Indole Glucosinolates Research Articles

Glucosinolate Abundance and Composition in Brassicaceae Influence Sequestration in a Specialist Flea Beetle

The horseradish flea beetle Phyllotreta armoraciae exclusively feeds on Brassicaceae, which contain glucosinolates as characteristic defense compounds. Although glucosinolates are usually degraded by plant enzymes (myrosinases) to toxic isothiocyanates after ingestion, P. armoraciae beetles sequester glucosinolates. Between and within brassicaceous plants, the glucosinolate content and composition can differ drastically. But how do these factors influence sequestration in P. armoraciae? To address this question, we performed a five-day feeding experiment with three Arabidopsis thaliana lines that differ four-fold in glucosinolate content and the composition of aliphatic and indolic glucosinolates. We quantified the amounts of ingested, sequestered, and excreted glucosinolates, and analyzed the changes in glucosinolate levels and composition in beetles before and after feeding on Arabidopsis. P. armoraciae accumulated almost all ingested glucosinolate types. However, some glucosinolates were accumulated more efficiently than others, and selected glucosinolates were modified by the beetles. The uptake of new glucosinolates correlated with a decrease in the level of stored glucosinolates so that the total glucosinolate content remained stable at around 35 nmol/mg beetle fresh weight. Beetles excreted previously stored as well as ingested glucosinolates from Arabidopsis, which suggests that P. armoraciae regulate their endogenous glucosinolate level by excretion. The metabolic fate of ingested glucosinolates, i.e. the proportions of sequestered and excreted glucosinolates, depended on glucosinolate type, content, and composition in the food plant. Overall, P. armoraciae sequestered and excreted up to 41% and 31% of the total ingested aliphatic and indolic glucosinolates from Arabidopsis, respectively. In summary, we show that glucosinolate variability in Brassicaceae influences the composition but not the level of sequestered glucosinolates in P. armoraciae beetles.

Both Biosynthesis and Transport Are Involved in Glucosinolate Accumulation During Root-Herbivory in Brassica rapa.

The optimal defense theory predicts that plants invest most energy in those tissues that have the highest value, but are most vulnerable to attacks. In Brassica species, root-herbivory leads to the accumulation of glucosinolates (GSLs) in the taproot, the most valuable belowground plant organ. Accumulation of GSLs can result from local biosynthesis in response to herbivory. In addition, transport from distal tissues by specialized GSL transporter proteins can play a role as well. GSL biosynthesis and transport are both inducible, but the role these processes play in GSL accumulation during root-herbivory is not yet clear. To address this issue, we performed two time-series experiments to study the dynamics of transport and biosynthesis in local and distal tissues of Brassica rapa. We exposed roots of B. rapa to herbivory by the specialist root herbivore Delia radicum for 7 days. During this period, we sampled above- and belowground plant organs 12 h, 24 h, 3 days and 7 days after the start of herbivory. Next, we measured the quantity and composition of GSL profiles together with the expression of genes involved in GSL biosynthesis and transport. We found that both benzyl and indole GSLs accumulate in the taproot during root-herbivory, whereas we did not observe any changes in aliphatic GSL levels. The rise in indole GSL levels coincided with increased local expression of biosynthesis and transporter genes, which suggest that both biosynthesis and GSL transport play a role in the accumulation of GSLs during root herbivory. However, we did not observe a decrease in GSL levels in distal tissues. We therefore hypothesize that GSL transporters help to retain GSLs in the taproot during root-herbivory.

In Silico Study of the Radical Scavenging Activities of Natural Indole-3-Carbinols.

Enzymatic hydrolysis of indole glucosinolates in the human body yields the Indole-3-carbinol family of compounds (I3Cs). I3Cs are implicated in the self-healing processes of the human body and thus used as over the counter dietary supplements characterized as potential antioxidants. In this study, the antioxidant activity of natural indole-3-carbinol derivatives were investigated against the HOO• radical by using thermodynamic and kinetic calculations. It was found that natural I3Cs containing only C8-H and/or N1-H bonds such as indole-3-carbinol, 4-methoxy-indole-3-carbinol, and 1-methoxy-indole-3-carbinol exhibit weak antioxidant properties. However, the presence of OH groups in the indole ring significantly increases the radical scavenging in aqueous as well as lipid media. In particular the HOO• scavenging activity of 1-hydroxyl-indole-3-carbinol far exceeds that of Trolox, 2.0 times in aqueous medium and 352.9 times in lipid (pentyl ethanoate) medium. This suggests that 1-hydroxyl-indole-3-carbinol is one of the most powerful known antioxidants in lipid environments, pointing at potential dietary application in preventive and regenerative medicine.

