論文の公開元へMolecular mechanisms of adaptation to low calcium conditions in Arabidopsis thaliana
概要
Plants are basis of existence of humanity. In a historical view, improvement of agriculture has preceded economic growth. Understanding of plant physiology and genetics is essential to make the agriculture further efficient in the future, thus, I believe that accumulation of knowledge on plant contribute to promotion of human welfare.
Plant growth depends on absorption of essential elements from soils and photosynthesis in leaves. Insufficient supply of essential elements causes deficiency symptoms negatively affecting agriculture. Seventeen essential elements including calcium (Ca) are described in plants. In plants, Ca is mainly transported from roots to shoots through xylem along transpiration stream. Transpiration rate per leaf is higher in old leaves and lower in new and small leaves, and also in organs where no or little stomata is, such as fruits. In addition, Ca is difficult to be translocated through phloem because of strong binding to pectin in cell wall. Due to these characteristics, even when Ca in soil is sufficient, Ca deficient symptoms often appear in new tissues accompanying necrosis: blossom-end rot in tomato; tip burn in Chinese cabbage. These deficiency symptoms reduce their commercial values. Breeding of low-Ca tolerant crops is a possible and promising solution for this problem, however, knowledge on low-Ca adaptation mechanisms in plants required for efficient molecular breeding is still limited.
In my thesis, I clarified a molecular mechanism of low-Ca adaptation in plants through genetical and physiological analyses of low-Ca sensitive mutants in Arabidopsis thaliana. In Chapter 1, I revealed that synthesis of callose, one of the cell wall polysaccharide, prevented cell death under low-Ca in A. thaliana. I found that the wild-type plants accumulated callose in response to low-Ca condition, depending on Glucan synthase like (GSL) 10. I also demonstrated that the inhibition of callose synthesis enhanced cell death lesion. From these results, I established that callose synthesis is indispensable process to prevent cell death for low-Ca adaptation. In Chapter 2, I clarified that redundant contributions of several GSL genes were required for the suppression of cell death and adaptation to low-Ca. I found that gsl1-5 and gsl8-11 mutants also exhibited the reduced callose accumulation under low-Ca. Further, I demonstrated that the GSL1 and GSL10 additively contributed to suppression of cell death under low-Ca. These results established that several GSL genes contribute to the ectopic callose accumulation, suppression of cell death, and low-Ca adaptation in A. thaliana. In Chapter 3, I demonstrated a potential involvement of defense responses in the development of cell death under low Ca.
Chapter 1: Callose synthesis suppresses cell death induced by low calcium condition in Arabidopsis thaliana
Necrosis in new leaves is one of the characteristics of Ca deficiency symptoms in plants. Mechanisms of necrosis induced by low-Ca condition or adaptation remains unclear.
A low-Ca sensitive mutant, lcs3, cannot develop true leaf under low-Ca. Before my study in Ph.D., it had been demonstrated in the laboratory of Plant Nutrition and Fertilizers at the University of Tokyo that the causal gene of lcs3 (hereafter referred to as gsl10-5) is GSL10. In my master thesis, I have demonstrated that A. thaliana GSL10 complemented a yeast -1,3 glucan (callose) synthase mutant, establishing that GSL10 encodes callose synthase.
In this Ph.D. study, I performed detailed physiological characterization and transcriptome analysis of gsl10-5. Observation of cell death by trypan blue staining revealed that gsl10-5 developed cell death in new leaves under a low-Ca condition and the introduction of GSL10 alleviated cell death phenotype in gsl10-5, showing that GSL10 has a function of prevention of cell death under low-Ca conditions. Callose staining with aniline blue revealed that the wild-type plants accumulated ectopic callose in response to low Ca and that gsl10-5 accumulated less callose than the wild-type plants. Callose accumulation in gsl10-5 was recovered by introduction of the wild-type gene, showing that GSL10 is a responsible gene of the ectopic callose accumulation induced by low Ca. These results suggest that callose synthesis is required for the adaptation to low Ca and prevention of cell death. To test this, the wild-type plants were grown under low-Ca condition in the presence of callose synthase inhibitors, 2-deoxy-D-glucose (DDG) or caspofungin. Shoot growth of the wild-type plants under low Ca with DDG or caspofungin were inhibited comparable to that of gsl10-5 under low-Ca condition. Application of DDG enhanced the cell death and reduced callose accumulation in the wild type under low-Ca condition. These results showed that callose synthesis is required for the prevention of cell death under low-Ca. To get insight into the transcriptome change(s) in GSL10-mediated low-Ca adaptation, RNA-sequencing analysis was performed. The results showed similar expression profiles between wild-type plants treated with low Ca and gsl10-5 grown under normal condition. Especially, the expressions of genes related to cell wall and defense responses are commonly altered, suggesting the requirement of GSL10 for the alleviation of both cell wall damage and defense responses caused by low Ca. These results demonstrate that callose synthesis is essential for the prevention of cell death and is a key part of plant strategy to survive under low-Ca condition.
