CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis

Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.     

Chemical defenses of crucifers: elicitation and metabolism of phytoalexins and indole-3-acetonitrile in brown mustard and turnip

The metabolism of the cruciferous phytoalexins brassinin and cyclobrassinin, and the related compounds indole-3-carboxaldehyde, glucobrassicin, and indole-3-acetaldoxime was investigated in various plant tissues of Brassica juncea and B. rapa. Metabolic studies with brassinin showed that stems of B. juncea metabolized radiolabeled brassinin to indole-3-acetic acid, via indole-3-carboxaldehyde, a detoxification pathway similar to that followed by the "blackleg" fungus (Phoma lingam/Leptosphaeria maculans). In addition, it was established that tetradeuterated brassinin was incorporated into the phytoalexin brassilexin in B. juncea and B. rapa. On the other hand, the tetradeuterated indole glucosinolate glucobrassicin was not incorporated into brassinin, although the chemical structures of brassinins and indole glucosinolates suggest an interconnected biogenesis. Importantly, tetradeuterated indole-3-acetaldoxime was an efficient precursor of phytoalexins brassinin, brassilexin, and spirobrassinin. Elicitation experiments in tissues of Brassica juncea and B. rapa showed that indole-3-acetonitrile was an inducible metabolite produced in leaves and stems of B. juncea but not in B. rapa. Indole-3-acetonitrile displayed antifungal activity similar to that of brassilexin, was metabolized by the blackleg fungus at slower rates than brassinin, cyclobrassinin, or brassilexin, and appeared to be involved in defense responses of B. juncea.     

Purification and characterization of a nitrilase from Brassica napus

In germinating seedlings of Brassica napus glucosinolate levels decrease and are potentially degraded to nitriles by a myrosinase. Little is known about the metabolism of glucosinolate aglycone products and the objective of this work was to investigate nitrilase activity and carry out a purification of the enzyme from seedlings of B. napus . A nitrilase capable of converting phenylpropionitrile to phenylpropionic acid was purified to apparent homogeneity from seedlings of B. napus . The protein has a molecular mass of approximately 420 kDa made up of 38 kDa subunits. The pI of the native protein was found to be 4.6. Under denaturing conditions on an isoelectric focusing (IEF) gel a major and minor protein was observed with pI in the range of 5.4-5.9, suggesting the presence of isoforms. Apart from the potential role of the nitrilase in indole-3-acetic acid (IAA) synthesis a developmental study with seedlings indicates that the increase in activity observed may be linked to the in vivo degradation of glucosinolates.     

Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low-seed,high-leaf glucosinolate oilseed mustard with uncompromised defence and yield

Glucosinolate content in the two major oilseed Brassica crops—rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 μmoles/g of seed dry weight, DW), making the protein-rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9-based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR-edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR-edited lines displayed a defence response at par or better than that of the wild-type line. The GTR-edited lines were equivalent to the wild-type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low-seed, high-leaf glucosinolate lines with an uncompromised defence and yield.     

Effects of previous cropping and fungicide timing on the development of light leaf spot (Pyrenopeziza brassicae), seed yield and quality of winter oilseed rape (Brassica napus)

Light leaf spot, caused by Pyrenopeziza brassicae, was assessed regularly on double-low cultivars of winter oilseed rape during field experiments at Rothamsted in 1990-91 and 1991-92. Previous cropping and fungicide applications differed; seed yield and seed quality were measured at harvest. In each season, both the initial incidence of light leaf spot and the rate of disease increase were greater in oilseed rape crops sown after rape than those sown after cereals. The incidence of diseases caused by Phoma lingam or Alternaria spp. was also greater in second oilseed rape crops. In 1991-92 there was 42% less rainfall between September and March than in 1990-91, and much less light leaf spot developed. However, P. lingam and Alternaria spp. were more common. Only fungicide application schedules including an autumn spray decreased the incidence of light leaf spot on leaves, stems and pods, as indicated by decreased areas under the disease progress curves (AUDPC) and slower rates of disease increase. Summer sprays decreased incidence and severity of light leaf spot on pods only. In 1990-91, all fungicide treatments which included an autumn spray increased seed and oil yields of cv. Capricorn but only the treatment which included autumn, spring and summer sprays increased yields of cv. Falcon. No treatment increased the yields of cv. Capricorn or cv. Falcon in 1991-92. Fungicide applications decreased glucosinolate concentrations in the seed from a crop of cv. Cobra severely infected by P. brassicae in 1990-91, but did not increase yield.     

Benzylglucosinolate degradation in heat-treated Lepidium sativum seeds and detection of a thiocyanate-forming factor

Lepidium sativum seeds were dry heated at 125° for varying periods, and also for 30 min at various temperatures. Autolysates were then analysed for benzylglucosinolate degradation products. Whilst heating for 4 hr 20 min at 125° was sufficient to prevent formation of benzyl thiocyanate, just over 7.5 hr at 125° was required before benzyl isothiocyanate also ceased to be produced. This indicates the presence of a discrete, thiocyanate-forming factor in L. sativum seeds, separate from thioglucosidase. After 7.5 hr at 125°, benzyl cyanide continued to be formed, proving that it can be obtained (in relatively small amounts) directly from the glucosinolate even without the influence of any thioglucosidase. In general, isothiocyanate was the more favoured product of glucosinolate degradation following heat treatment of seeds, until the point of thioglucosidase inactivation was approached when nitrile formation took over. It is suggested that the thiocyanate-forming factor is an isomerase causing Z-E isomerization of the glucosinolate aglucone, but that only those glucosinolates capable of forming particularly stable cations are then able to undergo E-aglucone rearrangement to thiocyanate.     

