Mechanistic Study Of The Regulation Of Juvenile-to-adult Phase Transition By DEL3 In Arabidopsis

The juvenile-to-adult phase transtion is a key developmental process in plants. It regulates reproductive competence, plant architecture and biomass production, the biosynthesis of plant secondary metabolites, biotic and abiotic response, and other developmental processes and economically important traits. Therefore, it is of great importance to study the molecular mechanisms underlying juvenile-to-adult transition in plants. Previous studies have shown that miR156-SPLs pathway is the master regulatory pathway controlling juvenile-to-adult transition in plants; however gene networks upstream and downstream of miR156-SPLs pathway still remain to be explored.In order to identify upstream factors regulating miR1256 expression, we performed a forward genetic screen in a M2 population of an EMS-mutagenized Arabidopsis seeds. We identified a mutant with delayed juvenile-to-adult phase transition phenotypes, which we named del3 (delayed juvenile-to-adult phase transition mutant 3,). In addition to producing later abaxial trichomes on the abaxial side of leaves compared with wild-type plants, del-3 also exhibited pleiotropic phenotypes including small overall stature, slow growth rate, yellowish, crinkled and serrated leaves, and distorted phyllotaxy of siliques.Map-based cloning analysis narrowed this mutation down to two markers between T22A6(B) and F23E 13(B) on chromosome IV. Analysis of mutant phenotype of candidate genes revealed that del3 showed a similar phenotype to cia1-2, an allelic mutant to atase2, which encodes a key purine biosynthetic enzyme,amidophosphoribosyltransferase, ATasse2 (AT4G34740). Resequencing of this candidate gene showed that there was a G-to-A mutation at the 725th base in the open reading frame, thus causing the replacement of a glycine at position 242 by an aspartic acid. Genetic complementation test confirmed that del3 and cia1-2 were allelic. Our results here demonstrated that the juvenile-to-adult phase change phenotype of deli was truly due to the mutation in the AT4G34740 gene.qRT-PCR analysis of the expression of genes in the miR156-SPLs pathway showed that the expression of pri-MIR156A and pri-MIR156C were significantly up-regulated in del3, whereas that of miR 172 and SPL3 was significantly down-regulated, and the expression of miR156?SPL9?SPL13?pri-MIR172B remained unchanged compared with that in WT. To test if DEL3 regulates the transcription of miR156A and SPL3, we crossed the miR156A and SPL3 transcriptional GUS reporter line pMIR156A::GUS and pSPL3::rSPL3-GUS to del3 to generate homozygous plants.GUS staining of pMIR156A::GUS and pSPL3::rSPL3-GUS lines in wild-type and del3 background demonstrated that DEL3 regulates the transcription of miR156A and SPL3. Genetic analysis indicated that miR172 over-expression transgeneic plants(Ubi10::MIR172B) and toe1/toe2 double mutant could partially rescue the delayed juvenile-to-adult phase transition phenotype of del3, suggesting that downregulation of miR172 is partially responsible for the delayed vegetative phenotype of del3, and miR172 acts downstream of DEL3.DEL3 is a chloroplast-localized protein encoded by a nuclear gene. It regulates early chloroplast development and purine biosynthesis. Loss-of-function mutation in DEL3, such as cia1-2 and dg169, led to abnormal chloroplast development and defects in photosynthesis. Previous studies indicated that levels of pri-MIR156A,pri-MIR156C, and mature miR156 were significantly elevated in the ch1-4 mutant with defect in chrolophyll b biosynthesis and reduced level of sugar resulted from impaired photosynthesis. In this study, we found that the defects in photosynthesis in del3 could also affect the expression of pri-MIR156A, pri-MIR156C, but not the level of mature miR156. Exogenous addition of sugar could only partially recover the delayed vegetative phase change phenotype of del3. These results suggest that DEL3 can modulate the miR156-SPLs pathway not only by the chloroplast development-photosynthesis- sugar signaling pathway but also by other means.The ratio of miR156/pri-MIR156 and miR172/pri-MIR172 in the del3 mutant was significantly lower than that in WT. This phenomenon also applies to other 9 randomly selected miRNAs. This implies that del3 is defective in miRNA processing.The pleiotropic phenotypes as manifested by del3 are also reminiscent of the typical to phenotype of other mutants defective in miRNA processing. Further genetic analysis showed that hyl1-2 and se-1, two mutants defective in miRNA processing,could significantly rescued the delayed juvenile-to-adult phase transition phenotype of del3, implying that hyl1-2 and se-1 are epistatic to del3.Based on above results, we proposed two processes that DEL3 acts in the regulation of juvenile-to-adult phase change in Arabidopsis: 1. DEL3 is involved in the regulation of chloroplast development and it regulates the expression of pri-MIR156A and pri-MIR156C through its effect on the chloroplast development-photosynthesis-sugar signaling pathway. Due to the defects in chloroplast development and photosynthesis in del3, the repression of miR156 transcription is alleviated by the lack of sugar, leading to elevated levels of pri-MIR156A and pri-MIR156C; 2. DEL3 is responsible for purine biosynthesis and metabolism, it somehow modulates nuclear miRNA processing through its involvement in the chloroplast-nuclear retrograde signaling pathway. Althought the expression of pri-MIR156A and pri-MIR156C was elevated in deli, the mature level of miR 156 remained stable due to defects in miRNA processing. However,the primary transcripts of miR172 remain relatively stable in del3, defects in miRNA processing correspondingly led to the downregualtion of the mature miR172 in del3.In conclusion, our study demonstrated that DEL3 modulates miR156-SPLs pathway by its effects on chloroplast development, photosynthesis and sugar signaling pathway, purine biosynthesis and metabolism, and miRNA processing pathway, thus regulating the juvenile-to-adult phase transition in Arabidopsis.