PmAGAMOUS recruits polycomb protein PmLHP1 to regulate single-pistil morphogenesis in Japanese apricot

Abstract

Japanese apricot (Prunus mume Sieb. et Zucc.) is a traditional fruit tree with a long history. Multiple pistils (MP) lead to the formation of multiple fruits, decreasing fruit quality and yield. In this study, the morphology of flowers was observed at four stages of pistil development: undifferentiated stage (S1), pre-differentiation stage (S2), differentiation stage (S3), and late differentiation stage (S4). In S2 and S3, the expression of PmWUSCHEL (PmWUS) in the MP cultivar was significantly higher than that in the single pistil (SP) cultivar, and the gene expression of its inhibitor, PmAGAMOUS (PmAG), also showed the same trend, indicating that other regulators participate in the regulation of PmWUS during this period. ChIP-qPCR showed that PmAG could bind to the promoter and the locus of PmWUS, and H3K27me3 repressive marks were also detected at these sites. The SP cultivar exhibited an elevated level of DNA methylation in the promoter region of PmWUS, which partially overlapped with the region of histone methylation. This suggests that the regulation of PmWUS involves both transcription factors and epigenetic modifications. Also, the gene expression of Japanese apricot LIKE HETEROCHROMATIN PROTEIN (PmLHP1), an epigenetic regulator, in MP was significantly lower than that in SP in S2-3, contrary to the trend in expression of PmWUS. Our results showed that PmAG recruited sufficient PmLHP1 to maintain the level of H3K27me3 on PmWUS during the S2 of pistil development. This recruitment of PmLHP1 by PmAG inhibits the expression of PmWUS at the precise time, leading to the formation of one normal pistil primordium.

AGAMOUS-LIKE24 controls pistil number in Japanese apricot by targeting the KNOTTED1-LIKE gene KNAT2/6-a

“…Based on their proximity to AtKNAT2 and AtKNAT6 in the phylogenetic tree, these genes were named PmKNAT2/6-a and PmKNAT2/6-b, respectively; since overexpression of KNAT2 in Arabidopsis expands AGAMOUS ( AG ) expression in carpels and ovules and forms ectopic carpel structures ( Ragni et al 2008 ). And in our previous study, we demonstrated that AG may be involved in the formation of Japanese apricot multi-pistil, so the gene function of PmKNAT2 in P. mume is well worth studying in depth ( Shi et al 2023 ). To further analyze the role of PmKNAT2/6-a and PmKNAT2/6-b in pistil number determination in Japanese apricot, reverse transcription quantitative PCR (RT-qPCR) experiments were conducted on flower buds from the pistil differentiation stage of 2 pistil varieties, ‘Longyan’ (‘LY’, single-pistil variety) and ‘Dayu’ (‘DY’, multi-pistil variety) ( Fig.…”

“…In a previous study, we identified the epigenetic factor PmLHP1 with the function of recognizing and recruiting H3K27me3 and its important role in the pistil number differentiation process in Japanese apricot ( Shi et al 2023 ). Since the Class I KNOTTED1-like homeobox ( KNOX ) genes in Arabidopsis were enriched in tri-methylated lysine 27 of histone H3 (H3K27me3) modifications ( Zhang et al 2007 ), we hypothesized that histone methylation is involved in the PmKNAT2/6-a-mediated multi-pistil construction process.…”

“…In a previous study, we found that PmLHP1 plays a key role in regulating floral number differentiation and that the Arabidopsis double mutant ag-11 lhp1 exhibits a multi-pistil phenotype. Heterologous expression of PmLHP1 rescued this phenotype, resulting in a normal single-pistil phenotype ( Shi et al 2023 ). To determine whether PmLHP1 is involved in the H3K27me3 histone modification process of PmKNAT2/6-a , RT-qPCR experiments were conducted using Arabidopsis heterologously expressing PmLHP1 ( Supplementary Figs.…”

“…Our analysis revealed that the motif of PmKNAT2/6-a was enriched at H3K27me3 modification sites, similar to observations in Arabidopsis ( Zhang et al 2007 ). PmLHP1 specifically recognizes and recruits H3K27me3 and represses a variety of genes involved in pistil number development ( Shi et al 2023 ). Western blot assays demonstrated higher expression levels of PmLHP1 and H3K27me3 during the critical period of pistil number differentiation in single-pistil Japanese apricot varieties, and their expression trends showed a positive association, suggesting the potential joint involvement of PmLHP1 and H3K27me3 in pistil number differentiation in Japanese apricot.…”

“…FISH experiments were performed according to the previous study and images were acquired using a Zeiss LSM 800 confocal microscope (Zeiss, Germany) ( Shi et al 2023 ). The excitation wavelength used for the fluorescence microscope to observe the 4′,6-diamidino-2-phenylindole (DAPI) channel was 405 nm, the exposure duration was 100 ms, and the gain was 1.5×.…”