The γ-Aminobutyric Acid (GABA) Synthesis Gene Regulates the Resistance to Water Core-Induced Hypoxia Stress for Pear Fruits

Abstract

Watercore is a physiological disorder which often occurs in Rosaceae fruits, and it causes hypoxia stress, promoting fruit decay. γ-aminobutyric acid (GABA) was reported as being involved in different abiotic stresses, and glutamate decarboxylase (GAD) is the key enzyme of GABA synthesis in plants. Our previous transcriptome analysis found that PpGAD2 was significantly induced in watercore fruit; however, the mechanism through which PpGAD2 regulates watercore-induced hypoxia stress resistance in pears is unclear. The present study found that the fruit pulp ethanol, malondialdehyde (MDA) and H₂O₂ content was significantly inhibited by exogenous GABA. The transcript abundance of PpGAD2 was significantly higher than that of other PpGADs in watercore fruit or healthy fruit. Tissue expression showed that the content of PpGAD2 in mature fruit was higher than in young fruit. Moreover, subcellular localization showed that PpGAD2 was located in the cytoplasm. Transient overexpression assays suggested that PpGAD2 had a role in GABA synthesis. Several CML (calmodulin–like) genes were also significantly increased in watercore fruit. Moreover, PpWRKY53 was significantly induced in watercore fruit, and the GUS activity assay showed that PpWRKY53 can significantly increase the activity of the PpGAD2 promoter. Taken together, these results demonstrate that PpGAD2 played an important role in GABA synthesis to increase plants’ resistance to hypoxia stress, and its activity may be affected by PpWRKY53 and several watercore-induced CML genes.

1. Introduction

Watercore is a very serious physiological disorder that frequently occurs in Rosaceae fruits, such as pear, apple and peach [1]. The symptoms of watercore are translucent fruit flesh and watery tissue. Our previous study suggested that watery tissue increased anaerobic respiration and caused the fruit to suffer from hypoxia stress [2]. Under hypoxia stress, plants experience a derangement of the cellular energy metabolism, causing a decrease in the pH in the cytoplasm, the inhibition of the ion transport pathway or nutrient acquisition, and the accumulation of toxic products from anaerobic respiration and reactive oxygen species (ROS) [3]. Pear watercore is most common in Japanese and Korean pear varieties with high internal quality. In recent years, Japanese and Korean sand pear varieties have been widely cultivated in Asia, and with the upgrading of cultivation technology, the proportion of high-quality fruit has increased significantly, leading to the increasing occurrence of sand pear watercore in production. However, watercore during pear production has not received enough attention, and it has caused great economic losses. Studying and understanding the hypoxia stress resistance mechanism in watercore fruit is urgent.

γ-aminobutyric acid (GABA) is a four-carbon non–protein amino acid. GABA has received a lot of research in the human and medical fields. For instance, GABA is an important neuroactive inhibitor in the nervous system, with effects such as reducing blood pressure, treating insomnia, alleviating anxiety, enhancing immunity and increasing memory [4]. In plants, many studies have shown that hypoxia can restore membrane potential and prevent an ROS-induced ion homeostasis imbalance; improve the defense ability of reactive oxygen species; remove chloroplast hydroxyl radicals and stabilize and protect chloroplast thylakoids; consume protons in cells to regulate the pH of the cytoplasm and alleviate the acidosis caused by hypoxia stress; and improve carbon recovery in the TCA cycle and promote the production of ATP [5]. Salvatierra et al. found that exogenous GABA application transiently increased the hypoxia tolerance of a sensitive genotype Prunus rootstock root [6]. GABA also regulates phenolic compound accumulation and enhances the antioxidant system in germinated hull-less barley under NaCl stress [7]. In plants, the GABA shunt pathway plays a key role in many regulatory mechanisms under stress conditions, such as endogenous signaling response molecules or metabolite accumulation [8]. The GABA shunt pathway is widely distributed in eukaryotes and prokaryotes and is an important metabolic pathway for GABA. In the cytoplasm, glutamate is first catalyzed by GAD for the irreversible production of GABA, GABA is subsequently converted into succinate acid by GABA transaminase (GABA-T). Succinate acid enters into the mitochondria and is catalyzed by means of succinate hemialdehyde dehydrogenase (SSADH) oxidation. Subsequently, succinate enters into the TCA cycle. Polyamine degradation is also one of the pathways used to generate GABA [9]. Compared with the GABA shunt pathway, polyamine degradation produced less GABA content. To date, GAD has been reported in many crops that respond to various stresses. For instance, the cotton GhGAD6 responds to cadmium stress and increases the GABA content to relieve cadmium stress-induced oxidative damage [10]. Moreover, the activation of glutamate decarboxylase (GAD) requires the involvement of Ca²⁺. Among many signal transduction pathways, Ca²⁺ is a multifunctional second messenger that regulates and activates many downstream responses of plants to various stresses. When plants are subjected to abiotic stress, Ca²⁺ in the cytoplasm will accumulate rapidly, resulting in the formation of a concentration difference of Ca²⁺ inside and outside the cell, which then generates a Ca²⁺ signal [11]. Different external stress stimuli induce plants to produce corresponding specific Ca²⁺ signals. Therefore, different calcium signal receptor proteins are required to recognize, decode and transmit the signals to the downstream, so that the downstream effector factors respond to the sensed specific Ca²⁺ signals, and then cause physiological and biochemical changes in plants to adapt to external adversity [11]. At present, Ca²⁺ receptor proteins in plants are mainly composed of three families: calmodulin (CaM)/calmodulin-like protein (CML), Ca²⁺-dependent protein kinase (CDPK), and calmodulin B-like protein (CBL). CaM is ubiquitous in eukaryotes, while CML, CDPK and CBL are unique to plants and some protozoa. CaM itself has no catalytic activity; however, after binding with Ca²⁺, it can interact with a downstream CaM-binding protein (CaMBP) to activate the function of these interacting proteins, and GAD is the CaMBP [12]. Our previous study reported that in watercore fruit, several GAD genes were significantly induced to up-regulated [2]. These PpGADs may play a key role in regulating fruit to resist hypoxia stress.

