Mutation in BrFLS encoding flavonol synthase induced anthocyanin accumulation in Chinese cabbage.

KEY MESSAGE: BrFLS mutation promoted anthocyanin accumulation in Chinese cabbage, which was verified in four allelic mutants. Chinese cabbage is a major vegetable crop in Eastern Asia. Anthocyanin-rich vibrantly colored varieties are increasingly favored by consumers for their higher nutritional and aesthetic value compared to the typical green varieties of Chinese cabbage. Herein, we identified an anthocyanin accumulation mutant aam1 from a mutant library of EMS-mutagenized Chinese cabbage DH line 'FT', which appeared partial purple on leaves, bolting stems and floral buds. This anthocyanin accumulation trait was genetically controlled by a recessive nuclear gene, and through MutMap mapping and KASP genotyping, BraA10g030950.3C was identified as the candidate causal gene with a G202 to A202 non-synonymous SNP variation in exon 1. Three additional mutants allelic to aam1 were obtained via screening of similar-phenotype mutants from the mutant library, namely aam2/3/4, where the causal SNPs reside in the same gene as aam1, corroborating that the mutation of BraA10g030950.3C caused anthocyanin accumulation. BraA10g030950.3C encodes a flavonol synthase that catalyzes dihydroflavonols substrate into flavonols and is homologous to Arabidopsis FLS1 (AT5G08640), named BrFLS. Compared to wildtype, the expression level of BrFLS was significantly reduced in the mutants, while BrDFR, which is involved in the anthocyanin biosynthesis and competes with FLS for the common substrate dihydroflavonols, was increased. The flavonol synthase activity decreased, and dihydroflavonol 4-reductase activity was elevated. Differentially accumulated flavonoid metabolites were detected between wildtype and aam1, which were enriched primarily in flavonol and anthocyanin pathways. Our results revealed that mutations in the BrFLS gene could contribute to anthocyanin accumulation and provide a new target for Chinese cabbage color modification.

The dihydroflavonol 4-reductase BoDFR1 drives anthocyanin accumulation in pink-leaved ornamental kale.

KEY MESSAGE: Overexpression and virus-induced gene silencing verified BoDFR1 conferred the anthocyanin accumulation in pink-leaved ornamental kale. Leaf color is an essential trait in the important horticultural biennial plant ornamental kale (Brassica oleracea var. acephala). The identity of the gene conferring this striking trait and its mode of inheritance are topics of debate. Based on an analysis of F1, F2, BC1P1, and BC1P2 ornamental kale populations derived from a cross between a pink-leaved P28 and white-leaved D10 line, we determined that the pink leaf trait is controlled by a semi-dominant gene. We cloned two genes potentially involved in anthocyanin biosynthesis in ornamental kale: Bo9g058630 and Bo6g100940. Based on their variation in sequence, we speculated that Bo9g058630, encoding the kale dihydroflavonol-4 reductase (BoDFR1) enzyme, plays a critical role in the development of the pink leaf trait. Indeed, an InDel marker specific for BoDFR1 completely co-segregated with the pink leaf trait in our F2 population. We then generated the 35Spro: DFR-GUS overexpression vector, which we transformed into D10. Overexpression of BoDFR1 indeed restored some anthocyanin accumulation in this white-leaved parental line. In addition, we targeted BoDFR1 in P28 using virus-induced gene silencing. Again, silencing of BoDFR1 resulted in a substantial decrease in anthocyanin accumulation. This work lays the foundation for further exploration of the mechanism underlying anthocyanin accumulation in pink-leaved ornamental kale.

Induction and Priming of Plant Defense by Root-Associated Insect-Pathogenic Fungi.

