GENETIC DISSECTION OF PINE1 CONTROLLED STEM ELONGATION AND NEW INSIGHTS INTO LONG DAY FLOWERING IN ORYZA SATIVA

Rice flowering is promoted by exposure to short days (SD) and occurs through the coordination of two developmental processes: the shoot apical meristem (SAM) produces an inflorescence while, along the stem, internodes elongate to allow heading. Recently, PREMATURE INTERNODE ELONGATION1 (PINE1), a C2H2 zinc f inger transcription factor, was described as the responsible for stem elongation during flowering and under submergence in deepwater rice. PINE1 is highly expressed when the stem is compact and it is rapidly downregulated as the plant needs to elongate internodes, which occurs during flowering induction in normal paddy rice as well as under submergence in deepwater rice. pine1 mutants shows constitutive internode elongation since germination. In this thesis, morphological studies with EdU staining assay were performed, revealing that the premature internode elongation is due to intercalary meristem (IM) development followed by cell elongation. Premature internode elongation was associated with pine1 mutants of wheat and maize as well, thus showing a conserved role of PINE1 among these species. Rice pine1 mutants are highly responsive to gibberellins (GA) as their internodes rapidly elongate proportionally to the exogenous GA concentration, contrary to WT stems whose growth is unaffected. To understand how PINE1 may be involved in the GA signalling pathway, we have generated single and double knockout mutants with CRISPR-Cas9 of pine1 and four well-characterized genes belonging to GA biosynthesis, perception and signalling pathways: GIBBERELLIN INSENSITIVE DWARF1 (GID1), GIBBERELLIN INSENSITIVE DWARF2 (GID2), SEMI-DWARF (SD1) AND SLENDER RICE1 (SLR1). Two novel gain-of-function dominant mutations in SLR1 were also identified. The novel dominant slender mutants (named slrD) lacks two aminoacids close to the DELLA motif and might be crucial for its degradation, causing SLR1 overaccumulation and dwarf phenotype. Double mutants pine1-gid1, pine1-gid2 and pine1-slrD, even preserving an overall dwarf aspect similarly to gid1, gid2 and slrD respectively, can develop an active IM similarly to pine1 single mutants, as demonstrated with EdU staining assay. RNA sequencing of slrD and slr1 revealed that when a loss-of-function of PINE1 is also present, the transcriptomic profile radically changes. These results suggest that PINE1 activity is crucial for complete silencing of IM, and that PINE1 may act synergistically with SLR1 for chromatin remodelling and repression of its target genes. PINE1 ability to remodel chromatin was assessed by the demonstration of the existence of a trimeric complex between PINE1, a TOPLESS protein and a histone deacetylase. Rice is short-day plant, but in temperate regions, the selection of loss-of-function alleles of some long-day f lowering repressors allows rice cultivation even where growing season is limited and characterized by long day (LD) photoperiod. In the present study, we took advantage of a mutagenized Volano (a typical Italian rice variety) population to identify novel genes or alleles associated with the regulation of flowering time in LD. MutMap is a rapid gene identification method based on whole-genome resequencing of pooled DNA from F2 segregating populations. In this study, it was applied to one F2 population derived from an early-flowering 5 line and to two additional populations derived from late-flowering lines. MutMap revealed that the early f lowering line resulted from a loss-of-function of PHOTOPERIOD SENSITIVITY 13 (OsSE13). The delay of f lowering was indeed caused by an aminoacidic substitution, not yet described, in the rice long day flowering promoter RICE FLOWERING LOCUS T 1 (OsRFT1) in one late flowering line, whereas the second late-flowering line presented a premature stop codon in the gene DROUGHT AND SALT TOLERANCE (OsDST), a gene whose loss-of-function mutant has already been previously reported to regulate drought and salt tolerance.