Brassica juncea, (L.) Czern. (L.) Czern.

Augustine, Rehna & Bisht, Naveen C., 2015, Biotic elicitors and mechanical damage modulate glucosinolate accumulation by co-ordinated interplay of glucosinolate biosynthesis regulators in polyploid Brassica juncea, Phytochemistry 117 (1), pp. 43-50 : 45-48

2.1. Effect of biotic elicitors on glucosinolate accumulation in B. juncea

The effect of various elicitor molecules (MeJA, SA, Glu and wounding) on the accumulation and profile of glucosinolates in B. juncea seedlings was studied in a time course experiment. There was a quantitative variation in both content and profile of glucosinolates in the treated samples as compared to the untreated control seedlings. Under MeJA treatment, there was a significant difference in total glucosinolate content ( Fig. 2 ). Total glucosinolate levels were elevated to a maximum of 1.8-fold (68.5 µmoles g ― 1 dry wt. against 37.2 µmoles g ― 1 dry wt. of the control) after 24 h of treatment. Upon SA addition, there was a significant decline in total glucosinolate content when analyzed after 6 h. However there was a marginal enhancement (up to 1.3 folds) after 24 h and 48 h post SA addition. Exogenously supplied Glu was also found to increase glucosinolate content. The total glucosinolate content was raised to 1.6-fold and further to 1.9-fold after 6 h and 24 h of Glu treatment respectively, although the levels declined after 48 h of treatment. Mechanical injury, given as wounding was found to have the most pronounced effect on total glucosinolate accumulation. After 6 h of wounding, total glucosinolates levels was enhanced by 1.6-fold which reached its peak after 24 h to 2.1 folds (79.5 µmoles g ― 1 dry wt.) followed by a decline to normal levels after 48 h.

Aliphatic glucosinolates constitute the major fraction of glucosinolate pool (ca. 90%) in B. juncea . When the pools of aliphatic and indole glucosinolates were analyzed independently, it was also found that although both aliphatic and indole glucosinolates were induced by these elicitors, indole glucosinolates had a more profound effect, contributing majorly toward increased levels of total glucosinolates. Interestingly, aliphatic glucosinolates were induced maximally by Glu and wounding whereas all the tested biotic elicitors significantly enhanced the accumulation of indole glucosinolates. Overall, MeJA treatment was found to be the strongest inducer of indole glucosinolates ( Fig. 2 ). Another significant observation was that, maximum accumulation of aliphatic glucosinolates occurred up to 24 h post treatment and declined thereafter. In contrast, indole glucosinolates showed a prolonged effect showing highest accumulation up to 48 h. In general, 24 h treatment was found to be optimum for enhancement of both aliphatic and indole glucosinolates in B. juncea seedlings.

Our results corroborated the hypothesis that glucosinolates are important components of plant defense against pest and diseases in B. juncea . Similar results were reported previously from other crops like B. rapa ssp. chinensis in which increased accumulation of total glucosinolates, particularly indole glucosinolates, was observed after 48 h of treatment with MeJA and methyl SA ( Wiesner et al., 2013). Baenas et al. (2014) showed that 8 day old sprouts of Brassica oleracea (broccoli), B. napus (rutabaga cabbage), B. rapa (turnip), and Raphanus sativus ( China rose radish and red radish) effectively accumulate higher levels of glucosinolates upon treatment with the phytohormones JA, MeJA and sugars. Our study demonstrates that although both aliphatic as well as indole glucosinolates can be effectively induced with biotic elicitors, indole glucosinolates showed prolonged and higher levels of induction suggesting a more significant role during pest and pathogen attack in B. juncea .