Plants evolved in close contact with a myriad of microorganisms, some of which formed associations with their roots, benefitting from carbohydrates and other plant resources. In exchange, they evolved to influence important plant functions, e.g. defense against insect herbivores and other antagonists. Here, we test whether a fungus, Metarhizium brunneum, which is mostly known as an insect pathogen, can also associate with plant roots and contribute to above-ground plant defense. Cauliflower (Brassica oleracea var. botrytis) seeds were sown together with M. brunneum-inoculated rice grains, and the resulting plants subjected to leaf herbivory by the specialist Plutella xylostella. Activity of myrosinases, the enzymes activating glucosinolates, was measured before and after herbivory; larval consumption and plant weight at the end of experiments. Metarhizium brunneum clearly established in the plant roots, and after herbivory myrosinase activity was substantially higher in M. brunneum-treated plants than in controls; before herbivory, M. brunneum-treated and control plants did not differ. Leaf consumption was slightly lower in the M. brunneum-treated plants whereas total biomass and allocation to above- or below-ground parts was not affected by the Metarhizium treatment. Thus, M. brunneum associates with roots and primes the plant for a stronger or faster increase in myrosinase activity upon herbivory. Consistent with this, myrosinase function has been suggested to be rate-limiting for induction of the glucosinolate-myrosinase defense system. Our results show that M. brunneum, in addition to being an insect pathogen, can associate with plant roots and prime plant defense.

Expression of BoNOL and BoHCAR genes during postharvest senescence of broccoli heads.

BACKGROUND: Chlorophyll is the most abundant pigment on Earth, essential for the capture of light energy during photosynthesis. During senescence, chlorophyll degradation is highly regulated in order to diminish toxicity of the free chlorophyll molecule due to its photoactivity. The first step in the chlorophyll degradation pathway is the conversion of chlorophyll b to chlorophyll a by means of two consecutive reactions catalyzed by enzymes coded by NYC1 (NON-YELLOW COLORING 1), NOL (NYC1-LIKE) and HCAR. RESULTS: In this work, we studied the expression of NOL and HCAR genes during postharvest senescence of broccoli. We found that the expression of BoNOL increase during the first days of storage and then decrease. In the case of BoHCAR, its expression is maintained during the first days and then it also diminishes. Additionally, the effect of different postharvest treatments on the expression of these genes was also analyzed. It was observed that the expression of BoNOL is lower in the treatments performed with 1-methylcyclopropene (1-MCP), 6-benzylaminopurine (6-BAP) and modified atmospheres, while BoHCAR expression showed an increase in these same treatments, and a decrease in the treatment with ethylene. There were no variations in the expression of both genes in heat treatment, UV-C treatment and visible light treatment. CONCLUSIONS: These results suggest that both BoHCAR and BoNOL show a lower regulation of their expression than other genes involved in chlorophyll degradation during senescence. © 2020 Society of Chemical Industry.

Overexpressing broccoli tryptophan biosynthetic genes BoTSB1 and BoTSB2 promotes biosynthesis of IAA and indole glucosinolates.

Tryptophan is one of the amino acids that cannot be produced in humans and has to be acquired primarily from plants. In Arabidopsis thaliana (Arabidopsis), the tryptophan synthase beta subunit (TSB) genes have been found to catalyze the biosynthesis of tryptophan. Here, we report the isolation and characterization of two TSB genes from Brassica oleracea (broccoli), designated BoTSB1 and BoTSB2. Overexpressing BoTSB1 or BoTSB2 in Arabidopsis resulted in higher tryptophan content and the accumulation of indole-3-acetic acid (IAA) and indole glucosinolates in rosette leaves. Therefore, the transgenic plants showed a series of high auxin phenotypes, including long hypocotyls, large plants and a high number of lateral roots. The spatial expression of BoTSB1 and BoTSB2 was detected by quantitative real-time PCR in broccoli and by expressing the ß-glucuronidase reporter gene (GUS) controlled by the promoters of the two genes in Arabidopsis. BoTSB1 was abundantly expressed in vascular tissue of shoots and inflorescences. Compared to BoTSB1, BoTSB2 was expressed at a very low level in shoots but at a higher level in roots. We further investigated the expression response of the two genes to several hormone and stress treatments. Both genes were induced by methyl jasmonate (MeJA), salicylic acid (SA), gibberellic acid (GA), Flg22 (a conserved 22-amino acid peptide derived from bacterial flagellin), wounding, low temperature and NaCl and were repressed by IAA. Our study enhances the understanding of tryptophan biosynthesis and its regulation in broccoli and Arabidopsis. In addition, we provide evidence that TSB genes can potentially be a good tool to breed plants with high biomass and high nutrition.

Patterns of Expansion and Expression Divergence of the Polygalacturonase Gene Family in Brassica oleracea.

