Metabolomic and Transcriptomic Profiles in Diverse Brassica oleracea Crops Provide Insights into the Genetic Regulation of Glucosinolate Profiles.

Glucosinolates (GSLs) are plant secondary metabolites commonly found in the cruciferous vegetables of the Brassicaceae family, offering health benefits to humans and defense against pathogens and pests to plants. In this study, we investigated 23 GSL compounds' relative abundance in four tissues of five different Brassica oleracea morphotypes. Using the five corresponding high-quality B. oleracea genome assemblies, we identified 183 GSL-related genes and analyzed their expression with mRNA-Seq data. GSL abundance and composition varied strongly, among both tissues and morphotypes, accompanied by different gene expression patterns. Interestingly, broccoli exhibited a nonfunctional AOP2 gene due to a conserved 2OG-FeII_Oxy domain loss, explaining the unique accumulation of two health-promoting GSLs. Additionally, transposable element (TE) insertions were found to affect the gene structure of MAM3 genes. Our findings deepen the understanding of GSL variation and genetic regulation in B. oleracea morphotypes, providing valuable insights for breeding with tailored GSL profiles in these crops.

Developing multifunctional crops by engineering Brassicaceae glucosinolate pathways.

Glucosinolates (GSLs), found mainly in species of the Brassicaceae family, are one of the most well-studied classes of secondary metabolites. Produced by the action of myrosinase on GSLs, GSL-derived hydrolysis products (GHPs) primarily defend against biotic stress in planta. They also significantly affect the quality of crop products, with a subset of GHPs contributing unique food flavors and multiple therapeutic benefits or causing disagreeable food odors and health risks. Here, we explore the potential of these bioactive functions, which could be exploited for future sustainable agriculture. We first summarize our accumulated understanding of GSL diversity and distribution across representative Brassicaceae species. We then systematically discuss and evaluate the potential of exploited and unutilized genes involved in GSL biosynthesis, transport, and hydrolysis as candidate GSL engineering targets. Benefiting from available information on GSL and GHP functions, we explore options for multifunctional Brassicaceae crop ideotypes to meet future demand for food diversification and sustainable crop production. An integrated roadmap is subsequently proposed to guide ideotype development, in which maximization of beneficial effects and minimization of detrimental effects of GHPs could be combined and associated with various end uses. Based on several use-case examples, we discuss advantages and limitations of available biotechnological approaches that may contribute to effective deployment and could provide novel insights for optimization of future GSL engineering.

Glucosinolates and Biotic Stress Tolerance in Brassicaceae with Emphasis on Cabbage: A Review.

Glucosinolates (GSLs) and GSL-associated genes are receiving increasing attention from molecular biologists due to their multifunctional properties. GSLs are secondary metabolites considered to be highly active in most Brassica species. Their importance has motivated the discovery and functional analysis of the GSLs and GSL hydrolysis products involved in disease development in brassicas and other plants. Comprehensive knowledge of the GSL content of Brassica species and the molecular details of GSL-related genes will help elucidate the molecular control of this plant defense system. This report provides an overview of the current status of knowledge on GSLs, GSL biosynthesis, as well as hydrolysis related genes, and GSL hydrolysis products that regulate fungal, bacterial, and insect resistance in cabbage and other brassicas.

Further insight into decreases in seed glucosinolate content based on QTL mapping and RNA-seq in Brassica napus L.

KEY MESSAGE: The QTL hotspots determining seed glucosinolate content instead of only four HAG1 loci and elucidation of a potential regulatory model for rapeseed SGC variation. Glucosinolates (GSLs) are amino acid-derived, sulfur-rich secondary metabolites that function as biopesticides and flavor compounds, but the high seed glucosinolate content (SGC) reduces seed quality for rapeseed meal. To dissect the genetic mechanism and further reduce SGC in rapeseed, QTL mapping was performed using an updated high-density genetic map based on a doubled haploid (DH) population derived from two parents that showed significant differences in SGC. In 15 environments, a total of 162 significant QTLs were identified for SGC and then integrated into 59 consensus QTLs, of which 32 were novel QTLs. Four QTL hotspot regions (QTL-HRs) for SGC variation were discovered on chromosomes A09, C02, C07 and C09, including seven major QTLs that have previously been reported and four novel major QTLs in addition to HAG1 loci. SGC was largely determined by superimposition of advantage allele in the four QTL-HRs. Important candidate genes directly related to GSL pathways were identified underlying the four QTL-HRs, including BnaC09.MYB28, BnaA09.APK1, BnaC09.SUR1 and BnaC02.GTR2a. Related differentially expressed candidates identified in the minor but environment stable QTLs indicated that sulfur assimilation plays an important rather than dominant role in SGC variation. A potential regulatory model for rapeseed SGC variation constructed by combining candidate GSL gene identification and differentially expressed gene analysis based on RNA-seq contributed to a better understanding of the GSL accumulation mechanism. This study provides insights to further understand the genetic regulatory mechanism of GSLs, as well as the potential loci and a new route to further diminish the SGC in rapeseed.

