@phdthesis{oai:sucra.repo.nii.ac.jp:00018697,
 author = {NAGAHAGE ISURA SUMEDA PRIYADARSHANA},
 month = {},
 note = {v, 102 p., Plants are sensitive to environmental changes that may lead to making changes in developmentally and physiologically to acclimate it. The mechanism of perceiving environmental changes by plants is well studied so far and that knowledge has been utilized to breed stress tolerant plants, high-quality yield plants, and biofuel production to compensate human needs. There is a tight relationship between environmental changes and plant senescence. Senescence is a highly organized process and essential set of functions to nutrient remobilization between senescence materials to newly generating tissues of the plant. Besides to natural senescence, pre-mature senescence is also induced in response to environmental stress or changes of light-dark diurnal rhythm. Thus, to elucidate the molecular mechanisms of how induction of senescence is taken place, a number of studies have been conducted so far. Indeed, a number of genes including transcriptional factors are isolated as key regulators of promotion or repression of senescence. However, how plants induce senescence in response to environmental statuses is currently still elusive.
NAC domain transcriptional factor family is a plant-specific transcriptional factor family. It has been known to be involved in various developmental and physiological functions such as seed development, environmental stress responses, flowering time management, and embryo development. In addition, Some NAC domain transcription factors such as VNI2, ORE1, ORS1, and ANAC046 are reported to be associated with regulation of senescence.
VNI2 is originally isolated as an interacting factor with another NAC domain transcriptional factor VND7, a master regulator of xylem vessel differentiation. VNI2 inhibits the transcriptional activities of VND7. Additionally, VNI2 is expressed in various types of cells suggesting that VNI2 plays pivotal roles in various biological events as well as xylem vessel formation. Here, to understand the biological roles of VNI2 further, we screened interacting factors, characterize the interacting factors and then try to understand the role of interaction in stress responses.
I focused on two NAC domain transcriptional factors, ATAF2 and ANAC102, isolated as interacting proteins with VNI2 through Yeast two-hybrid screening. To verify their transcriptional activities, I carried out a transactivation assay using protoplast cells. ATAF2 showed transcriptional activation activity, while ANAC102 neither show transcriptional activity nor repression activity. Thus, further studies were carried out by focusing on ATAF2 and VNI2.
To verify the transcriptional activities of ATAF2, the transient assay was performed. Obtained data suggested that ATAF2 could be a bi-functional transcriptional factor. Moreover, I found that the ATAF2 upregulates expression of ORE1, a key regulator of senescence. Although ATAF2 is known to be associated with biotic stress responses, it is unclear that ATAF2 is also involved in the promotion of senescence. Thus, I investigated the relationship between ATAF2 and senescence. The transient assay showed that ATAF2 has transcriptional activity on some senescence regulatory transcriptional factors including ORS1 and ANAC046 as well as ORE1. In addition, loss of function mutants （ataf2） and overexpression lines （ATAF2-OX） showed delaying and accelerating senescence phenotypes, respectively under a dark condition. Consistent the phenotypes of the loss of and the gain of function lines, the expression level of senescence marker genes were elevated in ATAF2-OX while low-level expression was observed in ataf2 under the long day condition, suggesting that ATAF2 promotes senescence in response to the light/dark condition via regulation of these genes.
To unveil the biological roles of VNI2 in response to environmental statuses, I investigated VNI2-ATAF2 protein complex. The transient assay showed that the transcriptional activity of ATAF2 on ORE1 was significantly inhibited by VNI2. Genetic interaction results based on vni2, ataf2, vni2ataf2 mutant lines under the long-day condition showed that senescence promotion phenotype of vni2 can be cancelled under the ataf2 background suggesting that VNI2 has ATAF2 dependent function in senescence. However, such a phenotype difference was not observed under the continuous light condition. Thus, it is possible that VNI2-ATAF2 interaction leads to senescence in the light-dependent pathway. In addition, ORE1 and ORS1 have been highly induced in 55 days old vni2 plant lines together with wild-type plants but a significantly low expression of them in ataf2 plant line. The vni2ataf2 line showed intermediate expression level that is comparable with chlorophyll content and its phenotype. By contrast, senescence marker genes expression was significantly high in vni2 mutant but low in wild-type, suggesting that there might be an interaction of VNI2 with ORE1 and ORS1, and VNI2 effectively inhibits them to results in lower expression of their downstream target genes.
ORE1 was identified as VNI2 interacting protein in large-scale screening and the molecular mechanism between ORE1 and VNI2 was not characterized so far. Here, I showed that senescence marker genes SAG12 and SAG29 can be transcriptionally activated by ORE1 under the transient assay system. Co-transfection of ORE1 together with VNI2 showed that ORE1 transcriptional activity on SAG12 and SAG29 could be inhibited by VNI2 suggesting that VNI2 regulates ORE1 too.
Besides, I found that the expression level of VNI2 is strongly activated by ATAF2 and ORE1, suggesting that the existence of a feedback mechanism to fine tune the light-dependent senescence pathway involving ATAF2, ORE1 and VNI2. VNI2 is known to be a very fragile protein and its function as a transcriptional inhibitor at a different level of light-dependent senescence pathway explains that short-term inhibition at partially and temporally makes a significant contribution on natural senescence as well as dark-induced senescence in the process of fine-tuning., ACKNOWLEDGEMENTS　 ii
TABLE OF CONTENTS 　iii
LIST OF FIGURES　 iv
Summary ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 1
Chapter 1 ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・4
Chapter 2 ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・11
2.1 Introduction ・・・・・・・・・・・・・・・・・・・・・・・・12
2.2 Materials and methods ・・・・・・・・・・・・・・・15
2.3 Results ・・・・・・・・・・・・・・・・・・・・・・・・・・・・20
2.4 Discussion ・・・・・・・・・・・・・・・・・・・・・・・・・23
2.5 Figures・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 25
Chapter 3 ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 35
3.1 Introduction ・・・・・・・・・・・・・・・・・・・・・・・・ 36
3.2 Materials and methods ・・・・・・・・・・・・・・・・ 38
3.3 Results ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・41
3.4 Discussion ・・・・・・・・・・・・・・・・・・・・・・・・・・ 45
3.5 Figures・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 49
Chapter 4 ・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 60
4.1 Introduction ・・・・・・・・・・・・・・・・・・・・・・・・61
4.2 Materials and methods ・・・・・・・・・・・・・・ 63
4.3 Results ・・・・・・・・・・・・・・・・・・・・・・・・・・・ 65
4.4 Discussion ・・・・・・・・・・・・・・・・・・・・・・・67
4.5 Figures・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 69
Chapter 5 ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 79
References ・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 86, 主指導教員 : 山口雅利, text, application/pdf},
 school = {埼玉大学},
 title = {Biological roles of an NAC domain transcription factor, VNI2, in response to environmental statuses in Arabidopsis},
 year = {2018},
 yomi = {ナガハゲ エスラ スメダ プリヤダルシャナ}
}