*2.3. Gene Expression Changes in Response to Green Garlic Volatiles and DADS*

In order to further clarify the potential mechanism of action of green garlic volatiles and DADS treatments on cucumber ROS and antioxidant enzyme activities, the expression levels of six related genes (*CscAPX, CsGPX, CsMDAR, CsSOD, CsCAT, CsPOD*) were examined with real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of these six genes were significantly changed in cucumber leaves after the treatments (Figure 4).

**Figure 4.** Changes in gene relative expression in response to green garlic volatiles and DADS. *CscAPX*, cucumber cytosol ascorbic acid peroxidase gene; *CsGPX*, cucumber glutathione peroxidase gene; *CsMDAR*, cucumber gene of scavenge MDA; *CsSOD*, *CsCAT* and *CsPOD*, cucumber genes of SOD, CAT, POD. \* *p* < 0.05; \*\* *p* < 0.01; ANOVA, followed by Tukey test and *t*-test. Data are means ± standard errors (*n* = 3, three biological replicates).

The green garlic volatiles and DADS treatments increased the expression levels of *CsGPX, CsMDAR*, and *CsPOD* genes and decreased the expression levels of *CscAPX, CsCAT* and *CsSOD* (Figure 4). Compared with the control group, the expression level of *CsGPX* was significantly decreased by 2-fold (*p* < 0.05) in the treatments with 12- and 18-bulbs of green garlic. The expression level of *CsPOD* in cucumber co-cultured with 12-bulbs of green garlic was increased almost 4-fold when compared with the control (*p* < 0.01) (Figure 4). However, the expression level of *CsCAT* was decreased in cucumbers that were co-cultured with green garlic, especially after 18-bulb treatment, and DADS

treatment (Figure 4). The *CscAPX* expression level was decreased by green garlic volatiles and the DADS treatments (Figure 4). The expression of *CsMDAR* was increased by 2-fold when compared with the control (*p* < 0.05) (Figure 4).

#### **3. Discussion**

Garlic organosulfur compounds are biosynthesized for defensive purposes such as protection against abiotic stressors and are formed quickly once the plant tissues are damaged [24]. Fresh garlic only contains alliin, a derivative of cysteine. When the cells of fresh garlic cell are crushed, allinase can convert alliin to allicin. Similarly, the DADS content of the cut green garlic segments were higher than that of the whole garlic plants (Table 1), which suggested that the damaged green garlics can volatilize more DADS. In general, DATS, DADS, and VDTs are the major sulfides derived from the garlic extracts. When garlic oil is extracted by steam distillation or simultaneous distillation, the resulting extracts mainly contain DADS and DATS [25]. When garlic was treated by high temperature aging, crushing, and roasting, the compounds identified by SPME-GC/MS were also DADS and DATS [26]. DADS (97.85%), DAS (0.01%), and DATS (0.01%) were found to be the predominant flavor components of garlic samples extracted by HS-SPME using a 50/30-micron DVB/CAR/PDMS fiber [16]. In cut green garlic segments, we can detect two sulfur compounds, DADS and DAS, while in whole green garlic, only DADS can be detected (Figure 1). The green garlic allelochemicals were determined by fiber-HS-SPME-GC-MS in this study. The analytical performances of needle trap micro-extraction (NTME) coupled with GC-MS were evaluated by analyzing a mixture of twenty-two representative breath volatile organic compounds (VOCs) belonging to different chemical classes (i.e., hydrocarbons, ketones, aldehydes, aromatics and sulfurs), which confirmed the reliability of this method [27,28]. Previous studies were mainly focused on the identification of garlic compounds produced from garlic cloves, which might not be identical to those seen this study, because we collected the volatile compounds of green garlic plants

By calculation and comparison, in general, the amount of DADS released by green garlic was approximately 0.08 mg/g. The four treatments of 0, 6, 12 and 18 bulbs of green garlic in the mini-greenhouse, corresponded to the DADS concentrations of 0, 0.025, 0.046 and 0.057 μmol/L per cucumber plant (Table S1). This means that the DADS concentration of a 6-bulb treatment in a mini-greenhouse is approximately equivalent to 50 mL of 1 mmol/L DADS treatment (0.024 μmol/L per cucumber plant). It has been reported that lower concentrations (0.01–0.62 mmol/L) of DADS can significantly promoted root growth, whereas higher levels (6.20–20.67 mmol/L) will have inhibitory effects [6]. Therefore, considering the effect and cost, 1 mmol/L DADS concentration is suitable for use.