Microevolution of Pieris butterfly genes involved in host plant adaptation along a host plant community cline.

Herbivorous insects have evolved counteradaptations to overcome the chemical defences of their host plants. Several of these counteradaptations have been elucidated at the molecular level, in particular for insects specialized on cruciferous host plants. While the importance of these counteradaptations for host plant colonization is well established, little is known about their microevolutionary dynamics in the field. In particular, it is not known whether and how host plant diversity shapes diversity in insect counteradaptations. In this study, we examine patterns of host plant use and insect counteradaptation in three Pieris butterfly species across Japan. The larvae of these butterflies express nitrile-specifier protein (NSP) and its paralogue major allergen (MA) in their gut to overcome the highly diversified glucosinolate-myrosinase defence system of their cruciferous host plants. Pieris napi and Pieris melete colonize wild Brassicaceae whereas Pieris rapae typically uses cultivated Brassica as a host, regardless of the local composition of wild crucifers. As expected, NSP and MA diversity was independent of the local composition of wild Brassicaceae in P. rapae. In contrast, NSP diversity correlated with local host plant diversity in both species that preferred wild Brassicaceae. Both P. melete and P. napi revealed two distinct major NSP alleles, which shaped diversity among local populations, albeit with different evolutionary trajectories. In comparison, MA showed no indication for local adaptation. Altogether, MA appeared to be evolutionary more conserved than NSP, suggesting that both genes play different roles in diverting host plant chemical defence.

The BrGI Circadian Clock Gene Is Involved in the Regulation of Glucosinolates in Chinese Cabbage.

Circadian clocks integrate environmental cues with endogenous signals to coordinate physiological outputs. Clock genes in plants are involved in many physiological and developmental processes, such as photosynthesis, stomata opening, stem elongation, light signaling, and floral induction. Many Brassicaceae family plants, including Chinese cabbage (Brassica rapa ssp. pekinensis), produce a unique glucosinolate (GSL) secondary metabolite, which enhances plant protection, facilitates the design of functional foods, and has potential medical applications (e.g., as antidiabetic and anticancer agents). The levels of GSLs change diurnally, suggesting a connection to the circadian clock system. We investigated whether circadian clock genes affect the biosynthesis of GSLs in Brassica rapa using RNAi-mediated suppressed transgenic Brassica rapa GIGENTEA homolog (BrGI knockdown; hereafter GK1) Chinese cabbage. GIGANTEA plays an important role in the plant circadian clock system and is related to various developmental and metabolic processes. Using a validated GK1 transgenic line, we performed RNA sequencing and high-performance liquid chromatography analyses. The transcript levels of many GSL pathway genes were significantly altered in GK1 transgenic plants. In addition, GSL contents were substantially reduced in GK1 transgenic plants. We report that the BrGI circadian clock gene is required for the biosynthesis of GSLs in Chinese cabbage plants.

Arabidopsis assemble distinct root-associated microbiomes through the synthesis of an array of defense metabolites.

Plant associated microbiomes are known to confer fitness advantages to the host. Understanding how plant factors including biochemical traits influence host associated microbiome assembly could facilitate the development of microbiome-mediated solutions for sustainable plant production. Here, we examined microbial community structures of a set of well-characterized Arabidopsis thaliana mutants disrupted in metabolic pathways for the production of glucosinolates, flavonoids, or a number of defense signalling molecules. A. thaliana lines were grown in a natural soil and maintained under greenhouse conditions for 4 weeks before collection of roots for bacterial and fungal community profiling. We found distinct relative abundances and diversities of bacterial and fungal communities assembled in the individual A. thaliana mutants compared to their parental lines. Bacterial and fungal genera were mostly enriched than depleted in secondary metabolite and defense signaling mutants, except for flavonoid mutations on fungi communities. Bacterial genera Azospirillum and Flavobacterium were significantly enriched in most of the glucosinolate, flavonoid and signalling mutants while the fungal taxa Sporobolomyces and Emericellopsis were enriched in several glucosinolates and signalling mutants. Whilst the present study revealed marked differences in microbiomes of Arabidopsis mutants and their parental lines, it is suggestive that unknown enzymatic and pleiotropic activities of the mutated genes could contribute to the identified host-associated microbiomes. Notwithstanding, this study revealed interesting gene-microbiota links, and thus represents valuable resource data for selecting candidate A. thaliana mutants for analyzing the links between host genetics and the associated microbiome.

