**5. Defense Response of GLS to Insects and Aphids**

Insects and aphid not only have a wide range of species and rapid reproduction, but also can cause wounds to the plant when feeding, leading to the invasion of pathogenic bacteria, and then cause secondary damage. Moreover, the GLS synthesis and response mechanisms following insect and aphid herbivory are qualitatively and quantitatively different [126]. Agerbirk et al. (2001) observed no correlation between *B. vulgaris* ssp. *arcuata* GLS content and resistance against *Phyllotreta nemorum* [127], while Kroymann et al. (2003) observed a positive correlation between GLS content and damage caused by *Psylliodes chrysocephala* [128]. According to Ulmer et al (2006), total GLS levels did not influence *Ceutorhynchus obstrictus* larval growth or development; however, high levels of specific GLS, such as p-hydroxybenzyl and 3-butenyl GLS, were associated with increased development time or reduced weight [129]. After *Brevicoryne brassicae* herbivory, Myrosinase binding protein (MBP), myrosinase associated protein (MyAP), and myrosinase transcripts, and the synthesis of indolyl and aliphatic GLS, particularly 3-hydroxypropyl and ITC, are induced [103,130,131].

By comparing the larval instar weights and mortality of cabbage stem flea beetle (*P. chrysocephala*) larvae, after feeding on different species, Döring et al. (2020) observed that aliphatic GLS contents increased in the infested turnip rape, and aliphatic and benzenic GLS decreased in infested Indian rape [132]. Although larval weight was not correlated with total GLS, it was positively correlated with progoitrin and 4-hydroxyglucobrassicin contents. Furthermore, decreasing the side chain length of aliphatic GLS and the degree of hydroxylation of butenyl GLS could increase the extent of feeding by adult flea beetles [87].

Numerous intermediate synthetic genes participate in GLS resistance to insects and aphids. For instance, Mewis et al. (2006) observed that GLS accumulation caused by *B. brassicae* and *Myzus persicae* required functional NPR1 and ETR1 genes [113]. After *Myzus persicae* feeding and aphid saliva treatment, a set of O-methyltransferases involved in the synthesis of aphid-repellent GLS were significantly up-regulated based on qRT-PCR analyses of 78 genes. However, ITC production was not correlated with these gene expression level, suggesting that aphid salivary components trigger a defense response in *Arabidopsis* that is independent of the aphid-deterrent GLS [133]. In addition, aphid attack

could increase indolyl GLS concentrations three-fold [134]. Using a combination of QTL fine-mapping and microarray-based transcript profiling methods, CYP81F2 was revealed to facilitate defense against *B. brassicae* but not resistance against herbivory by larvae from four lepidopteran species [135]. By comparing the survival of the *Bemisia tabaci* MEAM1 and *B. tabaci* MED following exposure to sinigrin and myrosinase, Hu et al. (2020) reported that exposure to the toxic hydrolysates of GLS hydrolysates and myrosinase is greater for MED than for MEAM1 [136].

#### **6. Conclusions and Future Research Outlook**

Cruciferous vegetables are the most important leafy vegetables; however, the cultivation of cruciferous plants is affected by various fungi, bacteria, aphids, and other pest insects. At present, the prevention and control of these diseases and insect pests mainly focus on chemical agents, and the dosage of the chemical pesticides is also increasing, which not only leads to excessive pesticide residues on cruciferous plants, causing great damage to the environment, but also threatens people's health. Understanding how host-plant characteristics influence the physiological and behavioral responses is essential for the development of resistant cruciferous germplasms. A large number of studies have shown that GLS, esters, and flavonoids are closely related to Cruciferae disease resistance [137,138]. As an important secondary metabolite in cruciferous vegetables, GLS are closely related to biotic and abiotic stresses. Numerous studies have demonstrated a positive relationship between GLS content and disease and insect resistance [57,88,99,106,107,111,112,116,128,134] (Table 1). Consequently, in future cruciferous vegetable breeding activities, varieties with high GLS contents can be selected appropriately to improve plant disease resistance and reduce pesticide use. The degradation products (isothiocyanate and thiocyanate) of GLS are involved in the resistance to a variety of fungi, bacteria, insects, and soil-borne pests [8,71,72]; the aqueous extracts of cruciferous leaves also contain ITC, which can restrict the growth of a variety of fungi, bacteria, and pests [84,85,110]. Moreover, the resistance of these degradation products to pests and diseases is a broad-spectrum resistance, and thus can be used to develop botanical pesticides.