Many stress-associated transcription factors, such as WRKY, NAC, ERF, etc., were found to vary in expression level under hypoxia stress. For instance, transcription factors including MYB, MYB-related, bZIP, bHLH and WRKYs employ calcium signaling and sugar metabolism pathways to induce resistance to hypoxia stress in cucumber seedlings [13]. Tang et al. observed that the interplay between ERF members and the two WRKYs increased the adaptation to hypoxia stress in Arabidopsis induced by submergence [14]. In persimmon fruit, DkNAC7 regulates de-astringency by activating DkERF9 and DkPDC2, encoding pyruvate decarboxylase under hypoxia conditions [15]. In addition, many transcription factors belonging to IAA, WRKY, HB, and ZIPs demonstrated a higher expression in low-oxygen-concentration apples [16]. In the current study, we found that the PpGAD2 promoter contained the W-box cis-element and that a WRKY gene was significantly upregulated in watercore fruit. Whether this WRKY gene can regulate the expression of PpGAD2 needs to be further studied. ‘Akibae’ pear is a good-fruit-quality sand pear cultivar; however, it is highly susceptible to watercore. Here, we tried to dissect the molecular mechanism of a key GAD gene, PpGAD2, in regulating the resistance to watercore-induced hypoxia stress by participating in GABA synthesis, which might help us to rich the preventive measures to avoid watercore in pears.

2. Materials and Methods

2.1. Plant Materials and Treatment

The three cultivars (‘Akibae’, ‘Aikansui’ and ‘Housui’) were grafted onto Pyrus calleryana. The watercore fruits of ‘Akibae’ were collected about 125 days after flowering (DAF). ‘Akibae’ fruits treated with GABA at a concentration of 5 mM 100 days after flowering (DAF) were sampled two weeks later. Young leaves (third to fourth leaf from the top of the plant), mature leaves (second to third leaf from the bottom of the shoot), young fruits (60 DAF) and mature fruits (105 DAF) were collected for tissue gene expression. In each fruit sample, five healthy and uniform in appearance fruits were randomly collected from the outer crown of the tree with three replicates. All samples were immediately sampled and frozen in liquid nitrogen and stored at −80 °C for further analysis.

2.2. Measurement of Ethanol, H₂O₂, MDA, GABA Content and GAD Activity

The fruits ethanol content was measured using a test kit from Suzhou Comin Biotechnology. Ethanol was oxidized and dehydrogenated into acetaldehyde under the catalysis of ethanol dehydrogenase. At the same time, NAD was reduced to produce NADH, which caused WST-8 to turn orange under the action of 1-mPMS. We weighed out about 0.5 g of tissue, added 1 mL of distilled water for homogenization, centrifuged at 8000× g and 25 °C for 10 min, and removed the supernatant for testing. Then, we added the reaction solution in sequence. The content of ethanol was measured by measuring the change in the absorbance value at 450 nm.

