Phytohormone and integrated mRNA and miRNA transcriptome analyses and differentiation of male between hermaphroditic floral buds of andromonoecious Diospyros kaki Thunb

Abstract

Background: Persimmon (Diospyros kaki Thunb.) has various labile sex types, and studying its sex differentiation can improve breeding efficiency. However, studies on sexual regulation patterns in persimmon have focused mainly on monoecy and dioecy, whereas little research has been published on andromonoecy. In order to reveal the sex differentiation regulation mechanism of andromonoecious persimmon, we performed histological and cytological observations, evaluated OGI and MeGI expression and conducted phytohormones assays and mRNA and small RNA transcriptome analyses of the male and hermaphroditic floral buds of the andromonoecious persimmon 'Longyanyeshi 1'.

Results: Stages 2 and 4 were identified as the critical morphological periods for sex differentiation of 'Longyanyeshi 1' by histological and cytological observation. At both stages, OGI was differentially expressed in male and hermaphroditic buds, but MeGI was not. This was different from their expressions in dioecious and monoecious persimmons. Meantime, the results of phytohormones assays showed that high IAA, ABA, GA₃, and JA levels at stage 2 may have promoted male floral bud differentiation. However, high JA levels at stage 4 and high ZT levels at stages 2 and 4 may have promoted hermaphroditic floral bud differentiation. In these phytohormone biosynthesis and signaling pathways, 52 and 54 differential expression genes (including Aux/IAA, ARFs, DELLA, AHP, A-ARR, B-ARR, CYP735A, CRE1, PP2C, JAZ, MYC2, COI1, CTR1, SIMKK, ACO, and MPK6) were identified, respectively. During the development of male floral buds, five metacaspases genes may have been involved in pistil abortion. In addition, MYB, FAR1, bHLH, WRKY, and MADS transcription factors might play important roles in persimmon floral bud sex differentiation. Noteworthy, miR169v_1, miR169e_3, miR319_1, and miR319 were predicted to contribute to phytohormone biosynthesis and signaling pathways and floral organogenesis and may also regulate floral bud sex differentiation.

Conclusion: The present study revealed the differences in morphology and phytohormones content between male and hermaphroditic floral buds of 'Longyanyeshi 1' during the process of sex differentiation, and identified a subset of candidate genes and miRNAs putatively associated with its sex differentiation. These findings can provide a foundation for molecular regulatory mechanism researching on andromonoecious persimmon.

Keywords: Andromonoecy; Diospyros kaki; Phytohormone; Sex differentiation; mRNA; miRNA.

Fig. 1

Male and hermaphroditic ‘Longyanyeshi 1’ floral buds. a Three-flower cyme. b Male and hermaphroditic floral bud anatomy. St: stamen; DP: defective pistil; Pi: pistil; Ov: ovary

Fig. 2

External morphological changes of the ‘Longyanyeshi 1’ floral buds

Fig. 3

Internal morphological differences between male and hermaphroditic ‘Longyanyeshi 1’ persimmon floral buds. a Stamen and carpel primordia appeared in male and hermaphroditic floral buds. b Anther primordia appeared but neither ovaries nor ovules formed in the carpel bases. c Filament and anther compartments appeared. d Pistils were aborted. e Anther, style, stigma, ovary, and ovule primordia appeared. f Filament and anther compartments appeared and funicles formed. g Stamens and carpels developed normally. St: stamen; C: carpel; Ov: Ovule; Ow: ovary wall

Fig. 4

OGI and MeGI construction diagram. UTR: untranslated regions; IR: inverted repeat; FR: forward repeat

Fig. 5

Small RNAs accumulation on the OGI genomic sequence. Data are expressed as the mean ± standard error of three replications. Red and black letters indicate a significant difference between male and hermaphroditic floral buds at each developmental stage, based on an independent T-test at the P < 0.05 significance level

Fig. 6

Small RNA accumulation on the MeGI genomic sequence. Data are expressed as the mean ± standard error of three replications. Red and black letters indicate a significant difference between male and hermaphroditic floral buds at each developmental stage, based on an independent T-test at the P < 0.05 significance level

Fig. 7

Differential expression of MeGI. FC, fold change

Fig. 8

Phytohormone levels in male and hermaphroditic floral buds at various stages. a Indole-3-acetic acid (IAA). b Abscisic acid (ABA). c Gibberellin 3 (GA₃). d Jasmonic acid (JA). e Zeatin (ZT). Data are expressed as the mean ± standard error of three replications. Red and black letters indicate a significant difference between male and hermaphroditic floral buds at each developmental stage, based on an independent T-test at the P < 0.05 significance level

Fig. 9

Heat map of clustered DEGs at stages 2 (a) and 4 (b). The original expression values of the DEG FPKM (fragments per kilobase per million) were normalized by Z-score

Fig. 10

GO and KEGG pathway enrichment analyses of DEGs at stages 2 (a, b) and 4 (c, d)

Fig. 11

Numbers of differentially expressed TFs at stages 2 (a) and 4 (b)

Fig. 12

Heat map of DEGs related to phytohormone biosynthesis and signaling pathways at stages 2 (a) and 4 (b). The original expression values of the DEG FPKM (fragments per kilobase per million) were normalized by Z-score

Fig. 13

Small RNA length distribution and abundance in 12 libraries. X-axis, length of small RNA distribution; Y-axis, corresponding percentage of raw reads

Fig. 14

DEG and DEM validation at stages 2 (a, b, c) and 4 (d, e, f) by RT-qPCR