2.2. Analysis of glucosinolate profile after treatment with elicitors

Since different glucosinolates are known to be associated with different biological functions, glucosinolate profiles were also analyzed in the treated samples (Table 1). Gluconapin (GNA), the major aliphatic glucosinolate fraction in B. juncea , was found to be maximally affected by Glu and wounding treatments and increased by ca. 2.2-fold after 24 h of treatment (ca. 60.0 µmoles g ― 1 dry wt. against 26.6 µmoles g ― 1 dry wt. of control). The accumulation of GNA was unaltered under MeJA and down regulated under SA treatment initially (6 h) but showed a significant enhancement at 24 h. Sinigrin (SIN), the second major fraction, was also found to increase only upon wounding and remained unaltered under other treatments. Glucoiberin (IBE) level was found to be initially unaffected but enhanced later under MeJA and Glu treatments. For example, after 24 h of MeJA treatment, IBE concentration increased to ca. 3.0 µmoles g ― 1 dry wt. compared to untreated control (1.8 µmoles g ― 1 dry wt.). However the response to Glu was immediate, which increased the level of IBE by 1.5-fold after 6 h of treatment. 4-Pentenyl (GBN) and 4-methylthiobutyl (4MTB) glucosinolates which are generally found in mature plants and seeds were not detected in the seedling stage.

Among indole glucosinolates fractions, glucobrassicin (I3M) increased under MeJA treatment by a magnitude of 9.0-fold (7.5 µmoles g ― 1 dry wt. in treated samples against 0.8 µmoles g ― 1 dry wt. in the control) after 48 h. No other treatment could significantly enhance I3M levels. Another indole glucosinolate, 1MOI3M, which is well known for its insect deterrence properties, was drastically enhanced by about 24.0 folds (14.7 µmoles g ― 1 dry wt. against the wild type level of 0.6 µmoles g ― 1 dry wt.) in the MeJA treated seedlings after 48 h. SA, Glu and wounding also significantly increased accumulation of this glucosinolate by a maximum of 7.2-, 8.8- and 6.7-fold respectively after 48 h of treatment. One of the major fractions responsible for fungal pathogen deterrence, 4MOI3M, was also found to be responsive to MeJA and SA after 24 h of treatment. However, it was the least affected fraction among the detected indole glucosinolates in B. juncea in response to the tested elicitors.

Differential accumulation of glucosinolate fractions in response to signaling molecules and wounding might be attributed to the specific role of these individual glucosinolates in plant defense. No detailed information is available till date which elucidates the distinct role of individual glucosinolate in plant defense. Various reports suggested that indole glucosinolates have pronounced effects during plant–pathogen/pest interaction ( Bednarek et al., 2009; Clay et al., 2009; De Vos et al., 2008; Kim and Jander, 2007; Kim et al., 2008). A very recent study by Sotelo et al. (2015) showed that aliphatic glucosinolates like GNA, SIN and GBN have potent effects against two bacterial ( Xanthomonas campestris pv. campestris and Pseudomonas syringae pv. maculicola) and two fungal ( Alternaria brassicae and Sclerotinia scletoriorum ) Brassica pathogens, when tested under in vitro conditions. We therefore presume that higher amounts of these aliphatic glucosinolates present in Brassica species could be important for constitutive defense against specific pathogens of Brassicas. Thus defense response mediated by glucosinolates seems to be highly complex and depends on the pathogen/pest, crop-species, quantitative differences and the types of glucosinolate present ( Santolamazza-Carbone et al., 2014).

Jasmonates function as vital signaling molecules in plant defense, particularly against insect herbivores and necrotrophic pathogens ( Schaller and Stintzi, 2008). Guo et al. (2013) recently reported that JA mainly enhances the accumulation of I3M and 1MOI3M whereas 4MOI3M is the major indole glucosinolate that is responsive to SA treatment. In Arabidopsis , methoxylation of indol-3-ylmethyl (I3M) to 4-methoxy-indol-3-ylmethyl glucosinolate (4MOI3M) was reported to be suppressed by MeJA, which, in turn can result in over accumulation of I3M and 1MOI3M ( Mikkelsen et al., 2003; Wiesner et al., 2013). In the current study, a marginal enhancement of 4MOI3M under MeJA treatment in B. juncea seedlings could be due to over accumulation of its precursor I3M or 1MOI3M glucosinolates to a significant level.