Plant polygalacturonases (PGs) are closely related to cell-separation events during plant growth and development by degrading pectin. Identifying and investigating their diversification of evolution and expression could shed light on research on their function. We conducted sequence, molecular evolution, and gene expression analyses of PG genes in Brassica oleracea. Ninety-nine B. oleracea PGs (BoPGs) were identified and divided into seven clades through phylogenetic analysis. The exon/intron structures and motifs were conserved within, but divergent between, clades. The second conserved domain (GDDC) may be more closely related to the identification of PGs. There were at least 79 common ancestor PGs between Arabidopsis thaliana and B. oleracea. The event of whole genome triplication and tandem duplication played important roles in the rapid expansion of the BoPG gene family, and gene loss may be an important mechanism in the generation of the diversity of BoPGs. By evaluating the expression in five tissues, we found that most of the expressed BoPGs in clades A, B, and E showed ubiquitous expression characteristics, and the expressed BoPGs in clades C, D, and F were mainly responsible for reproduction development. Most of the paralogous gene pairs (76.2%) exhibited divergent expression patterns, indicating that they may have experienced neofunctionalization or subfunctionalization. The cis-elements analysis showed that up to 96 BoPGs contained the hormone response elements in their promoters. In conclusion, our comparative analysis may provide a valuable data foundation for the further functional analysis of BoPGs during the development of B. oleracea.

Pol III-Dependent Cabbage BoNR8 Long ncRNA Affects Seed Germination and Growth in Arabidopsis.

Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nt that are distributed widely in organisms and play many physiological roles. The BoNR8 lncRNA is a 272 nt long transcript yielded by RNA polymerase III in cabbage that was identified as the closest homolog of the AtR8 lncRNA in Arabidopsis. The BoNR8 lncRNA was expressed extensively in the epidermal tissue in the root elongation zone of germinated seeds, and its accumulation was induced by abiotic stresses, auxins and ABA. To investigate the correlation between the BoNR8 lncRNA and germination, BoNR8-overexpressing Arabidopsis plants (BoNR8-AtOX) were prepared. Three independent BoNR8-AtOX lines showed less primary root elongation, incomplete silique development and decreased germination rates. The germination efficiencies were affected strongly by ABA and slightly by salt stress, and ABA-related gene expression was changed in the BoNR8-AtOX lines.

Selenium spiked in soil promoted zinc accumulation of Chinese cabbage and improved its antioxidant system and lipid peroxidation.

Selenium (Se) and zinc (Zn) are necessary mineral nutrients for human body but millions of people have an inadequate intake of them, and eat food enriched with Se and Zn may minimize these problems. Chinese cabbage is an important food in people's daily life. The aim of this study was to evaluate the effects of single Se, Zn and their combination treatment in soil on their accumulation, antioxidant system and lipid peroxidation in roots and leaves of Chinese cabbage using soil pot culture experiment. When 0.5 mg kg-1 Se +30 mg kg-1 Zn and 1.0 mg kg-1 Se +30 mg kg-1 Zn were spiked in soils, Zn concentrations in roots and leaves of Chinese cabbage were significantly increased (p < 0.05) by 20.2%, 37.8% and 17.9%, 34.1% respectively compared to the treatment of 30 mg kg-1 Zn added, and the latter was significantly higher (p < 0.05) than that of former, indicating Se significantly promoted Zn accumulation. Almost all physiological indexes including POD, SOD, CAT, APX, GR, Chlorophyll a, Chlorophyll b, Carotenoids, MDA and Free proline in the treatments of Se or Zn spiked were significantly improved (p < 0.05) or basically unaffected compared to the control without Se or Zn added. The biomass change trends were similar with these indexes either. These results showed that the addition in soil of Se and Zn significantly increased their accumulation in Chinese cabbage without affected its formal growth. Particularly, the addition of Se promoted Zn accumulation. The conclusions were more important reference for the production practice of cash crop enriched of Se and Zn either.

Mutation of ACX1, a Jasmonic Acid Biosynthetic Enzyme, Leads to Petal Degeneration in Chinese Cabbage (Brassica campestris ssp. pekinensis).

