Analysis of Glucosinolate Content and Metabolism Related Genes in Different Parts of Chinese Flowering Cabbage

Glucosinolates (GSLs) are important secondary metabolites that play important defensive roles in cruciferous plants. Chinese flowering cabbage, one of the most common vegetable crops, is rich in GSLs and thus has the potential to reduce the risk of cancer in humans. Many genes that are involved in GSL biosynthesis and metabolism have been identified in the model plant Arabidopsis thaliana; however, few studies investigated the genes related to GSL biosynthesis and metabolism in Chinese flowering cabbage. In the present study, the GSL composition and content in three different organs of Chinese flowering cabbage (leaf, stalk, and flower bud) were determined. Our results showed that the total GSL content in flower buds was significantly higher than in stalks and leaves, and aliphatic GSLs were the most abundant GSL type. To understand the molecular mechanisms underlying the variations of GSL content, we analyzed the expression of genes encoding enzymes involved in GSL biosynthesis and transport in different tissues of Chinese flowering cabbage using RNA sequencing; the expression levels of most genes were found to be consistent with the pattern of total GSL content. Correlation and consistency analysis of differentially expressed genes from different organs with the GSL content revealed that seven genes (Bra029966, Bra012640, Bra016787, Bra011761, Bra006830, Bra011759, and Bra029248) were positively correlated with GSL content. These findings provide a molecular basis for further elucidating GSL biosynthesis and transport in Chinese flowering cabbage.

Glucosinolates with their Hydrolysis Products from Two Cruciferous Plants with Study of Antidiabetic Activity Based on Molecular Docking

Glucosinolates (Gls) are natural bioactive compounds that form metabolites called isothiocyanates (ITC) which have various therapeutic effects. This study aimed to isolate the glucosinolates of Carrichtera annua L.(DC) (CA) and Farsetia aegyptia Turra (FA) belonging to the Crucifereae family. Total Gls were isolated from the aqueous methanolic extract of the plants and further purified using an acidic aluminum oxide column. Some of the obtained Gls were identified via spectroscopic methods (UV, NMR, and MS) and the rest were hydrolyzed by myrosinase to the corresponding isothiocyanates (ITC) for identification by GC/MS. Only one Gls was identified in CA as 4-methylthio-3-butenyl Gls (MTBG) in addition to 6-methyl sulfonylhexyl isothiocyanates (ITC), while 6-methyl sulfonyl-6-hydroxy hexyl ITC, 4-pentenyl ITC, 3-methylthio propyl ITC, 5-hydroxy pentyl ITC and 4-methylsulphinyl butyl ITC were identified in FA. The Gls demonstrated high binding activity to α-glucosidase and amylase, good pharmacokinetic characteristics, and exerted no carcinogenetic effects.

Sulforaphane suppresses autophagy during the malignant progression of gastric carcinoma via activating miR-4521/PIK3R3 pathway

Objective Sulforaphane, which exerts an effective anti-cancer ability, is a phytochemical converted from cruciferous plants. Here, we aimed to identify whether sulforaphane could suppress autophagy during the malignant progression of gastric carcinoma and to explore the underlying mechanisms. Methods SGC7901 cells were transfected with miR-4521 mimics, inhibitor, and pcDNA3.1- PIK3R3, and treated with sulforaphane or autophagy inhibitor. Cell proliferation, apoptosis, and miR-4521 or PIK3R3 expression were detected. Results MiR-4521 over-expression suppressed LC3-II/I ratio and Beclin-1 expression but induced p62 expression in SGC7901 cells. MiR-4521 also reduced gastric carcinoma cell proliferation and promoted apoptosis in vitro. In the mechanical observation, we identified that miR-4521 directly targeted PIK3R3 to repress its expression, and PIK3R3 up-regulation partly antagonized miR-4521-mediated autophagy, proliferation, and apoptosis in gastric carcinoma cells. In addition, sulforaphane exerted effective anti-cancer functions by repressing autophagy and growth in tumor cells at a concentration-dependent way. MiR-4521 inhibition or PIK3R3 over-expression weakened the anti-cancer functions of sulforaphane in gastric carcinoma cells. Conclusion Consequently, miR-4521 suppressed autophagy during the malignant progression of gastric carcinoma by targeting PIK3R3. Thus, miR-4521 may be applied as a therapeutic target for sulforaphane in gastric carcinoma.

From the Soil to the Club in the Roots: Clubroot

Among the millions of microorganisms inhabiting the soils, some can be plant pathogens, meaning they can become a disease to plants. Some diseases are more well-known than others. This is the case of clubroot, a very atypical microorganism that infects cruciferous plants, such as cabbage, kale, canola, and the common research plant thale cress. In this article, I will tell you more about clubroot and clubroot disease because there is still a lot to discover about the pathogen and the disease. Maybe you will be part of our lab in the future and investigate a fascinating soil-borne pathogen.