The fruits H₂O₂ content was measured using a test kit from Suzhou Comin Biotechnology. H₂O₂ and titanium sulfate formed a yellow titanium peroxide complex with characteristic absorption at 415 nm. We weighed out about 0.5 g of tissue, added 1 mL of distilled water for homogenization, centrifuged at 8000× g and 25 °C for 10 min, and removed the supernatant for testing. Then, we added the reaction solution according to the order of the instructions. We allowed it to stand at room temperature for 5 min, poured it into a cuvette, and measured the absorbance value at 415 nm.

Fruits GAD activity was determined using a test kit from Shanghai Enzyme-linked Biotechnology. The purified plant GAD antibody was used to coat the microporous plate to prepare the solid-phase antibody. Firstly, we diluted the standard solution in the kit and created a standard curve. Then, according to the instructions, we performed the processes of temperature incubation, solution preparation, washing, and enzyme color development, the absorbance was measured with an enzyme marker at the wavelength of 450 nm, and the activity concentration of plant GAD in the sample was calculated through the standard curve.

The fruits’ GABA content was determined using a test kit from Shanghai Enzyme-linked Biotechnology. GABA reacted with hypochlorite and phenol in the alkaline solution to produce a blue-green substance. We weighed out about 0.1 g of the tissue sample and add it into the mortar, added 1 mL of the extract, homogenized it on ice, centrifuged it at 12,000 rpm and 4 °C or room temperature for 10 min, and removed the supernatant for testing. Then, we added the reaction solution in sequence, evenly mixed it, placed it in a boiling water bath (95–100 °C) for 10 min, and then placed it in an ice bath which was increased to room temperature to show the blue-green color. Then, adding 200 μL to a 96-well plate, the content of GABA in the sample was obtained by detecting the value of the colored substance at a 645 nm wavelength.

Fruits MDA was measured according to Li [17]. The content of malondialdehyde was determined by means of the thiobarbituric acid method. MDA can react with sulfur under acidic and high temperature conditions. The reaction of barbituric acid (TBA) produces reddish-brown trimethylene, which has its maximum light absorption at 532 nm and its minimum light absorption at 600 nm.

2.3. RNA Extraction and Sequencing

The detailed RNA-seq analysis process for watercore fruit and healthy fruit can be found in our previous work [18]. The high quality clean data was mapped to the reference genome of Pyrus bretschneideri. All RNA-seq data were uploaded to the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (GEO accession number: GSE164987). Differential gene expression levels were computed using the fragments per kilobase of exons per million mapped reads (RPKM) method. We used the DESeq2 package to analyze the differential gene expression. The differentially expressed genes (DEGs) were selected using the following criteria: |log₂ ^foldchange| ≥ 1 and corrected p < 0.05. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed using the cluster Profiler package. GO categories and KEGG pathways with false discovery rate (FDR) q values ≤ 0.05 were considered significantly enriched.

2.4. Quantitative Real-Time PCR (qRT-PCR) Analysis

Total RNA of pear fruit skin was extracted using the CTAB method [19]. First-strand cDNA was synthesized using the qPCR HiScript3 RT superMix kit from Vazyme Biotechnology, Nanjing, China. qRT-PCR was determined with a Bio-Rad CFX96 instrument (Bio-Rad, Waltham, MA, USA) by using ChamQ SYBR qPCR Master Mix (Vazyme Biotechnolog, Nanjing, China). Gene-specific primers were designed using the Primer5 (v5.0) software (Table S1), and the specificity and quality of each primer pair were checked through melting curve analysis and sequencing. We used the Livak [20] method to calculate relative gene expression levels.

2.5. Subcellular Localization of PpGAD2

The full CDS of PpGAD2 was ligated into the pCAMBIA1301-GFP vector. If sequence verification demonstrated it was accurate, it was then used for tobacco leaf transient transformation by means of Agrobacterium-mediated transformation according to our previously described methods [21]. Leaves transformed with a noncoding sequence vector were used as the control (CK).

2.6. Transient Transformation of Pear Fruits

To establish PpGAD2′s role in GABA synthesis, the PpGAD2-overexpressing vectors were mobilized into the A. tumefaciens strain EHA105 and then used to transiently transform three kinds of pear fruits (‘Akibae’, ‘Aikansui’ and ‘Housui’). Fruits transformed with a noncoding sequence vector were used as the control (CK). Transformed fruits were collected 5 days later for further analysis.

2.7. GUS Activity Assay

The PpGAD2 promoter was ligated into the pCAMBIA1301-GUS vector. The CDS of PpWRKY53 was cloned into the binary pCAMBIA1301 vector. The fusion constructs and the positive control were separately transferred into the A. tumefaciens strain EHA105 by means of heat shock. A. tumufaciens-mediated transformation was used for transient GUS expression in tobacco leaves. The leaves’ GUS activity was tested using the test kit (Shanghai Enzyme-linked Biotechnology).