Petal color, size, and morphology play important roles in protecting other floral organs, attracting pollinators, and facilitating sexual reproduction in plants. In a previous study, we obtained a petal degeneration mutant (pdm) from the 'FT' doubled haploid line of Chinese cabbage and found that the candidate gene for pdm, Bra040093, encodes the enzyme acyl-CoA oxidase1. In this study, we sought to examine the gene networks regulating petal development in pdm plants. We show that the mRNA and protein expression of Bra040093, which is involved in the jasmonic acid (JA) biosynthetic pathway, were significantly lower in the petals of pdm plants than in those of 'FT' plants. Similarly, the JA and methyl jasmonate (MeJA) contents of petals were significantly lower in pdm plants than in 'FT' plants and we found that exogenous application of these hormones to the inflorescences of pdm plants restored the 'FT' phenotype. Comparative analyses of the transcriptomes of 'FT', pdm and pdm + JA (pJA) plants revealed 10,160 differentially expressed genes (DEGs) with consistent expression tendencies in 'FT' vs. pdm and pJA vs. pdm comparisons. Among these DEGs, we identified 69 DEGs related to floral organ development, 11 of which are involved in petal development regulated by JA. On the basis of qRT-PCR verification, we propose regulatory pathways whereby JA may mediate petal development in the pdm mutant. We demonstrate that mutation of Bra040093 in pdm plants leads to reduced JA levels and that this in turn promotes changes in the expression of genes that are expressed in response to JA, ultimately resulting in petal degeneration. These findings thus indicate that JA is associated with petal development in Chinese cabbage. These results enhance our knowledge on the molecular mechanisms underlying petal development and lay the foundations for further elucidation of the mechanisms associated with floral organ development in Chinese cabbage.

Manipulation of glyoxalase pathway confers tolerance to multiple stresses in rice.

Crop plants face a multitude of diverse abiotic and biotic stresses in the farmers' fields. Although there now exists a considerable knowledge of the underlying mechanisms of response to individual stresses, the crosstalk between response pathways to various abiotic and biotic stresses remains enigmatic. Here, we investigated if the cytotoxic metabolite methylglyoxal (MG), excess of which is generated as a common consequence of many abiotic and biotic stresses, may serve as a key molecule linking responses to diverse stresses. For this, we generated transgenic rice plants overexpressing the entire two-step glyoxalase pathway for MG detoxification. Through assessment of various morphological, physiological and agronomic parameters, we found that glyoxalase-overexpression imparts tolerance towards abiotic stresses like salinity, drought and heat and also provides resistance towards damage caused by the sheath blight fungus (Rhizoctonia solani) toxin phenylacetic acid. We show that the mechanism of observed tolerance of the glyoxalase-overexpressing plants towards these diverse abiotic and biotic stresses involves improved MG detoxification and reduced oxidative damage leading to better protection of chloroplast and mitochondrial ultrastructure and maintained photosynthetic efficiency under stress conditions. Together, our findings indicate that MG may serve as a key link between abiotic and biotic stress response in plants.

Comparison of foliar silicon and selenium on cadmium absorption, compartmentation, translocation and the antioxidant system in Chinese flowering cabbage.

Silicon (Si) and selenium (Se) are beneficial for many higher plants when grown on stress conditions. However, the mechanisms underlying the differential effects between foliar Si and Se in alleviation of plant toxicity exposed to cadmium (Cd) stress are remained unclear. In this study, we investigated the discrepant mechanisms of foliar Si and Se on Cd absorption and compartmentation by roots, its translocation in xylem, and the antioxidant system within Chinese flowering cabbage (Brassica campestris L. ssp. chinensis var. utilis) under low and high Cd stress. Results showed that plant growth was significantly enhanced by foliar additions of Si or/and Se according to an increased plant tissue biomass at high Cd exposure. In addition, the foliar coupled addition of Si and Se showed little effects on the concentrations of Si or Se in plant tissues in comparison with the single addition of foliar Si or Se respectively. The foliar Si alone or combined with Se markedly reduced the Cd concentrations in plant shoots under two Cd treatments. This might be explained by the lower Cd concentrations in symplast and apoplast and the higher Cd concentrations in cell walls of plant roots, and the lower Cd concentrations in xylem sap. However, no great changes in these values were observed under the treatments of foliar Se alone. Moreover, the foliar additions of Si or/and Se all increased the antioxidant enzyme activities of SOD, CAT and APX in plant tissues, especially at high Cd dosage. No significant differences in the increasing degrees of these three antioxidant enzymes were found between the foliar Si and Se treatments. However, only the foliar Se alone or combined with Si markedly promoted the antioxidant enzyme activities of GR and DHAR in plant tissues. Our findings demonstrate that the alleviation of Cd toxicity by foliar Si maybe mainly responsible for inhibition of Cd absorption and its translocation to plant shoots, reinforcing its compartmentation into root cell walls, whilst enhancing the antioxidant enzyme system may be employed by foliar Se.