Transcriptomics analysis of genes induced by melatonin related to glucosinolates synthesis in broccoli hairy roots.

Glucoraphanin (GRA) is found in the seeds and vegetative organs of broccoli (Brassica oleracea L. var. italica Planch) as the precursor of anti-carcinogen sulforaphane (SF). The yield of GRA obtained from these materials is weak and the cost is high. In recent years, the production of plant secondary metabolites by large-scale hairy roots culture in vitro has succeeded in some species. Melatonin (MT) is a natural hormone which existed in numerous organisms. Studies have demonstrated that MT can improve the synthesis of secondary metabolites in plants. At present, it has not been reported that MT regulates the biosynthesis of glucoraphanin in broccoli hairy roots. In this study, the broccoli hairy roots that grew for 20 d were respectively treated by 500 µM MT for 0, 6, 12, 20 and 32. To explore the reason of changes in secondary metabolites and reveal the biosynthetic pathway of glucoraphanin at transcriptional level. Compared with 0 h, the yield of GRA under other treatments was increased, and the overall trend was firstly increased and then decreased. The total yield of GRA reached the highest at 12 h, which was 1.22-fold of 0 h. Then, the genome of broccoli as the reference, a total of 13234 differentially expressed genes (DEGs) were identified in broccoli hairy roots under treatment with 500 µM MT for 0, 6, 12, 20 and 32 h, respectively. Among these DEGs, 6266 (47.35%) were upregulated and 6968 (52.65%) were downregulated. It was found that the pathway of 'Glucosinolates biosynthesis (ko00966)' was enriched in the 16th place by Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the upregulated DEGs. The expression of key genes in the GRA biosynthesis pathway was upregulated at all time points, and a deduced GRA biosynthesis pathway map was constructed for reference.

Comparative transcriptomic analyses of glucosinolate metabolic genes during the formation of Chinese kale seeds.

BACKGROUND: To understand the mechanism of glucosinolates (GSs) accumulation in the specific organs, combined analysis of physiological change and transcriptome sequencing were applied in the current study. Taking Chinese kale as material, seeds and silique walls were divided into different stages based on the development of the embryo in seeds and then subjected to GS analysis and transcriptome sequencing. RESULTS: The main GS in seeds of Chinese kale were glucoiberin and gluconapin and their content changed with the development of the seed. During the transition of the embryo from torpedo- to the early cotyledonary-embryo stage, the accumulation of GS in the seed was accompanied by the salient decline of GS in the corresponding silique wall. Thus, the seed and corresponding silique wall at these two stages were subjected to transcriptomic sequencing analysis. 135 genes related to GS metabolism were identified, of which 24 genes were transcription factors, 81 genes were related to biosynthetic pathway, 25 genes encoded catabolic enzymes, and 5 genes matched with transporters. The expression of GS biosynthetic genes was detected both in seeds and silique walls. The high expression of FMOGS-OX and AOP2, which is related to the production of gluconapin by side modification, was noted in seeds at both stages. Interestingly, the expression of GS biosynthetic genes was higher in the silique wall compared with that in the seed albeit lower content of GS existed in the silique wall than in the seed. Combined with the higher expression of transporter genes GTRs in silique walls than in seeds, it was proposed that the transportation of GS from the silique wall to the seed is an important source for seed GS accumulation. In addition, genes related to GS degradation expressed abundantly in the seed at the early cotyledonary-embryo stage indicating its potential role in balancing seed GS content. CONCLUSIONS: Two stages including the torpedo-embryo and the early cotyledonary-embryo stage were identified as crucial in GS accumulation during seed development. Moreover, we confirmed the transportation of GS from the silique wall to the seed and proposed possible sidechain modification of GS biosynthesis may exist during seed formation.