2.8. Statistical Analysis

Error bars indicate the standard errors (SEs) of the means. The data in the experiment were compared by means of one-way analysis of variance (SPSS 17.0). Differences were determined using the Tukey test (p < 0.05) and indicated using different letters. Bar graphs were drawn using the scientific software of GraphPad Prism 7.0 (San Diego, CA, USA).

3. Results

3.1. Effect of Exogenous GABA Treatment on Pear Fruit

To investigate the effect of exogenous GABA treatment on ‘Akibae’ pear fruit, 5 mM GABA was administered to the fruits. After the treatment with exogenous GABA, the ethanol, MDA and H₂O₂ content was significantly decreased compared with watercore fruit (Figure 1). Specifically, the ethanol, MDA and H₂O₂ content decreased by 40.38%, 68.99% and 41.96%, respectively.

3.2. Expression Pattern of PpGADs in Watercore Fruit

In our previous research, the expression of three PpGADs was significantly increased, which correlated with the GABA content in watercore fruit [2]. To further evaluate the transcript abundance of PpGADs, we used PpGAD1 in healthy fruit as a control (CK) and compared the PpGADs expression level again. The result show that the transcript abundance of PpGAD2 was significantly higher than that of PpGAD1 and PpGAD3 both in healthy fruit and in watercore fruit, and so PpGAD2 was selected for this study (Figure 2).

3.3. Tissue-Specific Expression of the PpGAD2

Different tissues from the ‘Akibae’ pear, including young leaves, mature leaves, mature fruits and young fruits, were used to determine the expression levels of PpGAD2. The results show that the PpGAD2 gene is highly expressed in leaves, and it is predominantly expressed in young leave. Moreover, PpGAD2 expression in mature fruits was higher than that in young fruits (Figure 3).

3.4. PpGAD2 Was Localized in Cytoplasm

To further validate the potential location of PpGAD2, the subcellular PpGAD2 was investigated in tobacco leaves. After observation with confocal microscopy, GFP signals showed that PpGAD2 was located in the cytoplasmic area (Figure 4).

3.5. Transient Overexpression of PpGAD2 in Pear Fruits

To further validate the roles of PpGAD2 in GABA synthesis, PpGAD2 overexpression constructs were agroinfiltrated into pear fruits. Besides to ‘Akibae’, ‘Hosui’ and ‘Aikansui’ were also used to further verify gene function. The transcript level of PpGAD2 was significantly increased in the three pear cultivars. After the transient transformation of PpGAD2 in the three cultivars of pear fruits, they also had a significantly increased GABA content and GAD activity (Figure 5).

3.6. Transcriptome Changes in Calmodulin-Related Genes in Watercore Fruit

As shown in

Table 1. Expression levels of calmodulin-like genes in watercore fruit using RNA-Seq.

Gene Description	FC	ID
calmodulin-like protein 3	5.31	LOC108865441
calmodulin-like protein 11	5.03	LOC103960085
calmodulin-like protein 7-1	3.31	LOC103939317
calmodulin-like protein 7-2	1.40	LOC103953910

, an analysis of DEGs found that four calmodulin-like genes were significantly upregulated in watercore fruit (LOC108865441, LOC103960085, LOC103939317 and LOC103953910) (Table 1).

3.7. PpWRKY53 Regulate the PpGAD2 Promoter Activity

According to the RNA-Seq analysis of watercore fruit, a significantly up expressed WRKY gene (Fold change: 5.58) was identified as PpWRKY53 (ID: LOC103943771). The regulation of the PpGAD2 promoter using PpWRKY53 was determined in tobacco after co-transformation. The results show that the PpGAD2 exhibited increased GUS activity compared to CK, which indicated that PpWRKY53 may activate GUS transcription, as driven by the PpGAD2 promoter (Figure 6).

4. Discussion

Plant stress, often caused by different environmental conditions exceeding the tolerance limit, leads to lower plant reproduction rates due to increased ROS production [22]. hypoxia stress mainly occurs in natural environments when the root system is waterlogged or the fruit experiences physiological hypoxia. Due to the differences in the tissues of fleshy fruits, O₂ diffusion resistance will be caused, which creates an obvious oxygen gradient in the pulp tissues [23]. Long-term water soaking may aggravate the formation of an oxygen gradient and cause the insufficient exchange of CO₂ and O₂, which can cause fruit hypoxia. In production, GABA application is an effective approach that relieves multiple types of abiotic stress by inhibiting ROS generation [24]. In the current study, the content of ethanol, MDA and H₂O₂ was significantly inhibited by GABA (Figure 1). Thus, our study suggested that GABA can alleviate watercore-induced hypoxia stress.

