Conclusions and future perspectives

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oxaloacetate/malate transporter exporting reducing equivalents to the cytosol to prevent photo-inhibition (the malate valve) in Arabidopsis. Transport of dicarboxylates across the chloroplast envelope plays an important role in transferring carbon skeletons to the nitrogen assimilation pathway and exporting reducing equivalents to the cytosol to prevent photo-inhibition (the malate valve). Although bHLH146 was shown to interact with DIT1, it needs to be further investigated whether bHLH146 is involved in photo-inhibition. Further, two hormone-related proteins were also identified as bHLH146 interactors. 1-aminocyclopropane-1-carboxylate oxidase 2 (ACO2) is involved in ethylene biosynthesis and gibberellic acid-stimulated Arabidopsis (GASA), which plays a role in gibberellin (GA) response. The Arabidopsis loss-of-function aco2 mutant is impaired in 1-aminocyclopropane-1-carboxylic acid (ACC)-mediated reversion of the ABA-induced inhibition of seed germination (Linkies et al. 2009).

Overexpression of GASA4 suppressed ROS accumulation in plants, and the transgenic seeds were partially resistant to the NO donor sodium nitroprusside (SNP) (Rubinovich and Weiss 2010). Phenotypes of GASA4 overexpression lines occasionally exhibited meristem identity changes in which the plants underwent the transition to flowering and produced floral meristems, followed by a reversion to normal indeterminate inflorescence development.

Flowers of GASA4 overexpression lines exhibited mosaic floral organs, most frequently were ectopic carpel or stamen structures (Roxrud et al. 2007). Interestingly, similar phenotypes were also observed in bHLH146 overexpression lines (Fig. 47). As bHLH146 interacts with GASA4, it is possible that these phenotypes result from the pathway that is regulated by bHLH146-GASA4 complex. It is worth to notice that six of the proteins interact with bHLH146 are uncharacterized proteins. This indicates that bHLH146 may also be involved in yet unknown pathways and needs further investigation.

141 uncharacterized A. thaliana transcription factor bHLH146 and these data will contribute towards understanding its role in multiple processes.

Copy number variations, phylogenetic relationships of CBF/DREB genes between A. thaliana, rice and barley indicated that CBF/DREB genes are diverged between monocots and dicots.

The barley genome is rich in CBF/DREB1 subfamily genes but contains relatively few DREB2 subfamily genes. Expression analysis revealed that two DREB2 subfamily genes HvDREB1 and HvDRF1.3 were constitutively expressed under laboratory and field conditions, indicating that these DREB2 genes play a role as housekeeping genes in barley. In contrast, expression of three other analyzed CBF/DREB1 subfamily genes did not show a clear pattern under the given conditions. The results of this study demonstrate that the field stress conditions are more complex when compared to laboratory conditions. Therefore, the knowledge of gene expression obtained from laboratory conditions cannot always betransferred to complex field conditions under which agricultural production takes place.

Although the involvement of ALDH gene families in detoxification of aldehydes during stress conditions and their significance during different abiotic stress adaptation and tolerance has been reviewed in many plants, until now no reports are available on ALDH gene regulation in salt stress tolerant halophytes. The availability of the genome sequences of both the halophytes E. parvulum and E. salsugineum which are close relatives of A. thaliana helped in identifying the ALDH genes and studying their regulation. Sixteen and 17 ALDH genes were identified by genome-wide analysis from the halophyte models E. parvulum and E. salsugineum, respectively. Genomic organization, copy number, sub-cellular localization and expression profiles of ALDH genes are mainly conserved between A. thaliana, E. parvulum and E.

salsugineum. Except for the expression of ALDH7B4, ALDH3H1, and ALDH10A8, no major differences were observed which may contribute to salinity tolerance in Eutrema halophytes.

Transcripts of ALDH3H1 and ALDH7B4 increased in response to NaCl at higher salt levels in E. salsugineum than in A. thaliana, whereas ALDH10A8 showed a different expression pattern under high salt in A. thaliana and E. salsugineum. This indicates that the regulation of transcription may be better adapted to high salt in E. salsugineum than in A. thaliana. Then the focus was on the gene ALDH7B4, which showed osmotic responsive expression in both A.

thaliana and E. salsugineum. EsALDH7B4 promoter deletion analysis in transgenic A. thaliana plants revealed a conserved G-box motif which is important while a specific “TC” rich motif

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in the EsALDH7B4 promoter represses the gene expression in transgenic A. thaliana. Yeast one-hybrid screening identified more than 40 putative transcription factors that interact with the EsALDH7B4 promoter. Consistent with promoter deletion results, many members from bZIP, bHLH families interacting with the G-box motif were identified. The interaction between the “TC” rich motif and the identified MYB_like transcription factor from yeast one-hybrid screening needs to be further confirmed using independent, biochemical methods.

There has been no report yet on bHLH146. In this study, we have been trying to characterize bHLH146 from two aspects: molecular characterization including expression profiles, DNA binding ability and cellular localization; and functional characterization using “gain of function”

and “loss of function” mutants. In addition, a yeast two-hybrid screening was performed with the aim to get a clue in which cellular pathways bHLH146 may participate. Results from these studies so far showed bHLH146 does not bind to a G-box but is mainly localized in the nucleus and acts as a transcriptional repressor. bHLH146 can interact with other G-box binding bHLH proteins such as bHLH49, bHLH69 and bHLH76, which supports the hypothesis that bHLH146 regulates downstream gene expression in an indirect manner. Since bHLH49 and bHLH76 are CIB proteins and regulate FT (FLOWERING LOCUS T) transcription and floral initiation. Therefore, it is assumed that bHLH146 might also be involved in the same pathway together with these well-studied CIB proteins. In addition, several GTPase related proteins were identified as interactors, which implies that bHLH146 is involved in many regulatory processes. Seedlings of bHLH146 overexpression lines showed a short hypocotyl and had a large open apical hook angle in the dark, which suggests it might be involved in photomorphogenesis. bHLH146 overexpression lines also occasionally exhibited abnormal ectopic petal, carpel or stamen structures while progeny of bHLH146 T-DNA insertion lines are heterozygous plants and exhibit a non-Mendelian segregation, and only heterozygous plants were obtained. An explanation for these phenotypes could be that bHLH146 interacts with GASA4 and TPTC proteins. It is necessary to cross the bHLH146 T-DNA insertion lines and wild type plants to identify the reason for the non-Mendelian segregation. Further analysis of bHLH146 overexpression lines and silencing lines by artificial microRNA should provide a better understanding of the functions of bHLH146.

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