Identification of regulatory genes controlling cell wall invertase expression in Arabidopsis reproductive organs

In higher plants, sucrose (Suc) is synthesised in source leaves for translocation through phloem to non-photosynthetic organs (sinks). Upon reaching sink cells, Suc is often hydrolysed by cell wall invertase (CWIN) into glucose (Glc) and fructose (Fru), as building blocks, energy source and signalling molecules for growth and development. Despite the central roles of CWIN in assimilate partitioning, the upstream molecular machineries that control CWIN expression remain largely unknown. This PhD project therefore aims to identify the ‘master’ regulatory genes controlling CWIN expression in reproductive organs using Arabidopsis as a model. We hypothesised that CWIN gene expression is possibly regulated by some transcription factors (TFs) and small RNAs (sRNAs). As the first step, a suite of bioinformatics tools was employed to search for candidate genes controlling the expression of CWIN2 and CWIN4, which are predominantly expressed in Arabidopsis reproductive organs. The analyses identify 18 TF genes and one microRNA (miRNA) as putative regulators of CWIN2 or CWIN4. Among them, MYB21, ARF6, ARF8, AP3 and CRC were confirmed to be regulators for CWIN gene expression based on the finding that their mutants exhibited reduced mRNA level of CWIN2 or CWIN4. One miRNA, miR3932, displayed an inverse expression pattern and an imperfect sequence match with CWIN2, indicating its possible involvement in degrading CWIN2 RNA. Due to time constraint, further studies were focused on examining the roles of candidate TFs in modulating the expression of the CWIN genes. Measurement of GUS signal in the mutants of the confirmed TF genes transformed with ProCWIN2/4:GUS revealed that MYB21, ARF6 and ARF8 mainly control CWIN2 expression in anthers/pollen, whereas MYB21, ARF6, ARF8, AP3 and CRC appear to regulate CWIN4 expression in nectaries and/or petals. Indeed, over-expression of AP3 increased CWIN2 and CWIN4 expression in the inflorescence, Furthermore, increasing the expression of CWIN4 driven by ARF8, promoter partially recovered arf8-3 short silique phenotype. Finally, transient expression of ARF6 and CRC induced CWIN2 and CWIN4 transcript levels, respectively, in Arabidopsis leaf protoplasts. Based on the results obtained, we proposed a model on the molecular network controlling CWIN2 and CWIN4 expression by MYB21, ARF6, ARF8, AP3, CRC and possible hormone pathways. Understanding the regulatory mechanisms controlling CWIN gene expression fills a major knowledge gap in the area. It also provides an opportunity to genetically modify the expression of CWIN2 and/or CWIN4 or related genes in reproductive organs by manipulating their regulatory genes. This approach has the potential to significantly improve the partitioning of assimilates towards plant reproductive organs for achieving high crop yield.