Characterization and Identification of a Ripening-Related Gene AaPG18 in Actinidia arguta

Abstract

Actinidia arguta (A. arguta) is a kind of climacteric fruit that quickly softens and limits fruit shelf-life and commercial value. Therefore, it is of great significance to develop kiwifruit genotypes with an extended shelf-life of fruit. However, the ripening and softening mechanisms remain unclear in A. arguta. Here, we demonstrated that a key polygalacturonase (PG)-encoding gene AaPG18 was involved in A. arguta ripening through the degradation of the cell wall. Fruits were harvested at three developmental stages (S1, S2, and S3) for high-throughput transcriptome sequencing, based on which two candidate transcripts c109562_g1 and c111961_g1 were screened. The genome-wide identification of the PG gene family assigned c109562_g1 and c111961_g1 to correspond to AaPG4 and AaPG18, respectively. The expression profiles of candidate genes at six preharvest stages of fruit showed significantly higher expression levels of AaPG18 than AaPG4, indicating AaPG18 might be a key gene during fruit ripening processes. The subcellular localization displayed AaPG18 was located at the cytoplasmic membrane. The transient overexpression of AaPG18 in strawberry and the following morphological observation suggested AaPG18 played a key role in maintaining the stability of cell morphology. The homologous transient transformation in A. arguta "RB-4" proved the crucial function of AaPG18 in fruit ripening processes by causing the rapid redness of the fruit, which was an indicator of fruit maturity. All in all, our results identified AaPG18 as a key candidate gene involved in cell wall degeneration, which provides a basis for the subsequent exploration of the molecular mechanisms underlying the ripening and softening of A. arguta fruit.

Keywords: AaPG18; Actinidia arguta; fruit ripening; gene expression; gene function.

Figure 1

Physiological indexes investigation. (A) Firmness values of fresh fruit flesh at six preharvest stages. (B) Soluble solid contents of fresh fruit flesh at six preharvest stages. (C) Respiration intensities of fresh fruit flesh at six preharvest stages. At least three readings were used to plot one point. The data represent the error bars ± SE (Standard Error) of three biological replicates.

Figure 2

The Venn diagrams of differentially expressed genes (DEGs) and the expression levels of transcripts. (A) Venn diagrams of all DEGs among three comparisons including S2 vs. S1, S3 vs. S1, and S3 vs. S2. (B) Venn diagrams of upregulated DEGs among three comparisons including S2 vs. S1, S3 vs. S1, and S3 vs. S2. (C) Venn diagrams of upregulated DEGs with a 5-fold change among three comparisons including S2 vs. S1, S3 vs. S1, and S3 vs. S2. (D) Expression levels of two key transcripts including c109562_g1 and c111961_g1. (E) Annotations of c109562_g1 and c111961_g1. The green font indicates c109562_g1 and c111961_g1 were both annotated as polygalacturonase in three known online databases including KO, Swissprot, and MF.

Figure 3

The expression levels of AaPG4 and AaPG18 in fresh fruit flesh at six preharvest stages. Kiwifruit β-actin was selected as an internal control gene during qPCR. Values are presented as means ± SD for three replicates. The statistical significance of mean values is indicted by different letters.

Figure 4

Protein structure analysis and subcellular localization of AaPG18. (A) Deduced protein sequence of AaPG18 with 395 amino acids. (B) Schematic representation of AaPG18 protein structure. Two possible N-terminus transmembrane domains (orange boxes) were included. (C) Hydropathic profile of AaPG18 protein using the predicting tool ProtScale (https://web.expasy.org/protscale/, accessed on 25 November 2021). (D) Prediction of transmembrane structure using TMHMM-2.0 (http://www.cbs.dtu.dk/services/TMHMM/, accessed on 25 November 2021). (E) Subcellular localization of AaPG18 in Nicotiana benthamiana (N. benthamiana) leaves. Cells expressing AaPG18–GFP fusion gene exhibited GFP fluorescence signals which were distributed in the cytoplasmic membrane. Cells expressing an empty vector only with a GFP tag showed GFP fluorescence signals at the nucleus and membrane. Three observed fields including brightness, GFP, and mergence were captured. The magnified field showed GFP signals in different parts of cells. White and orange arrows indicate the presence of GFP signals in the cell membrane and nucleus, respectively.

Figure 5

Transient overexpression in fruits of strawberry “snow”. (A) Diagrammatic sketch of the transient injection on the left and right sides of the same fruit of strawberry. (B) Phenotype observation of strawberry three days after injection. (C) Morphological observation of the cell section of strawberry fruit infiltrated with pBI121–AaPG18. (D) Morphological observation of the cell section of strawberry fruit without any injection. (E) Morphological observation of the cell section of strawberry fruit infiltrated with pBI121 (empty vector) as the control. The scale bar: 200 μm.

Figure 6

Homologous overexpression in fruits of A. arguta “RB-4”. (A) Phenotype of the side infiltrated with pBI121–AaPG18 presenting an obvious red color. (B) Phenotype of the side infiltrated with pBI121 presenting no change. (C) Expression level of AaPG18 in “RB-4” fruits infiltrated with pBI121–AaPG18 and pBI121. Similar to strawberry fruit, the left and right sides of one fruit were injected with pBI121–AaPG18 and pBI121, respectively. Values are presented as means ± SD for three replicates. Statistical significance: *** p < 0.001.