Metabolite analysis of Arabidopsis CYP79A2 overexpression lines reveals turnover of benzyl glucosinolate and an additive effect of different aldoximes on phenylpropanoid repression.

Indole-3-acetaldoxime (IAOx) and phenylacetaldoxime (PAOx) are precursors for the growth hormones indole-3-acetic acid (IAA) and phenylacetic acid (PAA) and the defense compounds glucosinolates in Brassicales. Our recent work has shown that Arabidopsis transgenic lines overexpressing AtCYP79A2, a PAOx-production enzyme, accumulate the PAOx-derived compounds benzyl glucosinolate and PAA. Here we report that they also accumulate the benzyl glucosinolate hydrolysis products benzyl isothiocyanate and benzyl cyanide, which indicates that the turnover of benzyl glucosinolate can occur in intact tissues. Myrosinases or ß-glucosidases are known to catalyze glucosinolate breakdown. However, transcriptomics analysis detected no substantial increase in expression of known myrosinases or putative ß-glucosidases in AtCYP79A2 overexpressing lines. It was previously shown that accumulation of aldoximes or their derivatives represses the phenylpropanoid pathway. For instance, ref2 mutant having a defect in one of the aldoxime catabolic enzymes decreases phenylpropanoid production. Considering that AtCYP79A2 is not expressed in most organs under optimal growth condition, ref2 accumulates aliphatic aldoximes but not PAOx. Interestingly, overexpression of AtCYP79A2 in ref2 resulted in a further decrease in sinapoylmalate content compared to ref2. This indicates that accumulation of PAOx has an additive effect on phenylpropanoid pathway suppression mediated by other aldoximes.

The Arabidopsis Iron-Sulfur (Fe-S) Cluster Gene MFDX1 Plays a Role in Host and Nonhost Disease Resistance by Accumulation of Defense-Related Metabolites.

Until recently, genes from the iron-sulfur (Fe-S) cluster pathway were not known to have a role in plant disease resistance. The Nitrogen Fixation S (NIFS)-like 1 (NFS1) and Mitochondrial Ferredoxin-1 (MFDX1) genes are part of a set of 27 Fe-S cluster genes induced after infection with host and nonhost pathogens in Arabidopsis. A role for AtNFS1 in plant immunity was recently demonstrated. In this work, we showed that MFDX1 is also involved in plant defense. More specifically, Arabidopsis mfdx1 mutants were compromised for nonhost resistance against Pseudomonas syringae pv. tabaci, and showed increased susceptibility to the host pathogen P. syringae pv. tomato DC3000. Arabidopsis AtMFDX1 overexpression lines were less susceptible to P. syringae pv. tomato DC3000. Metabolic profiling revealed a reduction of several defense-related primary and secondary metabolites, such as asparagine and glucosinolates in the Arabidopsis mfdx1-1 mutant when compared to Col-0. A reduction of 5-oxoproline and ornithine metabolites that are involved in proline synthesis in mitochondria and affect abiotic stresses was also observed in the mfdx1-1 mutant. In contrast, an accumulation of defense-related metabolites such as glucosinolates was observed in the Arabidopsis NFS1 overexpressor when compared to wild-type Col-0. Additionally, mfdx1-1 plants displayed shorter primary root length and reduced number of lateral roots compared to the Col-0. Taken together, these results provide additional evidence for a new role of Fe-S cluster pathway in plant defense responses.

Influence of Genotype on High Glucosinolate Synthesis Lines of Brassica rapa.

This study was conducted to investigate doubled haploid (DH) lines produced between high GSL (HGSL) Brassica rapa ssp. trilocularis (yellow sarson) and low GSL (LGSL) B. rapa ssp. chinensis (pak choi) parents. In total, 161 DH lines were generated. GSL content of HGSL DH lines ranged from 44.12 to 57.04 µmol·g-1·dry weight (dw), which is within the level of high GSL B. rapa ssp. trilocularis (47.46 to 59.56 µmol g-1 dw). We resequenced five of the HGSL DH lines and three of the LGSL DH lines. Recombination blocks were formed between the parental and DH lines with 108,328 single-nucleotide polymorphisms in all chromosomes. In the measured GSL, gluconapin occurred as the major substrate in HGSL DH lines. Among the HGSL DH lines, BrYSP_DH005 had glucoraphanin levels approximately 12-fold higher than those of the HGSL mother plant. The hydrolysis capacity of GSL was analyzed in HGSL DH lines with a Korean pak choi cultivar as a control. Bioactive compounds, such as 3-butenyl isothiocyanate, 4-pentenyl isothiocyanate, 2-phenethyl isothiocyanate, and sulforaphane, were present in the HGSL DH lines at 3-fold to 6.3-fold higher levels compared to the commercial cultivar. The selected HGSL DH lines, resequencing data, and SNP identification were utilized for genome-assisted selection to develop elite GSL-enriched cultivars and the industrial production of potential anti-cancerous metabolites such as gluconapin and glucoraphanin.