GAD genes showed different expression patterns in various plant tissues and organs. In citrus, Liu et al. found that the transcript of CsGAD1 was mainly expressed in flowers, while CsGAD2 was mainly expressed in fruit juice sacs [25]. In soybean, the expression of GmGAD4 and GmGAD5 was detected in cotyledons, whereas GmGAD1 and GmGAD3 were mostly expressed in hypocotyls and roots [26]. In the current study, we found that PpGAD2 was mainly expressed in the leaves compared with young and mature fruits. However, the expression of PpGAD2 in mature fruits was significantly higher than in young fruits, which may suggest that PpGAD2 plays a more important role in mature fruits than in young fruits. Sand pears are mainly cultivated in the region south of the Yangtze River in China and mature in July to September. The fruit growth and development season in this region is vulnerable to extreme conditions, such as high temperature, high humidity or drought. Therefore, the high expression of genes in the mature stage may also improve the resistance of fruit. In plants, GAD catalyzes the irreversible conversion of glutamate to GABA, and GAD exists in the cytosolic system. A previous study found the SlGAD2 and SGAD3 expression levels are positively correlated with the GABA content during tomato fruit development [27]. In SlGAD mutant plants, the fruit GABA content was significantly decreased, suggesting that GABA biosynthesis in tomato fruits involves the decarboxylation of glutamate by GAD enzymes [28]. Rajani et al. reported the overexpression of AtGAD1 in maize, with the intent of increasing the synthesis of GABA [29]. In this study, the GABA content and GAD activity were significantly increased in the three sand pear cultivars when PpGAD2 was overexpressed. Together with the subcellular localization analysis of PpGAD2 (Figure 4), we suggest that PpGAD2 plays a key role in regulating GABA synthesis. Moreover, CaMs/CaMs-like is an important Ca²⁺ sensor that functions in plant resistance, interacting with GADs. Arabidopsis GAD1, 2, and 4 contain CaM-binding domains, and all have the potential to interact with CaMs [30]. AtCML8 interacts with AtGAD4 and functions by regulating GABA accumulation in Arabidopsis defense induced by (Z)-3-hexenol [31]. In the current study, we found that several CML genes were significantly induced in watercore fruit, suggesting that they may interact with PpGAD2 to respond to fruit hypoxia stress. The validation of the interaction mechanism of PpGAD2 and CMLs requires further study in the future.

In recent years, the transcription regulation mechanisms of GADs have been reported. One previous study found that OsMYB55 binds to the promoter regions of OsGAD3, and OsMYB55 overexpression resulted in an accumulation of GABA and resistance to high temperatures in rice [32]. FaMYB5 could repress the transcription levels of FaGAD and influences strawberry citric acid accumulation [33]. WRKY TFs contain a specific signature with a highly conserved WRKY domain and are involved in many biological functions, such as regulating plant growth or development, producing secondary metabolites and increasing tolerance to different abiotic stresses [34]. To date, there is no direct evidence to prove that WRKY genes directly regulate the expression of GAD. Mirabella et al. found that the GAD4 gene was up-regulated in wrky40 and wrky6 mutant Arabidopsis, induced by E-2-hexenal [35]. In the current study, we found that WRKY genes were significantly upregulated in watercore fruit, which was consistent with the expression of PpGAD2. The GUS activity assay also showed that they can regulate the expression of PpGAD2. Together with the results above, it is suggested that PpWRKY53’s response to hypoxia stress signals is regulated by PpGAD2 expression, increasing GABA synthesis, which improves fruit resistance to ROS.

5. Conclusions

Watercore is a physiological disorder that leads to pear fruits suffering from hypoxia stress, seriously affecting the internal quality and storage capacity of pears. GABA is a non-protein amino acid that can improve plant resistance. Therefore, it is necessary to study the regulation of GABA on sand pear resistance to watercore-induced hypoxia stress. This study proved that GABA can improve the resistance of fruits to hypoxia stress. Gene function verification showed that PpGAD2 is the key gene that catalyzes GABA synthesis. Its expression is directly regulated by the transcription factor PpWRKY53. Several PpCML genes were also significantly increased in watercore fruit which may interact with PpGAD2 and thus improve GAD activity in pear; however, these need to be further studied in the future. Our study represents a valuable gene resource in providing a theoretical basis for watercore-preventative technologies and resistant breeding.