## 4 Discussion
According to previous studies, both a low pH (<5.0) and Fe\({}^{2+}\) were able to assist TGG in producing nitriles with or without specifier proteins in vitro [40; 41]. From a food nutrition perspective, it is recommended to harvest younger sprouts to provide more ITCs, which is also supported by our study on Chinese kale sprouts [24]. In the future, greater attention should be given to improving the ITC content and proportion using genetic and chemical methods. The RNA-seq data revealed that the biosynthesis of aliphatic GSL was not activated significantly until 3 d. As is well known, chloroplasts are required for the side-chainelongation of aliphatic GSL in _Arabidopsis_[42]. Our further analysis also showed that many genes, such as _BolC4t27842H_, _BolC3t19882H_, and _BolC5t31588H_, related to the development of the chloroplasts were highly expressed at 3 d and 5 d but not at 1 d (Table S3). Therefore, it was not surprising that these aliphatic GSLs biosynthetic genes were activated after chloroplast formation, accompanied by a slight rise in GRA and GER in older broccoli sprouts. Different from aliphatic GSL, the biosynthesis of indole GSL was up-regulated after seed germination. These results verified a previous speculation that indole GSL accumulation in sprouts developing from 3 d to 11 d was possibly due to the activation of indole GSL biosynthesis [21]. Our earlier study demonstrated an increase in TGG activity from 3 d to 5 d in broccoli sprouts [33]. Correspondingly, we also found the TGG levels were high enough for GSL hydrolysis in broccoli seeds [20]. In this study, there was no GSL detected in the ground sprout slurry, indicating GSL had been degraded completely. Besides classical TGG, some atypical TGGs such as BGLU28 and BGLU30 [37] might also have the capacity to hydrolyze GRA and GER directly. In addition to TGGs themselves, some specifier proteins also play important roles in the TGG-GSL pathway by redirecting the breakdown products of GSL. For example, the functional ESP from the Arabidopsis ecotype Ler could convert alkyl GSLs into corresponding simple nitriles [40]. Furthermore, many studies have also highlighted the essential role of ESPs in promoting the production of nitriles from GRA in broccoli florets and sprouts [13; 23; 43]. Recently, we isolated four copies of _BoLESPs_ and found _BolESP2_ was the most abundant isoform [24]. Silencing _BolESP2_ not only effectively increased the total ITCs but also decreased the total epithionitriles in Chinese kale. So, we suspected that the two _BolLSEPs_ might play important roles in promoting nitrile production in broccoli sprouts. However, it is well known that ESP activity can be suppressed by EPITHOSPECIFIER MODIFIER1 (ESM1) [44]. This suggests that the effect of the _BolESPs_ may have been partially suppressed by the highly expressed _BoLESM1s_ after seed germination, resulting in the production of ITC. Except for ESPs, prior research has confirmed the involvement of NSPs as cofactors in nitrile formation in _Arabidopsis_, notably NSP1, NSP2, and NSP5 [25; 41; 45]. The results of the up-regulation of several _BolNSEPs_ strongly suggested that these _BolNSP_ genes likely promoted nitrile production during the hydrolysis of GSLs in broccoli sprouts. Similar to NSPs in protein structure, myrosinase-binding proteins (MBPs) also have jacalin domains. And they could form stable complexes with TGGs in _Brassica napus_[46; 47]. A _BolMBP2_ (_BolC6t35814H_) was highly expressed during broccoli sprout development, especially at 3 d and 5 d (Table S3). Its biological function remains unknown but is possibly associated with GSL hydrolysis. The RNA-seq data not only explained that the major breakdown products in broccoli sprouts were nitriles rather than ITCs but also illustrated the complexity of the TGG-GSL system in the development of broccoli sprouts.