited UV-B-hypersensitive phenotypes during the vegetative stage, whilst HT of 30 ℃ resulted in complete abortion of pollen development during the developmental stage. These results suggest that autophagy exerts an essential role in the cell death pathway in plants exposed to UV-B and HT, and the degree of the importance for cells varies depending on the type of stress and growth stages during stress exposure.