Abstract:
The regulation of gene expression is a complex, multifaceted molecular process that controls development, growth and environmental adaptation. At its core, gene expression is regulated at the level of transcription, however, post-transcriptional mechanisms such as alternative pre-mRNA splicing contribute to its fine-tuning. While initially believed to be rare in plants, an increasing number of examples have demonstrated the importance of alternative splicing in diverse processes, including the context of flowering time control.
Plant reproductive success depends in part on the correct onset of flowering. Because of its importance, flowering is tightly regulated by several antagonistic floral inductive and repressive pathways. Plants integrate endogenous and exogenous signals to assess the overall environmental conditions and choose accordingly the best time to flower. Among the environmental signals perceived by plants, ambient temperature has received limited attention. One of the processes by which temperature can affect gene expression is through alternative splicing during mRNA maturation. The thermoregulation of alternative splicing and its contribution to flowering time regulation and morphogenesis in Arabidopsis thaliana are the focus of my dissertation.
In Chapter 2 I sought to clarify the role of FLM, a MADS domain transcription factor involved in flowering time regulation, which is characterized by temperature-dependent alternative splicing. FLM gives rise to two major isoforms whose role in the control of flowering has been discussed controversially. To investigate the specific contribution of these two isoforms I employed CRISPR/Cas9 gene editing to introduce targeted deletions in the FLM locus. These novel mutant lines lack exons specific for each main isoform, allowing a systematic analysis of their contribution to flowering time regulation. The results reported here support a central role for FLM-β, the most abundant isoform, in repressing the transition to flowering. Moreover, I show that FLM-δ, which has been proposed as a regulator of flowering induction in recent in vitro studies, does not promote flowering in vivo, likely because its endogenous expression levels do not reach the required activation threshold.
Plants are highly responsive to temperature fluctuations and are capable of modulating organogenesis and growth rate to adapt to novel conditions. In chapter 3 I describe the function of a novel, bona fide alternative splicing regulator that is essential for correct development and morphogenesis of Arabidopsis thaliana at low temperature. Loss of function mutations of this gene, which I named PORCUPINE (PCP), displayed severe meristem defects when grown at 16°C, whereas at 23°C no obvious phenotype was detectable. At any developmental stage, by solely changing the temperature, plant growth can be arrested or reinitiated. This behavior indicates the presence of a mechanism that allows rapid adaptation of growth and development to abrupt changes in the ambient temperature. The meristem defects detected in the mutants can be largely explained by the misregulation of two genes involved in maintaining the stem cell fate in the shoot apical meristem, WUSCHEL and CLAVATA3. These findings support the importance of temperature-dependent alternative splicing in plant morphogenesis and establish PCP as an important regulator of environmental adaptation.
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