| Plant phase transition is the transition from a plant growth stage to the next growth stage. The process of plant phase transition is divided into two parts-vegetative and reproductive growth stage.Vegetative phase transition refers to the process of plant from juvenile phase to adult phase which has close correlation with plant biomass, crop yield, plant architecture, biotic and abiotic response, and secondary metabolite production. Reproductive phase transition is vegetative-to-reproductive stage which is marked with bolting and flowering. It is suggested that miR156-SPLs pathway is the main regulatory pathway to regulate the vegetative growth stage and is also an important pathway for the regulation of plant reproductive growth stage. Therefore, researching on the function of upstream and downstream components of miR156-SPLs pathway can not only reveal the development of molecular regulatory mechanism of plant phase transition, but also provides the theoretical reference for high fruit yield, ideal plant architecture and resistance.In our study, a mutant of phase transition delay mutant (named dell) was screened in the M2 population by positive mutagenesis of Arabidopsis thaliana by means of forward genetics. After map-based cloning, candidate genes sequencing and allelic complementation assays, the mutant was determined to be a functional deletion allele of the FRY1 (also known as SAL1) gene.FRY1 gene encodes a protein having an inositol polyphosphate 1-phosphatase and a 3'(2'),5'-bisphosphate nucleotidase phosphatase activity. Besides, FRY1 function-deficient mutants have diverse phenotypes,such as drought resistance, leaf and root morphology. However, the relationship between the gene and the phase transition has not been reported.So, the dell mutant was investigated in our study by map-based cloning analysis, candidate gene sequencing, allelic complementation analysis, phase transition phenotype observation, phase transition-related pathway gene expression analysis, genetic relationship analysis, and drought resistance physiological index determination etc. The major results are as follows:1. The dell mutant mutations was identified to be located on chromosome 5 between the MGI19 and MSJ1 marker primers at a physical distance of 157 kb by map-based cloning analysis.2. Analysis of the gene function in the candidate region suggested that dell mutant gene-was AT5g63980 (FRY1 or SAL1). Gene cloning and sequencing revealed that the G in the end of the fifth intron of the dell mutant AT5g63980 gene was mutated to A. The dell mutant mRNA was extracted and cloning analysis of the gene showed that the fifth intron selective splicing site of the AT5g63980 gene in the dell shifted to the next AG site, and the fifth intron was 16 bp longer than that of WT.Correspondingly, the mature mRNA sequence of AT5g63980 was delimited by 16 bp in the dell mutant.The deletions resulted in frameshift and premature termination of translation, and the C-terminal domain of the DELI protein the mutant was missing.3. The mutant SALK 020882 of AT5g63980 gene was purchased at TAIR, and the FI plants were obtained by hybridization with dell. The phenotype analysis showed that SALK 020882, dell and F1 had a consistent defect phenotype. The above-mentioned allelic complementation determined dell as a functional deletion mutant of AT5g63980.4. Compared to WT, the dell mutant leaves were more round and the leaf aspect ratio of the same leaf position was smaller than that of WT, and the rate of leaf occurrence was slower than that of WT (1 leaf position). The leaf position of abaxial trichome appearance was later (1.98), and the number of rosette leaves was 4.41 more than that of WT, and the bolting and flowering delayed 11.78 days. These results showed that dell had vegetative phase transition and reproductive phase transition delay phenotype.5. The expression level of miR156 and its target gene SPL9 in dell mutant was not significantly different from that in WT, while the SPL3 gene was significantly down-regulated. In addition, the expression level of pri-M1R172B and miR172 in dell mutant were significantly lower than that in wild type. Flowering-related gene testing revealed that FUL, LFY, AP1, SOC1 were also down-regulated in the dell mutant. The pSPL3-GUS/del1 was obtained by crossing with the pSPL3::rSPL3-GUS reporter strain. GUS staining analysis showed that the ability of rSPL3-GUS staining was weakened in the dell mutant background, which was correlated with gene expression analysis of the SPL3 in the del1 mutant.6. miR172 and SPL3 were significantly down-regulated in dell based on gene expression analysis,then, genetic relationship analysis revealed that miR172 overexpression (Ubi10::MIR172B) and SPL3 overexpression (35S::rSPL3) were able to restore the dell phase transition delay phenotype, indicating that DELI acts upstream of miR172 and SPL3.7. The dehydration rate of dell mutant was significantly lower than that of WT at 11 time points from 0 to 300 min, and the survival rate after drought treatment was higher (10%) than that of WT. In addition,1/2MS medium added mannitol simulated drought resistance experiments found,dell mutants relative to the root length were longer than WT. The results of the above physiological indexes showed that the dell mutant was more resistant to drought stress than WT.In summary, we found that DEL1 (also known as FRY1, SAL1) has the function of regulating the phase transition and developmental process of Arabidopsis. Based on the accumulation of phase transition studies and the experimental results in this study, we propose the following processes: DEL1 regulates the key miRNA-miR172 during phase transition in an unknown manner. miR172 negatively regulates downstream genes (including SOC1, SPL3, LFY, API, FUL) through its target gene AP2-like gene, in which SPL3 transcription factor can also directly regulate flowering-related genes (LFY, API and FUL). Thus, DEL] regulates Arabidopsis thaliana phase transition and developmental process. |
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