Chapter 2: Contributions of GSL family to low-Ca adaptation in A. thaliana
Low-Ca sensitive mutants, lcs4, lcs5 and lcs6 cannot develop true leaves under low Ca as observed in gsl10-5. Before my study in Ph.D., in the laboratory, it had been revealed that the causal gene of lcs4 and lcs5 is GSL1 (lcs4 and lcs5 are hereafter referred to as gsl1-5 and gsl1-6, respectively) and that of lcs6 is GSL8 (lcs6 is hereafter referred to as gsl8-11). It had also been demonstrated that GSL1 and GSL8 complemented a yeast -1,3 glucan synthase mutant, suggesting GSL1 and GSL8 encodes a functional callose synthase.
In my Ph.D. study, I performed detailed physiological characterization of the gsl mutants. I analyzed the contributions of GSL genes to the low-Ca adaptation, the ectopic callose accumulation, and the suppression of cell death under low-Ca condition. To test whether GSL1 and GSL8 contribute to the callose accumulation under low Ca, I performed callose staining. gsl1-5 and gsl8-11 mutants showed the reduced ectopic callose accumulation compared to the wild-type plants, showing that callose accumulation is positively correlated with the adaptation to low-Ca condition. Under the low-Ca condition, double mutant of gsl1-5 and gsl10-5 showed enhanced growth inhibition and cell death compared to its parental single mutant lines. In the A. thaliana genome, 12 genes are annotated as putative callose synthase. I checked the low-Ca sensitivity of T-DNA insertion lines of other 9 members of GSL genes and GSL11 T-DNA line showed sensitivity to low Ca. These results suggest that the callose synthesized by GSL proteins additively contributes to the tolerance and the prevention of cell death under low-Ca in A. thaliana.
Chapter 3: Analysis of the involvement of callose in a low-Ca sensitive mutant lcs7
Before my study in Ph.D., in the laboratory, it had been revealed that a low-Ca sensitive mutant lcs7 exhibits growth inhibition under low-Ca condition similar to that of gsl mutants, and the low-Ca sensitivity of lcs7 is caused by a gain-of-function mutation in a transcription regulation factor. From microarray analysis, it had been shown that callose degrading enzyme genes were up-regulated in lcs7, implying the possibility that the reduction of callose is the cause of low-Ca sensitivity of lcs7. In addition, it had been shown that double mutants between lcs7 and gsl10-5 or gsl8-11 showed severe growth inhibition compared to single mutants.
In this Ph.D. study, I performed callose staining to test whether less callose accumulation is related with low-Ca tolerance in lcs7 as is observed in gsl mutants. There was no significant difference between the wild-type plants and lcs7, suggesting that the low-Ca sensitivity of lcs7 is not caused by the reduction of callose and that the enhanced low-Ca sensitivity of double mutant between lcs7 and gsl mutants is caused by additive effects of different low-Ca adaptation mechanisms. To speculate the reason for low-Ca sensitivity of lcs7, I re-analyzed the microarray data and performed gene ontology analysis. The results showed that the genes related to defense response and cell wall are enriched in differentially expressed genes in lcs7. These results suggest that the proper transcription regulation of defense responses and cell wall is crucial for the prevention of cell death under low Ca.
In this Ph.D. thesis, I concluded that A. thaliana adapts to low-Ca conditions by synthesizing callose, and that redundant contributions of GSL family are required for the suppression of the cell death. In addition, I suggested the involvement of defense response in cell death under low Ca. I believe this knowledge will contribute to the breeding of Ca deficiency tolerant crops and the improvement of agricultural productivity in the future.
