Identification of Kunitz-Type Inhibitor Gene Family of Populus yunnanensis Reveals a Stress Tolerance Function in Inverted Cuttings

Abstract

Plant protease inhibitors are a ubiquitous feature of plant species and exert a substantial influence on plant stress responses. However, the KTI (Kunitz trypsin inhibitor) family responding to abiotic stress has not been fully characterized in Populus yunnanensis. In this study, we conducted a genome-wide study of the KTI family and analyzed their gene structure, gene duplication, conserved motifs, cis-acting elements, and response to stress treatment. A total of 29 KTIs were identified in the P. yunnanensis genome. Based on phylogenetic analysis, the PyKTIs were divided into four groups (1,2, 3, and 4). Promoter sequence analysis showed that the PyKTIs contain many cis-acting elements related to light, plant growth, hormone, and stress responses, indicating that PyKTIs are widely involved in various biological regulatory processes. RNA sequencing and real-time quantitative polymerase chain reaction analysis showed that KTI genes were differentially expressed under the inverted cutting stress of P. yunnanensis. Transcriptome analysis of P. yunnanensis leaves revealed that PyKTI16, PyKTI18, and PyKTI19 were highly upregulated after inverted cutting. Through the GEO query of Populus transcriptome data, KTI genes played a positive defense role in MeJa, drought, time series, and pathogen stress. This study provided comprehensive information for the KTI family in P. yunnanensis, which should be helpful for the functional characterization of P. yunnanensis KTI genes in the future.

Keywords: KTI gene family; Populus yunnanensis; inverted cuttings; transcriptome.

Figure 1

Phylogenetic relationship of KTI gene family. The MEGA 11 with the neighbor-joining method was used to conduct the phylogenetic tree. Different background colors represented different groups of the KTI gene family.

Figure 2

Phylogeny during the evolution of angiosperms. Different colors represent the proportion of each species in the four groups.

Figure 3

Sequence alignment of functional sites of PDB and PyunKTI proteins of V. vinifera (5YH4) and Populus (Q5ZFE7) homologous proteins. A red background indicates complete amino acid sequence identity at the position; a yellow background indicates higher similarity.

Figure 4

Phylogenetic tree, conserved motif, and gene structure of the PyunKTI proteins. Different colors on the phylogenetic tree represent different groups of PyunKTI genes family. In the motif pattern, the motif numbers 1–15 are displayed in different colored boxes. In exon–intron analysis, the black lines represent introns, the green boxes represent the coding sequences, and the yellow boxes represent the non-coding sequences.

Figure 5

Chromosomal distribution of P. yunnanensis PyunKTI genes. Tandem replication genes are shown in red.

Figure 6

Synteny analysis of the PyunKTI genes in P. yunnanensis.

Figure 7

Synteny analysis of the PyunKTI genes in P. yunnanensis, P. trichocarpa, A. thaliana, and V. vinifera. All collinear genes were labeled gray, while the collinear KTI gene pairs were labeled in red.

Figure 8

Cis-regulatory element (CRE) analysis of PyunKTI genes family. The number of CREs in the promoter region of the PyunKTI genes. The number of each CRE was shown in the heatmap box, and white represents that there was no corresponding CRE. Different colors on the bar chart represent different types of CREs in the PyunKTI genes family.

Figure 9

Expression profile of PyunKTI genes from P. yunnanensis with upright and inverted cuttings. (A) PCA analysis of the PyunKTI genes in P. yunnanensis. (B) Volcano plot analysis of the PyunKTI genes in P. yunnanensis. (C) Heatmap analysis showing the expression patterns and coexpressed relationships of each KTI gene.

Figure 10

Inverted treatment increases the expression of KTI genes. (A–C) show the qPCR relative expression and transcriptome results of PyunKTI16, PyunKTI18, and PyunKTI19, respectively. JZ, JD, SZ, and SD denote July upright insertion, July inversion insertion, September upright insertion, and September inversion insertion, respectively. Pink represents the upright insertion results, green represents the inversion insertion results, and the broken line represents the transcriptome results.

Figure 11

KTI gene expression in the GEO database in the related transcriptome with P. trichocarpa as the reference gene. (A) NJ Phylogenetic tree of P. yunnanensis and P. trichocarpa. (B) GSE56864: Effect of Methyl Jasmonate on the poplar root transcriptome. 4Con and 8Con are the control roots; 3MeJA10 and 6MeJA10 are the Methyl Jasmonate treated roots. (C) GSE86960: A time series of autumn senescence leaves from P. tremula. Samples were collected from the growing season in July to the aging season in October. (D) GSE67697: Transcriptome analysis of poplar during leaf spot infection with Sphaerulina spp. Samples were collected at 0, 1, 3, 4, and 15 days after infection with the pathogen.

Figure 12

The grouping relationship between the KTI gene of P. yunnanensis and the KTI gene of P. trichocarpa and the expression pattern of the KTI gene in various tissue parts of P. trichocarpa.

Figure 13

KTI gene expression in the transcriptome of drought-treated P. trichocarpa as a reference gene in the GEO database. (A) GSE97463: Differential gene expression analysis of drought-responsive sense and antisense genes in Populus. Sample name starting with c represents the control group, and sample name starting with s represents the drought treatment group. (B) GSE79401: RNA-seq of drought-treated P. trichocarpa. Samples were collected at 0, 5, and 7 days for the drought treatment.