Apart from functioning as a carbon source, Glu also act as signaling molecule. Previous studies have also shown that sugars such as glucose and sucrose enhance glucosinolate accumulation in Brassica species ( Baenas et al., 2014; Wei et al., 2011). Glu treatment increased the aliphatic glucosinolate, GNA in Chinese kale and pak choi sprouts whereas levels of progoitrin, another aliphatic glucosinolate, were decreased. Of the major indole glucosinolates in Brassica sprouts, I3M and 1MOI3M showed a significant increase upon Glu treatment but not 4MOI3M ( Wei et al., 2011). Our results even though were in agreement with the fact that Glu enhances both aliphatic and indole glucosinolates, effect of Glu on specific glucosinolates fractions was different in B. juncea . GNA was found to over-accumulate under Glu treatment whereas SIN, the other major aliphatic glucosinolate showed no change as compared to the untreated control (Table 1). Among indole glucosinolates, 1MOI3M showed enhancement in 5% Glu treatment. Mechanical injury by wounding significantly raised 1MOI3M level in B. juncea as compared to increase in levels of other indole glucosinolates. Bodnaryk (1992) showed that mechanical wounding can increase indole glucosinolate levels. Wounding stimuli have seems to reciprocate during plant herbivore interaction through raising the level of 1MOI3M in B. juncea (Table 1). Thus differential accumulation of individual glucosinolates by a wide range of biotic stimuli, in all possibility, suggests their differential role in plant defense.

2.3. Effect of elicitors on the expression of transcriptional regulators of glucosinolate biosynthesis

Since glucosinolate accumulation is differentially enhanced by biotic elicitors tested above, we investigated the transcriptional regulation of glucosinolates biosynthesis under different stress conditions. We analyzed the steady state mRNA levels of reported MYB transcription factor genes which are known to be major regulators of both aliphatic and indole glucosinolates biosynthesis. Multiple homologs of genes encoding these transcription regulators have been identified in B. rapa (www.brasicadb.org/brad/; Cheng et al., 2011; Zang et al., 2009) and B. juncea ( Augustine et al., 2013b; Bisht et al., 2009). The expression levels of Brassica homologs of MYB28, MYB29, MYB34, MYB51 and MYB122 were analyzed using gene specific primers after 6 h, 24 h and 48 h of treatment.

Differential expression of glucosinolate regulators was observed in response to the tested biotic elicitors.After 6 h of MeJA treatment, indole glucosinolate regulators such as MYB34-1 and MYB122-2 showed pronounced induction whereas among the aliphatic glucosinolate regulators, only MYB29-1 showed enhancement in transcript levels ( Fig. 3 ). Other MYB genes showed a down regulation of their transcript levels during early time point of MeJA treatment. Under SA treatment, among the regulators of aliphatic glucosinolates, only MYB28-3 showed induction of gene expression after 6 h, whereas among the indole glucosinolate biosynthesis regulators, MYB51-1 and MYB122-1 showed up-regulation of their transcripts. Glu was found to induce expression of all the transcriptional regulators of aliphatic glucosinolate biosynthesis whereas the expression of indole glucosinolate regulators was suppressed or remained unaltered. Wounding was also found to induce the expression of most of the transcriptional regulators of both aliphatic and indole glucosinolates but in a differential manner. For instance, MYB28-1, MYB28-3, MYB34-1 and MYB51 were found to be up-regulated while the others showed no variation or reduction in expression levels after 6 h in response to wounding stimulus. Generally, the expression pattern of MYB gene homologs was in consonance with the altered glucosinolates profiles of treated seedlings.

