Search

Transient gene expression serves as a valuable tool for gene functional study in plants. Here we reported protoplast isolation and purification from the plantlet young leaves of Chinese kale (Brassica oleracae var. alboglabra Bailey), and their transient gene expression with polyethylene glycol (PEG)-mediated transformation and subcellular localization of phytoene desaturase (BaPDS1). The procedures of isolation andtransformation ofmesophyll protoplasts derived from Chinese kale were optimized, and the influencing factors were analyzed. The results showed that the optimal protocol of protoplast isolation and purification was initialized by digestion in enzyme solution (2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol) for 9 h. After filtered through 400 mesh and centrifuged at 179 ×g for purification, the total yield of protoplast reached as high as 6.04 × 107 protoplasts g−1 fresh weight (FW) and the viability of the protoplasts was up to 95%. A maximum transformation efficiency of approximately 30% measured by using green fluorescent protein (GFP) as a detecting gene was obtained when PEG4000 was at a final concentration of 40% and transformation time was set to 15 min. In addition, the subcellular localization of BaPDS1 in Chinese kale was targeted to the chloroplast, confirming the efficiency and reliability of this transient transformation system. Taken together, an efficient protoplast isolation, purification and transformation system in Chinese kale was established in this study, laying a foundation for future research in molecular biology and gene function in Chinese kale and other Brassica vegetables.

Glucosinolates are a diverse class of secondary metabolites that play a vital role in the plant defense response. Here, we investigated the function of the UDP-glucosyltransferase 74B1 (UGT74B1) gene in glucosinolate biosynthesis in Chinese kale. A 1401 bp coding sequence of BoaUGT74B1 was cloned from Chinese kale cultivar ‘Sijicutiao’. Among different organs, BoaUGT74B1 has the highest expression level in young seeds. Among different flower tissues, BoaUGT74B1 has the highest expression level in the pistil; the gene was downregulated in flower tissues as flowers matured. Subcellular localization showed that BoaUGT74B1 is located in chloroplasts. A purified ~50 kD fusion protein was obtained from Escherichia coli after prokaryotic expression of BoaUGT74B1. BoaUGT74B1 introduced into the white-flower cultivar ‘Sijicutiao’ and yellow-flower cultivar ‘Fuzhouhuanghua’ of Chinese kale by Agrobacterium-mediated transformation was strongly in both cultivars. Expression levels of BoaUGT74B1 in the overexpressed plants ‘Sijicutiao’ and ‘Fuzhouhuanghua’ were 1.53- to 2.93-fold and 3.07- to 5.30-fold higher, respectively than that of the wild type; the total indole glucosinolate content in these two overexpressed plants was 1.30- to 7.11-fold and 2.13- to 4.44-fold higher, respectively, compared with that in wild-type plants. Each indole glucosinolate accumulated in significant amounts in all overexpressed plants; 4‑methoxy glucobrassicin enhanced most in ‘Sijicutiao’ overexpressed plants, in concentrations up to 12.14-fold higher than the control; glucobrassicin enhanced most in ‘Fuzhouhuanghua’ overexpressed plants, reaching levels 22.76x that of its wild-type counterpart. Therefore, these findings indicate that BoaUGT74B1 plays an important role in the accumulation of indole glucosinolates in Chinese kale, and suggest the possible usefulness of this gene to improve the nutritional quality and resistance against biotic stress of Chinese kale.

The purpose of this study was to investigate the effect of glucose on glucosinolates, antioxidants and metabolic enzymes in Brassica sprouts. After glucose treatment, total glucosinolates, phenolics and anthocyanin contents and antioxidant activity were significantly enhanced in Chinese kale and pak choi sprouts, while only the anthocyanin and ascorbic acid contents were increased in radish sprouts. These results indicate that glucose treatment has selectively improved the nutritional compounds in different Brassica sprouts.

The contents of ascorbic acid, glucosinolates, sulforaphane, anthocyanins, total phenolics, the activity of myrosinase and phenylalanine ammonialyase (PAL) and the antioxidant activity of broccoli sprouts grown under 88 mM and 176 mM of sucrose and mannitol were investigated. The results showed that the contents of sulforaphane, ascorbic acid and anthocyanins in broccoli sprouts were significantly increased after treatment with 88 mM of sucrose compared with the control. The contents of glucosinolates and total phenolics, the activity of PAL and the antioxidant activity in broccoli sprouts treated with 176 mM sucrose were also significantly increased, whereas the activity of myrosinase was significantly reduced. On the other hand, the contents of glucosinolate, sulforaphane and total phenolics in broccoli sprouts were also significantly increased after treatment with 176 mM of mannitol, although the contents of sulforaphane were markedly reduced compared to those treated with 176 mM of sucrose. Sucrose might induce the production of health-promoting compounds through its role of signaling, generating osmotic pressure or serving as a substrate. These results indicate that sucrose treatment could improve the nutritional value of broccoli, and the sprouts growing under adequate concentration of sucrose could benefit our diet by producing more health-promoting compounds.

The effect of brassinosteroid (BR) on relative water content (RWC), stomatal conductance (g s ), net photosynthetic rate ( P N), intercellular CO 2 concentration (Ci), lipid peroxidation level, activities of antioxidant enzymes and abscisic acid concentration (ABA) in tomato ( Lycopersicon esculentum ) seedlings under water stress was investigated. Two tomato genotypes, Mill. cv. Ailsa Craig (AC) and its ABA-deficient mutant notabilis ( not ), were used. Water stress was achieved by withholding water and both the AC and not plants were treated with 1 μM 24-epibrassinolide (EBR) or distilled water as a control. The RWC, g s , Ci and P N were significantly decreased under water stress. However, EBR treatment significantly alleviated water stress and increased the RWC and P N. EBR application also markedly increased the activities of antioxidant enzymes (catalase, ascorbate peroxidase and superoxide dismutase) while it decreased g s , Ci and the contents of H 2 O 2 and malondialdehyde (MDA). Interestingly, ABA concentration in AC and not plants was markedly elevated after EBR treatment although the increasing rate and amplitude of ABA in not plants treated by EBR was significantly lower than those in AC plants. Our study suggested that amelioration of the drought stress of tomato seedlings may be caused by EBR-induced elevation of endogenous ABA concentration and/or the activities of antioxidant enzymes.