BoMyrosinase plays an essential role in sulforaphane accumulation in response to selenite treatment in broccoli

Sulforaphane, a naturally specialized metabolite, plays significant roles in human disease prevention and plant defense. Myrosinase(MY) is a key gene responsible for the catalysis of sulforaphane formation, but the molecular mechanisms through which MY regulates sulforaphane biosynthesis in plants remains largely unknown. Here, we discovered that the change of sulforaphane content in broccoli sprouts caused by exogenous selenite treatments is positively related to BoMY expression. BoMY overexpression in the Arabidopsis thaliana tgg1 mutants could dramatically increase myrosinase activity and sulforaphane content in the rosette leaves of 35S::BoMY/tgg1 and rescue its phenotypes.Moreover, an obvious increase of myrosinase activity and sulforaphane content was displayed in transgenic BoMY-overexpressed broccoli lines.In addition, a 2 033 bp promoter fragment of BoMY was isolated. Yeast one-hybrid(Y1H) library screening experiment uncovered that one bHLH transcription factor, BoFAMA, could directly bind to BoMY promoter to activate its expression, which was further evidenced by Y1H assay and dual-luciferase reporter assay. BoFAMA is a selenite-responsive transcription factor that is highly expressed in broccoli leaves;its protein is solely localized to nucleus. Additionally, genetic evidence suggested that the knockdown of FAMA gene in Arabidopsis thaliana could significantly decrease sulforaphane yield by inhibiting the expression of myrosinase genes. Interestingly, exogenous selenite supply could partially restore the low level of sulforaphane content in transgenic Arabidopsis FAMA-silencing plants. Our findings uncover a novel function of FAMAMY module in the regulation of selenite-mediated sulforaphane synthesis and provide a new insights into the molecular mechanism by which selenite regulates the accumulation of sulforaphane in plants.

Accurate pH decrease induced inactivation of myrosinase and polyphenol oxidase in salted radish for color improvement

Yellowing even browning of radish commonly occurred during the traditional pickling.This research aimed to determine the reason of yellowing of pickled radish,and propose the method for yellowing inhibition.The activity of endogenous enzymes in radish were analyzed,and the relationship between the activity and the browning was investigated.Myrosinase and polyphenol oxidase(PPO)were found as main potential enzymes resulting in browning of radish.During traditional salting processing,the myrosinase activity decreased,particularly at a high speed in the first 14 days,while the PPO activity did not decrease observably.The a*and b*values of salted radish were significantly correlated with myrosinase and PPO activity.Acid treatment was observed to be effective for browning inhibition of radish during soaking and storage,and the low pH conditions contributed to the preservation of original green color of radish.Stronger acidification during the first 3 d played a much more important role in browning inhibition than following weaker acidification treatment for 27 d.Low pH showed an inhibition effect on PPO activity,but myrosinase reactivity was more sensitive to pH changes than PPO.The myrosinase in the radish treated in the pickling solution at pH 2 was completely inactivated during the first 3 days.Further investigation of pH effect on improvement of salted radish color concluded that pH 2.4 was the optimal pH for the inactivation of myrosinase and PPO.Accordingly,short-term accurate acidification coupled with traditional salting treatment was originally proposed for effective browning inhibition of pickled radish.

Characterization of a myrosinase cDNA from Brassica parachinensis and its defense role against Plutella xylostella after suppression

In Brassicaceae, myrosinase catalyzes the hydrolysis of glucosinolate and plays an important role in anti-herbivore defense. We have cloned and characterized the full- length complementary DNA of myrosinase gene from Brassicaparachinensis that exhibits high sequence identity with myrosinase genes from other Brassica species. To investigate the role of this myrosinase in defense against the diamondback moth （Plutella xylostella）, we constructed an RNA-interference （RNAi） cassette expressing a double-stranded RNA that targeted myrosinase and transfected it into B. parachinensis. Myrosinase was sup- pressed in the resulting transgenic plants. Diamondback moth larvae feeding on transgenic plants had lower larval and pupal weights, longer pupal duration, and lower fecundity than those feeding on non-transgenic plants, suggesting that the diamondback moth has adapted to the glucosinolate-myrosinase defensive system. Therefore, the suppression of myrosinase is a potential approach for controlling the diamondback moth.

Effect of NaCl treatments on glucosinolate metabolism in broccoli sprouts

To understand the regulation mechanism of NaCI on glucosinolate metabolism in broccoli sprouts, the germination rate, fresh weight, contents of glucosinolates and sulforaphane, as well as myrosinase activity of broccoli sprouts germinated under 0, 20, 40, 60, 80, and 100 mmol/L of NaCI were investigated in our experiment. The results showed that glucoerucin, glucobrassicin, and 4-hydroxy glucobrassicin in 7-d-old broccoli sprouts were significantly enhanced and the activity of myrosinase was inhibited by 100 mmol/L of NaCI. However, the total glucosinolate content in 7-d-old broccoli sprouts was markedly decreased although the fresh weight was significantly increased after treatment with NaCI at relatively low concentrations （20, 40, and 60 mmol/L）. NaCI treatment at the concentration of 60 mmol/L for 5 d maintained higher biomass and comparatively higher content of glucosinolates in sprouts of broccoli with decreased myrosinase activity. A relatively high level of NaCI treatment （100 mmol/L） significantly increased the content of sulforaphane in 7-d-old broccoli sprouts compared with the control. These results indicate that broccoli sprouts grown under a suitable concentration of NaCI could be desirable for human nutrition.

Specialized endoplasmic reticulum-derived vesicles in plants:Functional diversity,evolution,and biotechnological exploitation

A central role of the endoplasmic reticulum(ER)is the synthesis,folding and quality control of secretory proteins.Secretory proteins usually exit the ER to enter the Golgi apparatus in coat protein complex II(COPII)-coated vesicles before transport to different subcellular destinations.However,in plants there are specialized ER-derived vesicles(ERDVs)that carry specific proteins but,unlike COPII vesicles,can exist as independent organelles or travel to the vacuole in a Golgi-independent manner.These specialized ERDVs include protein bodies and precursor-accumulating vesicles that accumulate storage proteins in the endosperm during seed development.Specialized ERDVs also include precursor protease vesicles that accumulate amino acid sequence KDEL-tailed cysteine proteases and ER bodies in Brassicales plants that accumulate myrosinases that hydrolyzes glucosinolates.These functionally specialized ERDVs act not only as storage organelles but also as platforms for signal-triggered processing,activation and deployment of specific proteins with important roles in plant growth,development and adaptive responses.Some specialized ERDVs have also been exploited to increase production of recombinant proteins and metabolites.Here we discuss our current understanding of the functional diversity,evolutionary mechanisms and biotechnological application of specialized ERDVs,which are associated with some of the highly remarkable characteristics important to plants.