The expression profile was also analyzed in the seedlings after 24 h of elicitor treatment at which response for both aliphatic and indole glucosinolates was observed. Among the aliphatic glucosinolate regulators, MYB29-1 expression was found to be enhanced after 24 h of MeJA treatment whereas the transcript levels of MYB34-1 and MYB122-2 homologs showed a decline ( Fig. 3 ). Under SA treatment, expression levels of some MYB genes were found to be enhanced after 24 h of treatment. While transcript levels of MYB29-1 and MYB122-1 showed a sharp increase, MYB28-2, MYB28-4, MYB28-5 and MYB34s levels were found to be unresponsive to SA application. Interestingly, under Glu treatment, the expression of all aliphatic glucosinolate regulators was higher while that of indole glucosinolate regulators remained suppressed. Expression profiling after wounding suggested that prolonged wounding for a period of 24 h up-regulates the expression of almost all indole glucosinolate regulating MYB genes, possibly reflecting their role during herbivory. Expression of all the MYB genes involved in aliphatic glucosinolate biosynthesis declined after 48 h of elicitor treatment. However, regulators of indole glucosinolate biosynthesis retained higher expression levels especially under MeJA and wounding even after 48 h of treatment. Expression of indole MYB s were very pronounced for a period of 48 h following mechanical injury by wounding. Among the indole glucosinolate regulators, MYB122-1 showed significant up-regulation 48 h post Glu treatment, which coincided with a higher accumulation of 1MOI3M at this stage.

The up-regulation of MYB29-1 after 24 h of treatment with MeJA was in agreement with AtMYB29 response as reported earlier ( Gigolashvili et al., 2007b). Enhanced accumulation of indole glucosinolates under MeJA treatment was well supported by the up-regulation of its transcriptional regulators. Previous studies in Arabidopsis and Brassica showed that upon treatment with MeJA, MYB34 and MYB51 genes encoding indole glucosinolate transcription regulators are up-regulated ( Dombrecht et al., 2007; Wiesner et al., 2013). However, in our study we found that MYB34-1 and MYB122-2 were the most abundant transcripts after 6 h of treatment, whereas MYB51-3 and MYB122-1 showed increased levels of expression at 48 h post MeJA treatment. In a recent study in Arabidopsis , Frerigmann and Gigolashvili (2014) reported that MYB34 plays a vital role upon JA signaling while MYB51 is the major regulator of indole glucosinolates upon SA signaling, and MYB122 plays only a minor role in JA induced glucosinolate biosynthesis.

Increased accumulation of aliphatic glucosinolates under Glu treatment was found to be mainly due to the up-regulation of MYB29-1 and MYB28-3 transcripts. It is therefore evident that although MYB29-1 plays a minor role in synthesis of basal levels of aliphatic glucosinolate, it is majorly responsible for stimuliinduced accumulation of glucosinolates in B. juncea . We also found that all the homologs of MYB28 and MYB29-1 were induced by Glu. MYB28 has been identified as one of the very rapidly up-regulated genes in response to Glu treatment in A. thaliana seedlings using microarray as well as functional genomic data ( Gigolashvili et al., 2007b; Zimmermann et al., 2004). MYB51 expression in Arabidopsis leaves is induced by wounding or touch, while MYB34 expression remains unaffected ( Gigolashvili et al., 2007a). Our study showed that all the indole glucosinolate regulators were induced by wounding but in a time-dependant manner in which, after 48 h, all the homologs showed increased expression. Among the aliphatic glucosinolates regulators, only MYB28-3 and MYB29-1 were found to be wound responsive. The differences between enhancement of glucosinolate levels and transcript up-regulation at different stages suggest the involvement of other transcriptional regulators and biosynthetic genes in glucosinolate biosynthesis. These need to be investigated to dissect the complete transcriptional network involved in the environmental regulation of glucosinolates.

Figures

Fig. 2. Accumulation of glucosinolates under various elicitor treatments in a time course experiment in B. juncea seedlings. Total aliphatic and indole glucosinolates content (µmoles g ―1 dry wt.) after 6 h, 24 h and 48 h of treatment, as estimated by HPLC. Bars are drawn based on the mean of three independent experiments and error bars represent standard deviation. Asterisks (⁄) indicate values significantly different from control at P <0.05. Letters OE and β represent significant difference at P <0.05 for aliphatic and indole glucosinolates, respectively.

Fig. 3. Expression profiling of MYB transcriptional regulators of aliphatic and indole glucosinolates biosynthesis in B. juncea seedlings. Heat map was constructed based on the relative expression value compared with the non-treated seedling at each time point, using UBQ9 as the reference gene. qRT-PCR was performed for three independent experiments for each target, and the data were averaged.