**4. Defense Response of GLS to Pests**

With global warming, the loss caused by pests is increasing. Meanwhile, because pests have migration ability, once the control is not effective, it will cause serious damage [109]. Hence, pest control has been a hot spot in agriculture. At present, pest control is mainly focused on chemical agents, but how *Brassicaceae* plants perceive and defend themselves from such threats remain poorly understood. Investigating the mechanisms via which *Brassicaceae* resist insect pests could facilitate efforts to improve crop productivity. Brown and Morra (1997) were the first to report that GLS-containing plants could control soil-borne plant pests [110]. Since then, numerous studies have demonstrated that GLS contents in tissues are positively correlated with damage caused by *Pieris rapae* and *Spodoptera littoralis* [111,112], but negatively correlated with the damage caused by slugs [86]. Furthermore, GLS accumulation induced by *Spodoptera exigua* required functional NPR1 and ETR genes [113].

In another study, the weights of *Trichoplusiani* and *Manduca sexta* on the TGG1 and TGG2 double myrosinase mutants were significantly higher than in wild-type *Arabidopsis* [27]. Similarly, *Mamestra brassicae* larvae gained less weight and exhibited stunted growth when fed on MINELESS (lacking myrosin cells) plants compared to when fed on wild-type plants, with the myrosinase activity in the wild-type seedlings reducing; however, the levels of indol-3-yl-methyl, 1-methoxy-indol-3-yl-methyl, and total GLS in both the wild-type and MINELESS seedlings increased [114]. Conversely, *M. brassicae* and *P. rapae* weighed more on the high-sinigrin concentration plants than in low-sinigrin concentration plants; however, their weights decreased in the high-sinigrin, high-glucoiberin, and high-glucobrassicin genotypes; furthermore, development time increased under high glucobrassicin concentrations [115].

By testing the GLS and phenolic concentrations trends in *Brassica nigra* (L.) Koch before and after herbivory by *Pratylenchus penetrans* Cobb and the larvae *Delia radicum* L., Van et al. (2005) observed that the total GLS levels were affected by herbivory by the two root feeders [116]. Besides, *Spodoptera litura Fabricius* was more affected by induced GLS responses than *Plutella xylostella* L. [117]. In addition, following a comparison of GLS levels and the expression profiles of GLS biosynthesis genes before and after *Plutella xylostella*

infestation, Liu et al. (2016) observed a difference in the proportions of stereoisomers of hydroxylated aromatic GLS between G-type (pest-resistant) and P-type (pest-susceptible) *Barbara vulgaris* [56]. Using *m*/*z* 60 as a marker of Al-ITC formation from the sinigrin GLS, Van et al. (2012) analyzed the GLS profiles and volatile organic compound emissions in five *Brassicaceae* species before and after artificial injury or infestation by cabbage root fly larvae (*D. radicum*). According to the results, *m*/*z* 60 in *B. nigra*, *B. juncea*, and *B. napus* was primarily emitted directly after artificial injury or root fly infestation, sulfide and methanethiol emissions from *B. nigra* and *B. juncea* increased after infestation, and *B. oleracea* and *Brassica carinata* exhibited increases in fig *m*/*z* 60 emissions following larval damage [118].

Long-term feeding on GLS-free *Brassicaceae* diets hardly affects *P. xylostella* oviposition preference and larvae survival; thus, high GLS content varieties are likely to be more susceptible to damage by *P. xylostella* than lower GLS content varieties [119]. Similarly, Chen et al. (2020) generated single or double mutant gss1 and gss2 lines using the CRISPR/Cas9 system and analyzed their resistance to *P. xylostella* [120]. According to the results of the bioassays, when fed on their usual artificial diet, there were significant reductions in egg hatching rates and final larval survival rate of the single mutant gss2 lines when compared with the original strain or mutant gss1 lines, and the absence of GSS1 or GSS2 reduced the survival rate of *P. xylostella* and prolonged the duration of the larval stage. In addition, feeding by *Spodoptera littoralis*, *Pieris brassicae*, and *P. rapae* led to upregulation of the aliphatic GLS pathway [121–123], and the GLS contents were negatively correlated with *P. brassicae* damage. Furthermore, methyl jasmonate (MeJA) can enhance resistance to *P. brassicae* by inducing GLS accumulation [124,125].