The glutathione S-transferase (PxGST2L) may contribute to the detoxification metabolism of chlorantraniliprole in Plutella xylostella(L.)

The diamondback moth (Plutella xylostella L.), is an economic pest of cruciferous plants worldwide, which causes great economic loss to cruciferous plants production. However, the pest has developed resistance to insecticides. One of such insecticides is chlorantraniliprole. The study of the mechanisms underlying resistance is key for the effective management of resistance. In this study, a comparative proteomics approach was used to isolate and identify various proteins that differed between chlorantraniliprole-susceptible and -resistant strains of P. xylostella. Eleven proteins were significantly different and were successfully identified by MALDI-TOF-MS. Metabolism-related proteins accounted for the highest proportion among the eleven different proteins. The function of the PxGST2L protein was validated by RNAi. Knockdown of PxGST2L reduced the GST activity and increased the toxicity of chlorantraniliprole to the diamondback moth. The resistance ratio of diamondback moth to chlorantraniliprole was reduced from 1029 to 505. The results indicated that PxGST2L is partly responsible for chlorantraniliprole insecticide resistance in DBM. Our finding contributes to the understanding of the mechanism underlying resistance to chlorantraniliprole in the DBM, to develop effective resistance management tactics.

Identification of a Sulfatase that Detoxifies Glucosinolates in the Phloem-Feeding Insect Bemisia tabaci and Prefers Indolic Glucosinolates

Cruciferous plants in the order Brassicales defend themselves from herbivory using glucosinolates: sulfur-containing pro-toxic metabolites that are activated by hydrolysis to form compounds, such as isothiocyanates, which are toxic to insects and other organisms. Some herbivores are known to circumvent glucosinolate activation with glucosinolate sulfatases (GSSs), enzymes that convert glucosinolates into inactive desulfoglucosinolates. This strategy is a major glucosinolate detoxification pathway in a phloem-feeding insect, the silverleaf whitefly Bemisia tabaci, a serious agricultural pest of cruciferous vegetables. In this study, we identified and characterized an enzyme responsible for glucosinolate desulfation in the globally distributed B. tabaci species MEAM1. In in vitro assays, this sulfatase showed a clear preference for indolic glucosinolates compared with aliphatic glucosinolates, consistent with the greater representation of desulfated indolic glucosinolates in honeydew. B. tabaci might use this detoxification strategy specifically against indolic glucosinolates since plants may preferentially deploy indolic glucosinolates against phloem-feeding insects. In vivo silencing of the expression of the B. tabaci GSS gene via RNA interference led to lower levels of desulfoglucosinolates in honeydew. Our findings expand the knowledge on the biochemistry of glucosinolate detoxification in phloem-feeding insects and suggest how detoxification pathways might facilitate plant colonization in a generalist herbivore.

Characterization of Xanthomonas campestris pv. campestris in Algeria

Xanthomonas campestris pv. campestris (Xcc) causes the black rot of cruciferous plants. This seed-borne bacterium is considered as the most destructive disease to cruciferous crops. Although sources of contamination are various, seeds are the main source of transmission. Typical symptoms of black rot were first observed in 2011 on cabbage and cauliflower fields in the main production areas of Algeria. Leaf samples displaying typical symptoms were collected during 2011 to 2014, and 170 strains were isolated from 45 commercial fields. Xcc isolates were very homogeneous in morphological, physiological and biochemical characteristics similar to reference strains, and gave positive pathogenicity and molecular test results (multiplex PCR with specific primers). This is the first record of Xcc in Algeria. Genetic diversity within the isolates was assessed in comparison with strains isolated elsewhere. A multilocus sequence analysis based on two housekeeping genes (gyrB and rpoD) was carried out on 77 strains representative isolates. The isolates grouped into 20 haplotypes defined with 68 polymorphic sites. The phylogenetic tree obtained showed that Xcc is in two groups, and all Algerian strains clustered in group 1 in three subgroups. No relationships were detected between haplotypes and the origins of the seed lots, the varieties of host cabbage, the years of isolation and agroclimatic regions.

iTRAQ-based quantitative proteomics reveals ChAcb1 as a novel virulence factor in Colletotrichum higginsianum