Genome-Wide Identification and Characterization of Pectin Methylesterase Inhibitor Genes in Brassica oleracea.

The activities of pectin methylesterases (PMEs) are regulated by pectin methylesterase inhibitors (PMEIs), which consequently control the pectin methylesterification status. However, the role of PMEI genes in Brassica oleracea, an economically important vegetable crop, is poorly understood. In this study, 95 B. oleracea PMEI (BoPMEI) genes were identified. A total of 77 syntenic ortholog pairs and 10 tandemly duplicated clusters were detected, suggesting that the expansion of BoPMEI genes was mainly attributed to whole-genome triplication (WGT) and tandem duplication (TD). During diploidization after WGT, BoPMEI genes were preferentially retained in accordance with the gene balance hypothesis. Most homologous gene pairs experienced purifying selection with ω (Ka/Ks) ratios lower than 1 in evolution. Five stamen-specific BoPMEI genes were identified by expression pattern analysis. By combining the analyses of expression and evolution, we speculated that nonfunctionalization, subfunctionalization, neofunctionalization, and functional conservation can occur in the long evolutionary process. This work provides insights into the characterization of PMEI genes in B. oleracea and contributes to the further functional studies of BoPMEI genes.

Genome-Wide Identification, Molecular Evolution, and Expression Profiling Analysis of Pectin Methylesterase Inhibitor Genes in Brassica campestris ssp. chinensis.

Pectin methylesterase inhibitor genes (PMEIs) are a large multigene family and play crucial roles in cell wall modifications in plant growth and development. Here, a comprehensive analysis of the PMEI gene family in Brassicacampestris, an important leaf vegetable, was performed. We identified 100 BrassicacampestrisPMEI genes (BcPMEIs), among which 96 BcPMEIs were unevenly distributed on 10 chromosomes and nine tandem arrays containing 20 BcPMEIs were found. We also detected 80 pairs of syntenic PMEI orthologs. These findings indicated that whole-genome triplication (WGT) and tandem duplication (TD) were the main mechanisms accounting for the current number of BcPMEIs. In evolution, BcPMEIs were retained preferentially and biasedly, consistent with the gene balance hypothesis and two-step theory, respectively. The molecular evolution analysis of BcPMEIs manifested that they evolved through purifying selection and the divergence time is in accordance with the WGT data of B. campestris. To obtain the functional information of BcPMEIs, the expression patterns in five tissues and the cis-elements distributed in promoter regions were investigated. This work can provide a better understanding of the molecular evolution and biological function of PMEIs in B. campestris.

A Simple Method for On-Gel Detection of Myrosinase Activity.

Myrosinase is an enzyme present in many functional foods and spices, particularly in Cruciferous vegetables. It hydrolyses glucosinolates which thereafter rearrange into bioactive volatile constituents (isothiocyanates, nitriles). We aimed to develop a simple reversible method for on-gel detection of myrosinase. Reagent composition and application parameters for native PAGE and SDS-PAGE gels were optimized. The proposed method was successfully applied to detect myrosinase (or sulfatase) on-gel: the detection solution contains methyl red which gives intensive red bands where the HSO4- is enzymatically released from the glucosinolates. Subsequently, myrosinase was successfully distinguished from sulfatase by incubating gel bands in a derivatization solution and examination by LC-ESI-MS: myrosinase produced allyl isothiocyanate (detected in conjugate form) while desulfo-sinigrin was released by sulfatase, as expected. After separation of 80 µg protein of crude extracts of Cruciferous vegetables, intensive color develops within 10 min. On-gel detection was found to be linear between 0.031⁻0.25 U (pure Sinapis alba myrosinase, R² = 0.997). The method was successfully applied to detection of myrosinase isoenzymes from horseradish, Cruciferous vegetables and endophytic fungi of horseradish as well. The method was shown to be very simple, rapid and efficient. It enables detection and partial characterization of glucosinolate decomposing enzymes without protein purification.