CRISPR-Cas9-Mediated Gene Editing of MYB28 Genes Impair Glucoraphanin Accumulation of Brassica oleracea in the Field.

Discoveries in model plants grown under optimal conditions can provide important directions for crop improvement. However, it is important to verify whether results can be translated to crop plants grown in the field. In this study, we sought to study the role of MYB28 in the regulation of aliphatic glucosinolate (A-GSL) biosynthesis and associated sulfur metabolism in field-grown Brassica oleracea with the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene-editing technology. We describe the first myb28 knockout mutant in B. oleracea, and the first CRISPR field trial in the United Kingdom approved and regulated by the UK Department for Environment, Food & Rural Affairs after the reclassification of gene-edited crops as genetically modified organisms by the European Court of Justice on July 25, 2018. We report that knocking out myb28 results in downregulation of A-GSL biosynthesis genes and reduction in accumulation of the methionine-derived glucosinolate, glucoraphanin, in leaves and florets of field-grown myb28 mutant broccoli plants, whereas accumulation of sulfate, S-methyl cysteine sulfoxide, and indole glucosinolate in leaf and floret tissues remained unchanged. These results demonstrate the potential of gene-editing approaches to translate discoveries in fundamental biological processes for improved crop performance.

The flavor of Chinese kale sprouts is affected by genotypic variation of glucosinolates and their breakdown products.

Metabolic profiling of glucosinolates and their breakdown products in sprouts of 22 Chinese kale (Brassica oleracea var. alboglabra, BOA) varieties were investigated by using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Relationships between glucosinolate metabolites and flavor of Chinese kale sprouts were also analyzed. Results showed that compositions and contents of both glucosinolates and their breakdown products varied greatly among different varieties of Chinese kale sprouts. Gluconapin and 4,5-Epithio-pentanenitrile were the dominant glucosinolate and glucosinolate breakdown product in Chinese kale sprouts, respectively. Gluconapin and glucobrassicin were significantly related to bitterness (r = 0.577, 0.648, respectively; p < 0.05). BOA 1 and BOA 13, BOA 3 and BOA 10 are good candidates for future breeding programs since the former two varieties have light bitterness and pungency, and the latter two varieties contain high levels of glucosinolate breakdown products such as isothiocyanates and epithionitriles in sprouts.

RcTGA1 and glucosinolate biosynthesis pathway involvement in the defence of rose against the necrotrophic fungus Botrytis cinerea.

BACKGROUND: Rose is an important economic crop in horticulture. However, its field growth and postharvest quality are negatively affected by grey mould disease caused by Botrytis c. However, it is unclear how rose plants defend themselves against this fungal pathogen. Here, we used transcriptomic, metabolomic and VIGS analyses to explore the mechanism of resistance to Botrytis c. RESULT: In this study, a protein activity analysis revealed a significant increase in defence enzyme activities in infected plants. RNA-Seq of plants infected for 0 h, 36 h, 60 h and 72 h produced a total of 54 GB of clean reads. Among these reads, 3990, 5995 and 8683 differentially expressed genes (DEGs) were found in CK vs. T36, CK vs. T60 and CK vs. T72, respectively. Gene annotation and cluster analysis of the DEGs revealed a variety of defence responses to Botrytis c. infection, including resistance (R) proteins, MAPK cascade reactions, plant hormone signal transduction pathways, plant-pathogen interaction pathways, Ca2+ and disease resistance-related genes. qPCR verification showed the reliability of the transcriptome data. The PTRV2-RcTGA1-infected plant material showed improved susceptibility of rose to Botrytis c. A total of 635 metabolites were detected in all samples, which could be divided into 29 groups. Metabonomic data showed that a total of 59, 78 and 74 DEMs were obtained for T36, T60 and T72 (T36: Botrytis c. inoculated rose flowers at 36 h; T60: Botrytis c. inoculated rose flowers at 60 h; T72: Botrytis c. inoculated rose flowers at 72 h) compared to CK, respectively. A variety of secondary metabolites are related to biological disease resistance, including tannins, amino acids and derivatives, and alkaloids, among others; they were significantly increased and enriched in phenylpropanoid biosynthesis, glucosinolates and other disease resistance pathways. This study provides a theoretical basis for breeding new cultivars that are resistant to Botrytis c. CONCLUSION: Fifty-four GB of clean reads were generated through RNA-Seq. R proteins, ROS signalling, Ca2+ signalling, MAPK signalling, and SA signalling were activated in the Old Blush response to Botrytis c. RcTGA1 positively regulates rose resistance to Botrytis c. A total of 635 metabolites were detected in all samples. DEMs were enriched in phenylpropanoid biosynthesis, glucosinolates and other disease resistance pathways.