Many studies have shown that allelochemicals significantly inhibit the activities of antioxidant enzymes, increase the level of free radicals, lead to membrane lipid peroxidation and membrane potential changes, thereby reducing the scavenging effect on ROS and destroying the entire membrane system of plants [9]. ROS play an important role in plants signal transduction pathways, as key regulators of processes such as growth, development, response to biotic and environmental stimuli, and plant metabolism, especially H2O2 which is important for programmed cell death to resist disease [20]. Green garlic volatiles and DADS significantly decreased the O2 •− contents (Figure 3B). However, they significantly increased the H2O2 content in cucumber leaves (Figure 3C). Plants have complex antioxidant systems to deal with ROS damage, including enzymatic systems (such as SOD, CAT and POD) and non-enzymatic systems (such as MDA, GSH and ASA) [23]. Previous studies have shown that the activities of POD, CAT, SOD and MDA contents in the leaves and roots of tomato were increased after the treatment of DADS [14]. In this study, the activity of POD was increased, while that of CAT was decreased, and the MDA content was also decreased after when treated with DADS. SOD is the most important ROS scavenger by converting O2 •− into molecular oxygen and H2O2 [23]. H2O2 can be decomposed into molecular oxygen and H2O by APX GPX, CAT, or POD [20,29]. Green garlic volatiles and DADS promoted the activities of POD and reduced the contents of GSH and ASA and the activity of CAT, then led to the change of H2O2 content (Figure 3). The gene expression changes

were consistent with the changes of antioxidant enzyme and antioxidant substances. The green garlic volatiles decreased the expression of *CscAPX*, *CsGPX*, *CsCAT* and *CsPOD*. *SOD*, *CAT*, and *POD* genes play key roles in plant tissue antioxidant defenses [30]. *SOD* gene is the defense first line to against the ROS. Genes like *GPX*, *cAPX*, *MDAR*, *CAT* and *POD* work to further convert H2O2 into H2O and O2 through different reactions [23]. *GPX* utilizes glutathione (GSH) as an electron donor to reduce ROS [31]. The *POD* gene is another reported defense-related enzyme gene and 24 *POD* genes have been identified in transcriptome analysis after DADS treatment [14]. *CAT* gene directly decompose H2O2 into H2O and O2. *CAT* gene is indispensable for ROS detoxification [32]. APX enzymes catalyze the conversion of H2O2 into H2O and MDA using ascorbate [33]. *MDAR* helps to scavenge the MDA and generate dehydroascorbate (DHA) [34].

In summary, a scheme (Figure 5) showing green garlic volatiles effect on cucumber ROS, antioxidant enzymes, antioxidant substances and genes can be proposed. Firstly, green garlic volatiles decreased the expression of *CsSOD* gene, which made the O2 •− turn into molecular oxygen and H2O2. Then green garlic volatiles decreased the expression of *CscAPX, CsGPX, CsCAT* and *CsPOD* gene, which can promote the activity of POD and reduce the contents of GSH and ASA and the activity of CAT. Finally, green garlic volatiles increased the accumulation of H2O2. This increased accumulation of H2O2 might increase the disease resistance of cucumber. We will continue to work on the effects of green garlic volatiles on cucumber disease resistance in the future.

**Figure 5.** A scheme of green garlic volatiles effect on cucumber ROS, antioxidant enzymes, antioxidant substances and genes.