Biochemical Analysis of the Role of Leucine-Rich Repeat Receptor-Like Kinases and the Carboxy-Terminus of Receptor Kinases in Regulating Kinase Activity in Arabidopsis thaliana and Brassica oleracea.

Protein post-translational modification by phosphorylation is essential for the activity and stability of proteins in higher plants and underlies their responses to diverse stimuli. There are more than 300 leucine-rich repeat receptor-like kinases (LRR-RLKs), a major group of receptor-like kinases (RLKs) that plays an important role in growth, development, and biotic stress responses in higher plants. To analyze auto- and transphosphorylation patterns and kinase activities in vitro, 43 full-length complementary DNA (cDNA) sequences were cloned from genes encoding LRR-RLKs. Autophosphorylation activity was found in the cytoplasmic domains (CDs) of 18 LRR-RLKs; 13 of these LRR-RLKs with autophosphorylation activity showed transphosphorylation in Escherichiacoli. BRI1-Associated Receptor Kinase (BAK1), which is critically involved in the brassinosteroid and plant innate immunity signal transduction pathways, showed strong auto- and transphosphorylation with multi-specific kinase activity within 2 h of induction of Brassica oleraceae BAK1-CD (BoBAK1-CD) in E. coli; moreover, the carboxy-terminus of LRR-RLKs regulated phosphorylation and kinase activity in Arabidopsis thaliana and vegetative crops.

Evolutionary history and functional divergence of the cytochrome P450 gene superfamily between Arabidopsis thaliana and Brassica species uncover effects of whole genome and tandem duplications.

BACKGROUND: The cytochrome P450 monooxygenase (P450) superfamily is involved in the biosynthesis of various primary and secondary metabolites. However, little is known about the effects of whole genome duplication (WGD) and tandem duplication (TD) events on the evolutionary history and functional divergence of P450s in Brassica after splitting from a common ancestor with Arabidopsis thaliana. RESULTS: Using Hidden Markov Model search and manual curation, we detected that Brassica species have nearly 1.4-fold as many P450 members as A. thaliana. Most P450s in A. thaliana and Brassica species were located on pseudo-chromosomes. The inferred phylogeny indicated that all P450s were clustered into two different subgroups. Analysis of WGD event revealed that different P450 gene families had appeared after evolutionary events of species. For the TD event analyses, the P450s from TD events in Brassica species can be divided into ancient and recent parts. Our comparison of influence of WGD and TD events on the P450 gene superfamily between A. thaliana and Brassica species indicated that the family-specific evolution in the Brassica lineage can be attributed to both WGD and TD, whereas WGD was recognized as the major mechanism for the recent evolution of the P450 super gene family. Expression analysis of P450s from A. thaliana and Brassica species indicated that WGD-type P450s showed the same expression pattern but completely different expression with TD-type P450s across different tissues in Brassica species. Selection force analysis suggested that P450 orthologous gene pairs between A. thaliana and Brassica species underwent negative selection, but no significant differences were found between P450 orthologous gene pairs in A. thaliana-B. rapa and A. thaliana-B. oleracea lineages, as well as in different subgenomes in B. rapa or B. oleracea compared with A. thaliana. CONCLUSIONS: This study is the first to investigate the effects of WGD and TD on the evolutionary history and functional divergence of P450 gene families in A. thaliana and Brassica species. This study provides a biology model to study the mechanism of gene family formation, particularly in the context of the evolutionary history of angiosperms, and offers novel insights for the study of angiosperm genomes.

Regulation of FATTY ACID ELONGATION1 expression and production in Brassica oleracea and Capsella rubella.

