Genome-wide analysis of the GLP gene family and overexpression of GLP1-5-1 to promote lignin accumulation during early somatic embryo development in Dimocarpus longan.

Longan (Dimocarpus longan Lour.) is an economically important subtropical fruit tree. Its fruit quality and yield are affected by embryo development. As a plant seed germination marker gene, the germin-like protein (GLP) gene plays an important role in embryo development. However, the mechanism underlying the role of the GLP gene in somatic embryos is still unclear. Therefore, we conducted genome-wide identification of the longan GLP (DlGLP) gene and preliminarily verified the function of DlGLP1-5-1. Thirty-five genes were identified as longan GLP genes and divided into 8 subfamilies. Based on transcriptome data and qRT‒PCR results, DlGLP genes exhibited the highest expression levels in the root, and the expression of most DlGLPs was upregulated during the early somatic embryogenesis (SE) in longan and responded to high temperature stress and 2,4-D treatment; eight DlGLP genes were upregulated under MeJA treatment, and four of them were downregulated under ABA treatment. Subcellular localization showed that DlGLP5-8-2 and DlGLP1-5-1 were located in the cytoplasm and extracellular stroma/chloroplast, respectively. Overexpression of DIGLP1-5-1 in the globular embryos (GEs) of longan promoted the accumulation of lignin and decreased the H2O2 content by regulating the activities of ROS-related enzymes. The results provide a reference for the functional analysis of DlGLPs and related research on improving lignin accumulation in the agricultural industry through genetic engineering.

Genome-wide analysis of the SAUR gene family and function exploration of DlSAUR32 during early longan somatic embryogenesis.

The early auxin responsive small auxin up-regulated RNA (SAUR) family is an important gene family in the auxin signal transduction pathway. This study focused on the regulatory mechanism of DlSAUR genes during early somatic embryogenesis (SE) and its response to hormone treatment and abiotic stress. Mining of the available Dimocarpus longan Lour. (D. longan) genome sequence yielded 68 putative SAUR genes. Transcript profiles based on RNA-seq data showed that most of the 24 detected DlSAUR genes were highly expressed in the globular embryos (GE) (10) and most of them responded to heat stress and 2,4-D treatment. The results of qRT-PCR showed that most of DlSAUR genes were up-regulated under auxin inhibitor N-1-naphthylphthalamic acid (NPA) and auxin indole-3-acetic acid (IAA) treatments. Moreover, NPA could promote longan SE. The assay for ATAC-seq data analysis showed that chromatin accessibility of 19 of the 24 DlSAUR genes were open during early SE, and most DlSAUR genes differentially expressed during early SE were not associated with H3K4me1 signal enrichment. The DlSAUR32 was selected for subcellular localization and RNA-seq analysis, which encode a cell nuclear-localized protein. Dual-luciferase assays and transient transformation showed that the transcription factors (TFs) DlWRKY75-1 and DlWRKY75-2 might bind to the DlSAUR32 promoters to inhibition gene transcription. Transient overexpression of DlWRKY75-1 and DlWRKY75-2 decreased IAA content in N. benthamiana leaves. Thus, the regulatory network composed of DlSAUR32 and its related TFs may regulate the early longan SE and be involved in the auxin response regulatory pathway of longan.

Genome-wide analysis of the XTH gene family and functional analysis of DlXTH23.5/25 during early longan somatic embryogenesis.

Introduction: Xyloglucan endotransglucosylase (XET)/hydrolase (XTH) is a cell wall-modifying protein that affects cell expansion and loosening of the cell wall. Results: This study focused on the regulatory mechanism of DlXTH genes during early somatic embryogenesis (SE) and the heat stress response in longan. Mining of the available D. longan genome sequence yielded 25 putative XTH genes. Transcript profiles based on RNA sequencing (RNA-seq) data showed that most of the 17 detected DlXTH genes were highly expressed in the embryogenic callus (EC) (8) and globular embryo (GE) (8), and 13 of them responded significantly to heat stress. The assay for transposase-accessible chromatin sequencing (ATAC-seq) data analysis showed that in terms of chromatin accessibility, 22 of the 25 DlXTH genes were open during early SE, and most of the peak DlXTH genes with transcription differences during early SE were associated with high levels of H3K4me1. The most differentially expressed genes, DlXTH23.5 and DlXTH25, were selected for analysis. According to subcellular localization and quantitative real-time PCR (qRT-PCR) analysis, DlXTH23.5/25, which encode cell membrane-localized proteins, were expressed at the highest level in the GE and significantly responded to heat stress. Dual-luciferase assays and transient transformation showed that the transcription factors (TFs) DlWRKY31, DlERF1, and DlERF5 might bind to the DlXTH23.5/25 promoters to activate gene transcription. Transient overexpression of TFs and DlXTH23.5/25 induced XET activity in Nicotiana benthamiana leaves. Under heat stress in the longan EC, the XET activities and expression levels of TFs and DlXTH23.5/25 were significantly increased, and a high concentration of XET might inhibit longan SE. Discussions: Thus, the regulatory network composed of DlXTH23.5/25 and its related TFs may regulate early longan SE and participate in the regulatory pathway of longan under heat stress via cell wall repair through the action of XET.