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UMR 1332 Biologie du Fruit et Pathologie

Friday november 27 2020 - 9h30 - Videoconference - INRAE Villenave d'Ornon - Thesis defence Jiaojiao WANG

"Study of starch metabolism in tomato fruit by using forward and reverse genetic approaches"

Jiaojiao WANG

PhD Team Métabolisme (META)

PhD Supervisor : Yves GIBON 

Lieu :

Videoconference

Résumé :

Starch plays a central role during fruit development and ripening. Its degradation provides energy and sugars in ripening fruits whereas its accumulation responds to environmental stresses, which suggests a buffering role. Knowledge of starch metabolism in leaves has dramatically improved, mostly through the characterization of Arabidopsis mutants (starch-less or -excessive), but only very little is known in fruits. In this study, we used both forward and reverse genetic approaches to obtain tomato mutants with starch-excess fruits.

For applying forward genetics, we took advantages of the robotic platform and the large EMS mutant population of tomato (Micro-Tom, an ideal model for starch research), and performed a screen by using red fruits of nearly 3,000 individuals. Three starch-excess candidates were found and one (P17C12) could be confirmed as a homozygous mutant with a recessive starch-excess phenotype with Mendelian inheritance analysis. A large BC1F2 population was phenotyped and the starch-excess phenotype was mapped-by-sequencing. The sequence data obtained from the two bulks revealed that the mutation was likely located at the end of chromosome 1, a region that harbours 1 KO and 2 missense mutations as well as numerous mutations in non-coding regions. We decided to work with the KO, missense mutations and mutations in 5’ UTR and 3’ UTR first, which were a RecQ helicase 4 gene (RECQ4, Solyc01g103960), a mitochondrial carrier protein family gene (MCF, Solyc01g095510), a phospholipid-transporting ATPase gene (PTA, Solyc01g096930), a conserved peptide upstream open reading frame (CPuORF, Solyc01g105700), and a rubredoxin protein family gene (Solyc01g097910), respectively.

To evaluate those mutations, we utilised different bioengineering techniques adapted to each case. We created KO or missense homozygous mutants by using CRISPR/Cas9 for RECQ4, MCF and PTA candidate genes, and homozygous mutants with insertion/deletion mutations in the 5’ UTR for CPuORF, but none of them showed a starch-excess phenotype. For Rubredoxin, besides creating KO mutants and mutants with similar mutations in 3’ UTR with CRISPR/Cas9, we also overexpressed Rubredoxin under the control of the CMV-35S promoter as we found a significant increase of Rubredoxin mRNA in the 12 DAP (days after pollination) fruit of BC1F1 and S2 (P17C12), compared to the WT. The homozygous KO mutants could not grow through cotyledon stage, indicating that the lack of Rubredoxin is lethal for phototrophic plants. The mutants with mutations in 3’ UTR showed no changes in starch amount or mRNA levels of rubredoxin. Then, among the transformants overexpressing Rubredoxin, some showed 100 times higher expressions levels of Rubredoxin in leaves, but showed no significant starch increase in 20 DAP fruit. Finally, none of the five candidate genes checked here led to a starch-excess phenotype in fruits, implying that more investigations are required regarding fine-mapping and mining the possible candidate genes.

We also attempted to obtain starch-excess mutants via reverse genetics. We selected one alpha-amylase (AMY3.2) and three beta-amylases (BAM1.2, BAM3.2 and BAM9) to create the corresponding KO mutants by CRISPR/Cas9 techniques. We obtained homozygous KO mutants of AMY3.2 and BAM9, and heterozygous KO mutants of BAM1.2 and BAM3.2 in T1 generation. The mutants of two transgenic lines lacking AMY3.2 showed lower starch content in 20 DAP fruits. While the loss of BAM9 led to poor fertilization in mutants. Furthermore, in most of mutants, starch levels were remarkably high in the ripening 35 DAP fruit, accompanied with a significant high level of glucose in red ripe fruits. Then, in the red ripe fruit, mutants contained significant more glucose than WT fruits. Those results lead us to speculate that BAM9 may play important roles both in pollen viability and starch degradation during fruit ripening.

Jury members :

M. GIBON Yves - INRAE - PhD Supervisor
Mrs. DINANT Sylvie -INRAE - Reporter
M. ZOUINE Mohamed - ENSAT - Reporter
Mrs. COOKSON-WODRICH Sarah Jane - INRAE - Examiner
M. HERNOULD Michel -University of Bordeaux - Examiner
Mrs. ISSAKIDIS-BOURGUET Emmanuelle- Institute of Plant Sciences Paris-Saclay - Examiner
M. BALDET Pierre - INRAE - Invited