Pest invasion and disease infestation can increase GLS, especially indole GLS, in cruciferous plants [8,9,89–95,105,117,121–123,133,134]. In the case of rapeseed and other species that have low GLS, molecular biology techniques can be used to increase indole GLS production, which could improve resistance to diseases and insect, without increasing total GLS synthesis. Similar to other secondary metabolites, GLS synthesis is regulated by plant hormones. By controlling the amount of sulfur fertilizer applied and exogenous plant hormone treatments, such as JA, ET, MeJA, and SA, GLS synthesis can be modulated, and, in turn, disease resistance [57,105,125,126,139–142]. In addition, pathogen infection and insect herbivory can trigger the upregulation of the JA and ET biosynthesis genes, and increase defensive capacity via GLS accumulation [108]. Therefore, in subsequent cruciferous vegetable production activities, appropriate plant hormones could be sprayed as a novel pest management strategy to improve their stress resistance and minimize pesticide use (Figure 3).

**Table 1.** Correlation of the GLS components and their metabolites in corresponding pathogen, pest, and insect resistance.



**Table 1.** *Cont.*


**Table 1.** *Cont.*

**Figure 3.** Factors affecting the synthesis of glucosinolate in cruciferous plants.

Some studies have demonstrated that the different stereoisomer structures of hydroxylated aromatic GLS is one of the important factors influencing the varying disease resistance levels between non-cultivars and resistant cultivars [56]. Consequently, by determining and analyzing the GLS responsible for resistance in tolerant materials, chemical synthesis or biotechnology tools can be used to mass-produce the corresponding GLS for widespread application. The present review on the GLS responsible for disease and pest resistance in cruciferous vegetables and their underlying mechanisms could not only offers insights on how cruciferous plants could respond to increased biotic stress in the future but could also facilitate the development of novel disease and pest-resistant plants and the development of safe and high-yield cruciferous vegetable germplasms globally.

Despite there being many research studies on cruciferous plant resistance, which indicated the invasion of the diseases, insects and pests will lead to the synthetic form of the hormones and GLS, and total GLS, especially the indole GLS content, has a positive correlation with cruciferous resistance, but only a few studies have identified the specific resistance due to GLS and other metabolites. This may be related to the determination methods of GLS composition. At present, the conventional determination methods of GLS are HPLC (high performance liquid chromatography) and HPLC-MS; these two methods are not only expensive and difficult, but also often lack some standard samples. So, efficient and accurate GLS determination methods need to be improved or developed in subsequent research. Moreover, the synthesis mechanism and corresponding intermediate pathway of GLS are mainly focused on the model plant *A. thaliana*; studies on other species are thus few. The genomes and cultivation of most cruciferous vegetable patterns are different from *A. thaliana*. Compared with *Arabidopsis*, the genomes and cultivation patterns of Cruciferous plants are more complex. Although the genomes of many cruciferous plants have been sequenced, the genomes of the majority of species are still unknown, which has limited the

study on the anabolism of GLS and other disease-resistance-related substances. With the reduction of the cost of genome sequencing, transcriptome sequencing, and omics analysis, it is believed that people will have a new understanding of the mechanism of GLS against diseases and insects.

**Author Contributions:** Z.L., H.W., L.H. and J.Y. designed the research; S.L. and J.L. performed the data analysis and prepared the manuscript; J.X., G.Z. and L.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Agricultural University, grant number GSCS-2020-11, Natural Science Foundation of Gansu Province, grant number 20JR10RA537, Gansu Agricultural University doctoral Research Initiation fund, grant number GAU-KYQD-2019-30, and Special Project of Central Government Guiding Local Science and Technology Development, grant number ZCYD-2020-5.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
