Function Of CLE14, A Peptide Signal-encoding Gene, In Regulating Leaf Senescence In Arabidopsis

The peptides encoded by CLE(CLAVATA3/ENDOSPERM SURROUNDING REGION)family genes, as a type of plant hormone, play an important role in regulating the cell division and differentiation of plant meristems through ligand-receptor signaling pathways. It has been reported that CLE family genes control growth and development of plant roots, stems, flowers, and the division and differentiation of the meristem stem cells. Arabidopsis has 32 CLE genes, most of their functions are still unknown except a few members including CLV3, CLE8, CLE40, CLE41 and CLE45. In this study, we adopted the method of reverse genetics to study the function of CLE14 by observing and analyzing the phenotype of twoArabidopsis CLE14 T-DNA insertion mutants, combined with analysis of CLE14 expression patterns, CLE14 overexpression and complementation experiments. In addition, we try to obtain the cle14 knock-out mutants using TALEN technology. The main results are as follows:(1) We analyzed the CLE14 expression levels in young leaf, mature leaf, early senescence leaf and late senescence leaf via RT-PCR and qRT-PCR. The rusults showed that the CLE14 expression level was very low in non-senescence and early senescence leaves, but sharply increased in late senescence leaves, 7.1 times and 4.1 times higher than maturity and early senescence period respectively. We found that CLE14 was expressed more actively in the aging part of a leaf, especially in late senescence part. The expression pattern analysis experiments indicated that CLE14 is upregulated during leaf senescence and might be involved in leaf senescence in Arabidopsis.(2) We obtained two CLE14 T-DNA insertion lines via PCR identification, finding that their rosette leaves showed precocious senescence compared to wild type, and the phenotype of cle14-c, whose T-DNA insertion position was closer to coding region, was more obvious. RT-PCR experiments showed that CLE14 expression of the two T-DNA insertion lines was not completely knocked out, with a small amount of expression remained. Determination of chlorophyll content, Fv/Fm, ion leakage of cle14-c mutant and wild-type rosette leaves showed that the chlorophyll content of mutant leaves averaged 0.336 mg/g, significantly lower than that of the wild type, 0.543 mg/g. At the same leaf position, Fv/Fm of cle14 mutant is generally lower than wild type, and ion leakage rate is generally higher than wild type, the differences are more significant in the middle leaves. The above experiments indicate that the loss of CLE14 cause precocious senescence of Arabidopsis leaf.(3) We found that the rosette leaves of overexpression line showed delayed senescence compared to wild type by observing the phenotype of CLE14 overexpressing transgenic lines. Determination of chlorophyll content, Fv/Fm, ion leakage of CLE14 overexpression line and wild-type rosette leaves showed that the chlorophyll content of transgenic line leaves averaged 0.743 mg/g, significantly higher than that of the wild-type, 0.568 mg/g. At the same leaf position, Fv/Fm of the overexpression line is generally higher than wild type, and ion leakage rate is generally lower than wild type. When treating the detached Arabidopsis leaves with synthetic CLE14 peptide, leaf senescence was delayed to some extent, similar to the phenotype of CLE14 overexpression line. The above experiments indicate that overexpression of CLE14 in Arabidopsis delays leaf senescence.(4) We preformed a complementation test experiment by introducing the construction ProCLE14::CLE14 which promoted CLE14 expression with its native promoter to cle14 muants, finding that CLE14 restored the cle14 muant plants to wild type. The above experiment confirmed that loss of CLE14 expression in the cle14 mutant was the only cause of the precocious senescence phenotype.(5) We constructed a pair of TALEN plasmids aiming to knock out CLE14 and cloned them to a subcloning vector, obtaining the plant expression vector of TALEN. Gene mutation detection of transgenic positive plants showed that the mutation may occur on target genes of some samples, further sequencing analysis was needed to identify knockout plants.