Caracterización de la regulación transcripcional y función del gen SDH2-3 de Arabidopsis thaliana.

Our group has undertaken the study of mitochondrial function in plants focusing on complex II (succinate dehydrogenase, SDH) of Arabidopsis thaliana. This complex plays a pivotal role in the tricarboxylic acid (TCA) cycle and the electron transport chain, two fundamental processes in the energetic plant metabolism. We have described the existence of three nuclear genes coding for iron-sulfur proteins (SDH2), named SDH2-1, SDH2-2 and SDH2-3. Two of them, SDH2-1 and SDH2-2, are almost identical (96%) and share the same exon-intron structure. SDH2-3 on the other hand, has a different exonintron structure and has approximately 70% identity to the other two. Additionally, unlike SDH2-1 and SDH2-2 that are expressed in adult plants, SDH2-3 expression is confined to seed maturation and it decreases during germination, while SDH2-1 and SDH2-2 transcripts are low in seeds and they begin to accumulate after germination and during vegetative growth. We have described seed-specific cis-elements necessary for promoter activity (three ABA-responsive elements or ABRE and one RY element) and determined in vitro binding of seed-specific transcription factors to them (bZIP10, bZIP25 and bZIP53 binding to ABREs; ABI3 and FUS3 binding to RY). Moreover, we have determined that ABI3 and FUS3 are necessary for promoter activity in planta, as mutant lines of these factors showed decreased SDH2-3 levels in seeds. On the other hand, we have determined that SDH2-3 is important for seed germination, indicating a specific role during this developmental stage. The main focus of this thesis work is to study the seed-specific expression and the function of SDH2-3. It is worth noting that no reports of mitochondrial proteins bearing this singular expression pattern have been published. Therefore, we will determine essential regions of the SDH2-3 promoter and additional putative cis-elements controlling transcriptional regulation, transcription factors involved, and the function of this protein during seed maturation and postgerminative growth. Regarding the SDH2-3 promoter, during this thesis we were able to determine a minimal region necessary and sufficient for promoter activity, between -114 and +49 from the transcription start site. Moreover, the 5´UTR region (+1 to +49) is essential for SDH2-3 promoter activity, as determined by loss-of-function experiments. In addition, transient expression assays showed that ABI3 is able to activate SDH2- 3 transcription in vivo alone or in combination with bZIP factors bZIP10, bZIP25 or bZIP53. However, single bZIP factors were unable to activate the promoter, and only transfection with bZIP10 and bZIP53 was able to induce expression. Moreover, SDH2-3 transcript levels are significantly reduced in bzip53 mutant dry seeds. These results indicate the SDH2-3 promoter is activated by bZIP transcription factors and corroborate the importance of ABI3. On the other hand, we determined that SDH2-3 influences seed development and maturation, as lack of this protein resulted in decreased seed weight. Interestingly, protein content also showed a reduction in sdh2-3 mutants while lipid content did not show any biologically significant variation. This is an interesting feature since seed metabolism is directed during maturation towards the formation of seed proteins and lipids. Thus, the decrease in protein content would explain the lower total weight. Although it has been suggested that mitochondria plays a minor role during seed maturation, these results suggest that its role should be reconsidered, as it may carry out important metabolic tasks during this stage. Mitochondrial role during postgerminative growth is well characterized. Here we show that sdh2-3 mutants have impaired hypocotyl growth in the dark. Moreover, TTFA treatment (complex II inhibitor) abolishes hypocotyl growth and seedling establishment. All these results suggest an essential role for complex II during postgerminative growth and establishment. Wild seeds in nature generally germinate underground, in conditions where they lack direct sunlight. A seed with a non-functional SDH2-3 would be in disadvantage over wild-type seeds, which would elongate their hypocotyls further until they reach light in order to promote photoautotrophic growth. SDH2-3 gives an important advantage to the plant in energy-consuming processes such as germination and early stages towards seedling establishment. This work shows the importance that this nonessential gene can have in critical stages of plant development. The existence of a SDH2-3-like gene in the moss Physcomitrella patens has drawn our attention because SDH2-3 has been described as a seed-specific expressed gene in angiosperms, and mosses do not have seeds. We confirmed the SDH2-3-like gene is expressed and increases under osmotic stress, in contrast to saline stress, desiccation and high ABA content. Moreover, we determined that Physcomitrella SDH2-3 promoter lacks significant activity in Arabidopsis, either in seeds or vegetative tissue. These results indicate that the transcriptional regulation of this gene in Physcomitrella evolved in an independent way as compared to Arabidopsis.