MAIN CONCLUSION: The contribution of variations in coding regions or promoters to the changes in FAE1 expression levels have be quantified and compared in parallel by specifically designed swapping constructs. FATTY ACID ELONGATION1 (FAE1) is a key gene in control of erucic acid synthesis in plant seeds. The expression of FAE1 genes in Brassica oleracea and Capsella rubella, representatives of high and low erucic acid species, respectively, was characterized to provide insight into the regulation of very long-chain fatty-acid biosynthesis in seeds. Virtually, no methylation was detected either in B. oleracea or in C. rubella, suggesting that modification of promoter methylation might not be a predominant mechanism. Swapping constructs were specifically designed to quantify and compare the contribution of variations in coding regions or promoters to the changes in FAE1 expression levels in parallel. A significantly higher fold change in erucic acid content was observed when swapping coding regions rather than when swapping promoters, indicating that the coding region is a major determinant of the catalytic power of ß-ketoacyl-CoA synthase proteins. Common motifs have been proposed as essential for the preservation of basic gene expression patterns, such as seed-specific expression. However, the occurrence of variation in common cis-elements or the presence of species-specific cis-elements might be plausible mechanisms for changes in the expression levels in different organisms. In addition, conflicting observations in previous reports associated with FAE1 expression are discussed, and we suggest that caution should be taken when selecting a plant transformation vector and in interpreting the results obtained from vectors carrying the CaMV 35S promoter.

Characterization of S-nitrosoglutathione reductase from Brassica and Lactuca spp. and its modulation during plant development.

Cellular homeostasis of S-nitrosoglutathione (GSNO), a major cache of nitric oxide bioactivity in plants, is controlled by the NADH-dependent S-nitrosoglutathione reductase (GSNOR) belonging to the family of class III alcohol dehydrogenases (EC 1.1.1.1). GSNOR is a key regulator of S-nitrosothiol metabolism and is involved in plant responses to abiotic and biotic stresses. This study was focused on GSNOR from two important crop plants, cauliflower (Brassica oleracea var. botrytis, BoGSNOR) and lettuce (Lactuca sativa, LsGSNOR). Both purified recombinant GSNORs were characterized in vitro and found to exists as dimers, exhibit high thermal stability and substrate preference towards GSNO, although both enzymes have dehydrogenase activity with a broad range of long-chain alcohols and ω-hydroxy fatty acids in presence of NAD+. Data on enzyme affinities to their cofactors NADH and NAD+ obtained by isothermal titration calorimetry suggest the high affinity to NADH might underline the GSNOR capacity to function in the intracellular environment. GSNOR activity and gene expression peak during early developmental stages of lettuce and cauliflower at 20 and 30 days after germination, respectively. GSNOR activity was also measured in four other Lactuca spp. genotypes with different degree of resistance to biotrophic pathogen Bremia lactucae. Higher GSNOR activities were found in non-infected plants of susceptible genotypes L. sativa UCDM2 and L. serriola as compared to resistant genotypes. GSNOR and GSNO were localized by confocal laser scanning microscopy in vascular bundles and in epidermal and parenchymal cells of leaf cross-sections. The presented results bring new insight in the role of GSNOR in the regulation of S-nitrosothiol levels in plant growth and development.

Characterization of BcMF23a and BcMF23b, two putative pectin methylesterase genes related to pollen development in Brassica campestris ssp. chinensis.

Two homologous genes, Brassica campestris Male Fertility 23a (BcMF23a) and Brassica campestris Male Fertility 23b (BcMF23b), encoding putative pectin methylesterases (PMEs) were isolated from Brassica campestris ssp. chinensis (syn. Brassica rapa ssp. chinensis). These two genes sharing high sequence identity with each other were highly expressed in the fertile flower buds but silenced in the sterile ones of genic male sterile line system ('Bcajh97-01A/B'). Results of RT-PCR and in situ hybridization suggested that BcMF23a and BcMF23b were pollen-expressed genes, whose transcripts were first detected at the binucleate pollen and maintained throughout to the mature pollen grains. Western blot indicated that both of the putative BcMF23a and BcMF23b proteins are approximately 40 kDa, which exhibited extracellular localization revealed by transient expression analysis in the onion epidermal cells. The promoter of BcMF23a was active specifically in pollen during the late pollen developmental stages, while, in addition to the pollen, BcMF23b promoter drove an extra gene expression in the valve margins, abscission layer at the base of the first true leaves, taproot and lateral roots in seedlings.

The N-Glycan cluster from Xanthomonas campestris pv. campestris: a toolbox for sequential plant N-glycan processing.

N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N2M3FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the ß-xylosidase NixI (GH3), which is involved in the cleavage of the ß-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.