Tenualexin,Other Phytoalexins and Indole Glucosinolates from Wild Cruciferous Species

In general, the chemodiversity of phytoalexins, elicited metabolites involved in plant defense mechanisms against microbial pathogens, correlates with the biodiversity of their sources. In this work, the phytoalexins produced by four wild cruciferous species (Brassica tournefortii, Crambe abyssinica (crambe), Diplotaxis tenuifolia (sand rocket), and Diplotaxis tenuisiliqua (wall rocket)) were identified and quantified by HPLC with photodioarray and electrospray mass detectors. In addition, the production of indole glucosinolates, biosynthetic precursors of cruciferous phytoalexins, was evaluated. Tenualexin, (=2‐(1,4‐dimethoxy‐1H‐indol‐3‐yl)acetonitrile), the first cruciferous phytoalexin containing two MeO substituents in the indole ring, was isolated from D. tenuisiliqua, synthesized, and evaluated for antifungal activity. The phytoalexins cyclobrassinin and spirobrassinin were detected in B. tournefortii and C. abyssinica, whereas rutalexin and 4‐methoxybrassinin were only found in B. tournefortii. D. tenuifolia, and D. tenuisiliqua produced 2‐(1H‐indol‐3‐yl)acetonitriles as phytoalexins. Because tenualexin appears to be one of the broad‐range antifungals occurring in crucifers, it is suggested that D. tenuisiliqua may have disease resistance traits important to be incorporated in commercial breeding programs.     

Use of Brassicaceous seed meals to improve seedling emergence of tomato and pepper in Pythium ultimum infested soils

Growth room studies were conducted to determine the impact of Brassicaceae seed meals on the emergence of tomato and pepper seedlings in Pythium ultimum infested soils. Pasteurised Burch sandy loam soils were amended with intact and denatured seed meal of rape seed and mustard. Brassica juncea or Brassica napus intact seed meal increased the tomato and pepper seedling emergence. Interestingly, B. napus amended soils resulted in the same seedling emergence with B. juncea regardless of their relatively lower glucosinolate content compared to mustard-based seed meals. Seedling emergence in soils amended with intact Sinapis alba seed meal was significantly the lowest for both tomato and pepper seedlings. In contrast, seedling emergence was higher in soils amended with denatured than intact S. alba seed meals suggesting some glucosinolate-related inhibitory effect on seedling emergence of both crops. Glycine max seed meal amendment improved the seedling emergence better than the control but to a lower-level when compared to glucosinolate containing seed meals. This finding suggests that even though improvement of seedling emergence of tomato and pepper in P. ultimum infested soils can be achieved using Brassicaceae seed meals, it cannot be entirely attributed to glucosinolate-related processes. These studies demonstrate that intact B. napus and B. juncea seed meals can be used to improve tomato and pepper seedling emergence in P. ultimum infested soils.     

外源喷施萘乙酸对小白菜硫代葡萄糖苷的影响

本试验研究了不同浓度的萘乙（NAA）并结合单次喷施（NAA-1）和两次喷施（NAA．2）,对小白菜生长和硫代葡萄糖苷（简称硫苷）含量的影响。结果表明,与对照相比,不同的NAA处理浓度均显著增加了小白菜的鲜重。同时,NAA处理对总硫苷和单个硫苷含量产生了显著的影响。NAA-1处理时,总脂肪族硫苷、2．苯乙基硫苷和总硫苷在20mg·L-1时到达最大值;而总吲哚族硫苷在50mg·L-1时含量到达最高。NAA．2处理时,大部分单个硫苷和总硫苷在10mg·L-1处理即达到最大值。可见,在较低浓度两次喷施NAA试验中对大部分硫苷的诱导效果高于单次喷施NAA;但随着NAA处理浓度的提高,单次喷施NAA对硫苷的诱导效果较好。其中,吲哚-3-甲基硫苷含量在NAA-1处理,喷施50mg·L-1时显著高于其他处理：而2-苯乙基硫苷在单次和两次喷施NAA时,分别在20mg·L-1和10mg·L-达到了最大值。     

Exudates from Rice Callus Tissues Resulting from the Lack of Cuticle and Wax Layers

Nondialyzable melanoidins prepared from a glucose-glycine system were investigated as to their decolorization and degradation products on ozone treatment. Melanoidins were decolorized to degrees of 84 and 97% after ozonolysis at — 1°C for lOmin and 90min, respectively, and the mean molecular weight of melanoidins decreased from 7000 to 3000 after ozonolysis for 40 min. The major components of electrofocused melanoidins before and after ozone treatment had pis of 3.00 and 2.86, respectively, the pI 3.00 band being significantly affected.

IR measurement showed that the absorption at 1290 cm^?1 disappeared and that at 1720 cm^?1 newly appeared on ozonolysis, respectively, and the absorption at 1620 cm^?1 disappeared on acid hydrolysis after ozonolysis.

Furthermore, the major degradation products in the ether-soluble fractions obtained from ozone-treated melanoidins were identified as butanedioic acid, glycolic acid, 2-hydroxybutanoic acid and so on.

In the aqueous fraction, one of the major products was glycine, which was produced to the level of 1.05% on ozonolysis which increased to 5.75% per melanoidin on acid hydrolysis after ozonolysis. From these findings and the IR results, it is postulated that glycine was considerably incorporated into melanoidin molecules as the amide form.