Transcriptome analysis of floral sex reversal induced by high temperature in hermaphroditic papaya (Carica papaya L.)

Abstract:【Objective】High temperature generally induces hermaphrodite-to-male sex reversal in papaya, which leads to yield decline and is one of the main factors restricting the high-efficient production of papaya. At present, the molecular mechanism of hermaphrodite-to-male sex reversal induced by high temperature in papaya has not been fully elucidated. In this study, transcriptome analysis was carried out to collect the differentially expressed genes (DEGs) between the male (gynoecium abortion in in-duced by high temperature) and the hermaphroditic (functional gynoecium) flowers, and the contents of endogenous hormones were measured in both flower types. The study aimed to provide a new perspective for exploring the molecular regulation mechanism of floral sex reversal in hermaphroditic papaya. 【Methods】The male and hermaphroditic flowers (length＜5 mm) of hermaphroditic plants of GZY3-6 papaya cultivar were collected as the experimental materials at 11:00—13:00 (air temperature was 39- 40 ℃) on 16th July 2019. Total RNAs were extracted from the flower samples and reverse transcribed into cDNAs for the library construction. The cDNA libraries were sequenced using Illumina Novaseq 6000. Differential gene expression analysis was performed using the DESeq2 R package. StringTie and Ballgown were used to estimate the expression level of all transcripts and perform gene expression levels in FPKM. Genes with p- value＜0.05 and |log2(fold change)|≥1 were considered differentially expressed. GO and KEGG enrichment analyses of the DEGs were conducted using R package GOseq software and KOBAS software, respectively. 17 differently expressed flower development- and hormone-related genes were selected and their specific primers designed for qRT- PCR verification. PlantCARE was used to predict cis- acting elements on the promoters of these flower development- related genes, and TBtools software was used to visualize the results. Endogenous hormone contents in the male and the hermaphroditic flowers were determined by using high- performance liquid chromatography.【Results】A total of 27 793 genes were obtained, from which 517 DEGs were identified, of which 214 and 303 DEGs were up- and down-regulated in the male flowers, respectively. These DEGs were mainly enriched in GO terms such as transcription and transcriptional regulation, plant hormone response and signal transduction, cell differentiation and regulation of organ growth, plasma membrane component, oxidative stress, and KEGG pathways such as plant hormone signal transduction, plant pathogen interaction, and MAPK signaling pathway. Through the results of functional annotation, 70 DEGs related to plant hormone (including abscisic acid, auxin, cytokinin, ethylene, gibberellic acid, jasmonic acid, and salicylic acid) signaling transduction, response, biosynthesis and metabolism were screened out, of which 52 genes were down- regulated, and 18 genes up- regulated in the male flowers. Furthermore, ANT, ANT2, CIB1, HHO5, SAP, and ZIP21, which are involved in flower development, were down-regulated in the male flowers, and SAP gene was specifically expressed in the hermaphroditic flowers. The promoter sequence of ANT2 was not found in the papaya genome database. The promoters of ANT, CIB1, HHO5, SAP, and ZIP21 were found to contain abscisic acid-and stress-responsive cis-acting elements. In addition, the promoters of ANT, CIB1, and ZIP21 contained auxin- and jasmonic acid-responsive cis-acting elements; the promoter of HHO5 contained jasmonic acid- and salicylic acid-responsive cis-acting elements; and the promoter of SAP contains low temperature-responsive cis-acting element. The expression levels of the 17 selected-DEGs obtained by qRT-PCR matched well with the results of the transcriptome, which verified the reliability of the results of gene differential expression. The contents of ACC, IAA, tZ, SA, and JA in the hermaphroditic flowers were significantly higher than those in the male flowers, and the contents of ABA and GA3 had no significant difference between the two flower types.【Conclusion】A total of 517 DEGs were identified by transcriptome analysis of the male and the hermaphroditic flowers in papaya. They were enriched in plant hormone signal transduction, transcription regulation, plasma membrane components and other biological processes. 70 of the DGEs related to hormones and 6 DGEs related to flower development-related were screened in the male and the hermaphroditic flowers. In the male flowers, the down- regulation of ILL3, GH3.1, GH3.9, LOG3, LOG5, LOX2 and OPR3 genes and the up-regulation of SAMT1 led to the decrease in the accumulation of IAA, tZ, SA and JA, and resulted in the decrease in the expression of ANT, CIB1, and HHO5. TheP gene promoter contains low temperature- responsive cis- acting element, and high temperature might inhibit its expression. These changes in profile of genes induced by high temperature may result in hermaphrodite-to-male sex reversal in papaya.