LATE ELONGATED HYPOCOTYL potentiates resistance conferred by CIRCADIAN CLOCK ASSOCIATED1 to aphid by co-regulating the expression of indole glucosinolate biosynthetic genes.

CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are core components of the circadian clock in Arabidopsis thaliana that impacts plant response to biotic stresses. Their clock-regulating functions are believed to be partially redundant, and mutation of either gene leads to shortened periods of the circadian cycle. Our recent study has demonstrated that CCA1 promotes plant resistance to the green peach aphid (Myzus persicae) through modulation of indole glucosinolate biosynthesis, but the role of LHY remains to be elucidated. Here we showed that, similar to cca1-11, single mutant lhy-21 became more susceptible to aphid infestation. Damage to the cca1-11 lhy-21 double mutant by aphids was most pronounced, indicating that the defensive roles of CCA1 and LHY were not entirely redundant. Also, the cyclic expression pattern of key indole glucosinolate biosynthetic genes was considerably disturbed in both single mutants and this was more severe in the double mutant. Apparently, both CCA1 and LHY were necessary for circadian-regulated indole glucosinolate biosynthesis. Taken together, LHY-CCA1 coordination in transcriptional regulation of indole glucosinolate biosynthetic genes most likely contributed to plant defensive capacity against aphids.

Evolution of A bHLH Interaction Motif.

Intrinsically disordered proteins and regions with their associated short linear motifs play key roles in transcriptional regulation. The disordered MYC-interaction motif (MIM) mediates interactions between MYC and MYB transcription factors in Arabidopsis thaliana that are critical for constitutive and induced glucosinolate (GLS) biosynthesis. GLSs comprise a class of plant defense compounds that evolved in the ancestor of the Brassicales order. We used a diverse set of search strategies to discover additional occurrences of the MIM in other proteins and in other organisms and evaluate the findings by means of structural predictions, interaction assays, and biophysical experiments. Our search revealed numerous MIM instances spread throughout the angiosperm lineage. Experiments verify that several of the newly discovered MIM-containing proteins interact with MYC TFs. Only hits found within the same transcription factor family and having similar characteristics could be validated, indicating that structural predictions and sequence similarity are good indicators of whether the presence of a MIM mediates interaction. The experimentally validated MIMs are found in organisms outside the Brassicales order, showing that MIM function is broader than regulating GLS biosynthesis.

Lifetimes of the Aglycone Substrates of Specifier Proteins, the Autonomous Iron Enzymes That Dictate the Products of the Glucosinolate-Myrosinase Defense System in Brassica Plants.

Specifier proteins (SPs) are components of the glucosinolate-myrosinase defense system found in plants of the order Brassicales (brassicas). Glucosinolates (GLSs) comprise at least 150 known S-(ß-d-glucopyranosyl)thiohydroximate-O-sulfonate compounds, each with a distinguishing side chain linked to the central carbon. Following tissue injury, the enzyme myrosinase (MYR) promiscuously hydrolyzes the common thioglycosidic linkage of GLSs to produce unstable aglycone intermediates, which can readily undergo a Lossen-like rearrangement to the corresponding organoisothiocyanates. The known SPs share a common protein architecture but redirect the breakdown of aglycones to different stable products: epithionitrile (ESP), nitrile (NSP), or thiocyanate (TFP). The different effects of these products on brassica consumers motivate efforts to understand the defense response in chemical detail. Experimental analysis of SP mechanisms is challenged by the instability of the aglycones and would be facilitated by knowledge of their lifetimes. We developed a spectrophotometric method that we used to monitor the rearrangement reactions of the MYR-generated aglycones from nine GLSs, discovering that their half-lives (t1/2) vary by a factor of more than 50, from <3 to 150 s (22 °C). The t1/2 of the sinigrin-derived allyl aglycone (34 s), which can form the epithionitrile product (1-cyano-2,3-epithiopropane) in the presence of ESP, proved to be sufficient to enable spatial and temporal separation of the MYR and ESP reactions. The results confirm recent proposals that ESP is an autonomous iron-dependent enzyme that intercepts the unstable aglycone rather than a direct effector of MYR. Knowledge of aglycone lifetimes will enable elucidation of how the various SPs reroute aglycones to different products.