Colletotrichum higginsianum is an important hemibiotrophic fungal pathogen that causes anthracnose disease on various cruciferous plants. Discovery of new virulence factors could lead to strategies for effectively controlling anthracnose. Acyl-CoA binding proteins (ACBPs) are mainly involved in binding and trafficking acyl-CoA esters in eukaryotic cells. However, the functions of this important class of proteins in plant fungal pathogens remain unclear. In this study, we performed an iTRAQ-based quantitative proteomic analysis to identify differentially expressed proteins (DEPs) between a nonpathogenic mutant ΔCh-MEL1 and the wild-type. Based on iTRAQ data, DEPs in the ΔCh-MEL1 mutant were mainly associated with melanin biosynthesis, carbohydrate and energy metabolism, lipid metabolism, redox processes, and amino acid metabolism. Proteomic analysis revealed that many DEPs might be involved in growth and pathogenesis of C. higginsianum. Among them, an acyl-CoA binding protein, ChAcb1, was selected for further functional studies. Deletion of ChAcb1 caused defects in vegetative growth and conidiation. ChAcb1 is also required for response to hyperosmotic and oxidative stresses, and maintenance of cell wall integrity. Importantly, the ΔChAcb1 mutant exhibited reduced virulence, and microscopic examination revealed that it was defective in appressorial penetration and infectious growth. Furthermore, the ΔChAcb1 mutant was impaired in fatty acid and lipid metabolism. Taken together, ChAcb1 was identified as a new virulence gene in this plant pathogenic fungus.

Extracellular amylase is required for full virulence and regulated by the global post-transcriptional regulator RsmA in Xanthomonas campestris pathovar campestris

As with many phytopathogenic bacteria, the virulence of Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot disease in cruciferous plants, relies on secretion of a suite of extracellular enzymes that includes cellulase (endoglucanase), pectinase, protease and amylase. Although the role in virulence of a number of these enzymes has been assessed, the contribution of amylase to Xcc virulence has yet to be established. In this work, we investigated both the role of extracellular amylase in Xcc virulence and the control of its expression. Deletion of XC3487 (here renamed amyAXcc), a putative amylase-encoding gene from the genome of Xcc strain 8004, resulted in a complete loss of extracellular amylase activity and significant reduction in virulence. The extracellular amylase activity and virulence of the amyAXcc mutant could be restored to the wild-type level by expressing amyAXcc in trans. These results demonstrated that amyAXcc is responsible for the extracellular amylase activity of Xcc, and indicated that extracellular amylase plays an important role in Xcc virulence. We further found that the expression of amyAXcc is strongly induced by starch and requires activation by the global post-transcriptional regulator RsmA. RsmA binds specifically to the 5’-untranslated region (5’UTR) of amyAXcc transcripts, suggesting that RsmA regulates amyAXcc directly at the post-transcriptional level. Unexpectedly, in addition to post-transcriptional regulation, the use of a transcriptional reporter demonstrated that RsmA also regulates amyAXcc expression at the transcriptional level, possibly by an indirect mechanism.

Oximes and nitric oxide signalling in Medicago truncatula root system architecture

Nitric oxide (NO) is a widely recognized signalling molecule in plants. It affects almost every developmental step during the whole plant’s lifespan. Among all of its already described functions, NO is recognised to act synergistically with Indole-3-acetic acid (IAA), promoting the development of the secondary roots. Until now, only a few reductive NO synthesis pathways have been confirmed, whereas no oxidative pathway has been yet described. Experiments of our research group measured de novo synthesis of NO3- and NO2- in Pisum sativum and M. truncatula grown with NH4+ as the sole N source (unpublished data). This fact suggests the existence of an oxidative pathway for NH4+ in the Fabaceae family, which is proposed to be part of the signalling of the NH4+ toxicity and to participate in the alleviation mechanism. Due to their molecular configuration, oximes are very strong candidates for being the precursors of NO, and thus the first step into this nitrogen oxidation pathway. Among these oximes, Indole-3-acetaldoxime (IAOx) is particularly relevant since it is placed in the crossroad between IAA and indole glucosinolates. The role of IAOx in growth-signalling and root phenotype is poorly studied in cruciferous plants and mostly unknown in non-cruciferous plants. In this PhD thesis, we aim to demonstrate that IAOx is present in M. truncatula playing an important role of signalling during plant root development and also that this signalling is mediated by NO. For that purpose, we synthesized a set of pure IAOx and other indolic and non-indolic oximes and performed pharmacological approaches with the model legume M. truncatula. We analysed the root phenotype, quantified the indolic compounds in tissue (shoots and roots) and measured the Indole-3- acetaldehyde oxidase and IAOx dehydratase genes expression. Our data showed that all the oximes promoted the ‘superoot’ phenotype. All this matches with the hypothesis that IAOx exerts its signalling by liberating NO. This new knowledge is a step forward towards the discovery of an oxidative NO synthesis pathway in plants and throws light into the interplay between IAOx, IAA and nitrogen nutrition, which will be paramount for further field research in crop production.