טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentCohen Hagai
SubjectElucidation of the Regulatory Metabolic Networks Involved
in Methionine Biosynthesis in Arabidopsis Thaliana
Seeds
DepartmentDepartment of Biology
Supervisors Professor Gadi Schuster
Professor Rachel Amir
Full Thesis textFull thesis text - English Version


Abstract

Methionine is an essential sulfur-containing amino acid found at low levels in plant seeds, limiting their nutritional value as a protein source. Previous studies suggest that methionine can be synthesized de novo in seeds through the aspartate-family pathway via the activity of its main regulatory enzyme, CYSTATHIONINE γ-SYNTHASE (CGS), and/or from S-methylmethionine (SMM) that is transported from vegetative tissues into the seeds where it is re-converted back to methionine. Yet, the relative contribution of these two pathways to methionine synthesis in plant seeds is far from being fully understood. Revealing this issue is the main objective of the current study. Additionally, this research sets to obtain more knowledge on the relation between methionine metabolism to the Arabidopsis seed metabolome and transcriptome, and its involvement in the in vivo regulation of seed storage compounds. First, we generated transgenic RNAi::AtCGS seeds which accumulated up to two-fold more methionine apparently due to higher flux of transported SMM from vegetative tissues and enhanced inter-conversion of SMM to methionine within seeds by HOMOCYSTEINE S-METHYLTRANSFERASES (AtHMTs). These findings suggest compensative roles for the SMM cycle in maintaining methionine levels in seeds when its synthesis through the aspartate-family pathway is restricted. Such compensation did not occur in RNAi::AtHMT3 seeds that synthesized much less methionine, suggesting that the contribution of the SMM cycle to methionine synthesis at late developmental stages of seed filling in Arabidopsis is more significant than the synthesis de novo of methionine through the aspartate-family pathway. Next, we generated transgenic Arabidopsis seeds expressing a feedback-insensitive form of the Arabidopsis thaliana CGS, named ‘SSE’ seeds, in order to reveal if the aspartate-family pathway is active at late stages of seed development. The SSE seeds accumulated up to six-fold higher methionine levels. Using omics approaches, we demonstrated that higher methionine levels led to higher synthesis of most other amino acids through a yet unknown mechanism. These resulted in overaccumulation of storage compounds such as total protein and starch at the expanse of the seed relative water content, causing stronger desiccation processes during seed development. These processes triggered the induction of global transcriptomic and metabolic stress responses, which were found to be mediated through the metabolism and signaling cascades of the stress-associated hormones of ethylene and abscisic acid. As previous studies proposed that methionine may regulate the synthesis of seed storage proteins in vitro, we used SSE seeds, which contain higher methionine and total protein contents, to examine whether this also occurs in vivo. Proteomic analyses showed that higher methionine affects from a certain threshold the accumulation of several subunits of 12S-globulins and possibly also 2S-albumins, regardless of their methionine contents. The expression of most genes encoding these proteins did not correlate with their abundances, proposing that methionine may regulate them at both transcriptional and post-transcriptional levels. The seeds also had differential fatty acid composition and lower lipid contents, possibly due to fatty acid degradation through the peroxisomal β-oxidation pathway.