Plant surface metabolites as potent antifungal agents.

Triunsaturated fatty acids are substrates for the synthesis of the defense hormone jasmonate which plays roles in resistance to numerous fungal pathogens. However, relatively little is known about other potential roles of di-unsaturated and triunsaturated fatty acids in resistance to fungal pathogens - in particular those that can attack plants at the seedling stage. We examined the roles of polyunsaturated fatty acids (PUFAs) in Arabidopsis thaliana during attack by the necrotrophic pathogen, Botrytis cinerea. We found that PUFA-deficient Arabidopsis mutants (fad2-1, fad2-3 and fad3-2 fad7-2 fad8 [fad trip]) displayed an unexpectedly strong resistance to B. cinerea at the cotyledon stage. Preliminary analyses revealed no changes in the expression of defense genes, however cuticle permeability defects were detected in both fad2-1 and fad trip mutants. Analysis of B. cinerea development on the surface of cotyledons revealed arrested hyphal growth on fad2-3 and fad trip mutants and 28% reduction in fungal adhesion on fad2-3 cotyledons. Surface metabolite analysis from the cotyledons of PUFA mutants led to the identification of 7-methylsulfonylheptyl glucosinolate (7MSOHG), which over-accumulated on the plant surface. We linked the appearance of 7MSOHG to defects in cuticle composition and permeability of mutants and show that its appearance correlates with resistance to B. cinerea.

Differential expression of major genes involved in the biosynthesis of aliphatic glucosinolates in intergeneric Baemoochae (Brassicaceae) and its parents during development.

KEY MESSAGE: Thus study found the temporal and spatial relationship between production of aliphatic glucosinolate compounds and the expression profile of glucosinolate-related genes during growth and development in radish, Chinese cabbage, and their intergeneric hybrid baemoochae plants. Glucosinolates (GSLs) are one of major bioactive compounds in Brassicaceae plants. GSLs play a role in defense against microbes as well as chemo-preventative activity against cancer, which draw attentions from plant scientists. We investigated the temporal relationship between production of aliphatic Glucosinolate (GSLs) compounds and the expression profile of GSL related genes during growth and development in radish, Chinese cabbage, and their intergeneric hybrid, baemoochae. Over the complete life cycle, Glucoraphasatin (GRH) and glucoraphanin (GRE) predominated in radish, whereas gluconapin (GNP), glucobrassicanapin (GBN), and glucoraphanin (GRA) abounded in Chinese cabbage. Baemoochae contained intermediate levels of all GSLs studied, indicating inheritance from both radish and Chinese cabbage. Expression patterns of BCAT4, CYP79F1, CYP83A1, UGT74B1, GRS1, FMOgs-ox1, and AOP2 genes showed a correlation to their corresponding encoded proteins in radish, Chinese cabbage, and baemoochae. Interestingly, there is a sharp change in gene expression pattern involved in side chain modification, particularly GRS1, FMOgs-ox1, and AOP2, among these plants during the vegetative and reproductive stage. For instance, the GRS1 was strongly expressed during leaf development, while both of FMOgs-ox1 and AOP2 was manifested high in floral tissues. Furthermore, expression of GRS1 gene which is responsible for GRH production was predominantly expressed in leaf tissues of radish and baemoochae, whereas it was only slightly detected in Chinese cabbage root tissue, explaining why radish has an abundance of GRH compared to other Brassica plants. Altogether, our comprehensive and comparative data proved that aliphatic GSLs biosynthesis is dynamically and precisely regulated in a tissue- and development-dependent manner in Brassicaceae family members.