Plant responses to ozone: Effects of different ozone exposure durations on plant growth and biochemical quality of Brassica campestris L. ssp. chinensis

Determining plant reactions to atmospheric pollutants is important in predicting and planning for new horticultural production management programs and challenges in the future. A high level of ozone in the troposphere is considered the most phototoxic air pollutant, and has strong oxidative potential. This study aimed to provide understanding on the role of ozone stress on early vegetative growth stage Pak-Choi (Brassica campestris L. ssp. chinensis). An experiment was conducted within airtight bio-chambers under 150 ppb ozone concentration with different exposure durations: control, 1 h/day and 4 h/day. The leaf area was not affected by ozone treatment, but the dry matter correlated negatively with increasing ozone exposure durations. In photopigments, the lutein, lycopene and chlorophylls were increased at 1 h/day exposure, however, total carotenoids and β-carotene were not affected. However, for the 4 h/day exposure condition, all photopigment contents were significantly decreased in comparison to the 1 h/day exposure. The total aliphatic and total glucosinolate (GLS) were significantly increased at 1 h/day exposure, and then significantly decreased at 4 h/day exposure. However, the total indole and aromatic GLS were significantly increased after 4 h/day exposure. Therefore, these results provide evidence about the impact of ozone stress on the specific secondary plant metabolites of Pak-Choi and this needs to be taken in account when selecting urban production sites for vegetables.

Analysis of glucosinolates in ‘Nainaiqingcai’ mustard under the alpine cold climate

The composition and content of glucosinolates were investigated in ‘Nainaiqingcai’ mustard to reveal the diversity of the glucosinolates among the individual edible parts (leaves, petioles, and bolting stems). The results showed the significant differences of the composition and content of glucosinolates were found in the different edible parts of ‘Nainaiqingcai’ mustard. Eleven glucosinolates were detected in leaves of ‘Nainaiqingcai’ mustard, including six aliphatic glucosinolates, four indole glucosinolates and one aromatic glucosinolate. Only ten of the eleven glucosinolates were detected in petioles and bolting stems of ‘Nainaiqingcai’ mustard. The highest contents of glucosinolates in ‘Nainaiqingcai’ mustard was found in leaves. Aliphatic glucosinolates were predominant in the ‘Nainaiqingcai’ mustard, followed by indole and aromatic glucosinolates. Sinigrin was the predominant glucosinolate in the ‘Nainaiqingcai’ mustard. In summary, these findings provide a theoretical reference for the scientific consumption and a foundation for the further study of ‘Nainaiqingcai’ mustard in the future.

Brassinosteroids Antagonize Jasmonate-Activated Plant Defense Responses through BRI1-EMS-SUPPRESSOR1 (BES1).

Brassinosteroids (BRs) and jasmonates (JAs) regulate plant growth, development, and defense responses, but how these phytohormones mediate the growth-defense tradeoff is unclear. Here, we identified the Arabidopsis (Arabidopsis thaliana) dwarf at early stages1 (dwe1) mutant, which exhibits enhanced expression of defensin genes PLANT DEFENSIN1.2a (PDF1.2a) and PDF1.2b The dwe1 mutant showed increased resistance to herbivory by beet armyworms (Spodoptera exigua) and infection by botrytis (Botrytis cinerea). DWE1 encodes ROTUNDIFOLIA3, a cytochrome P450 protein essential for BR biosynthesis. The JA-inducible transcription of PDF1.2a and PDF1.2b was significantly reduced in the BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) gain-of-function mutant bes1- D, which was highly susceptible to S. exigua and B. cinerea BES1 directly targeted the terminator regions of PDF1.2a/PDF1.2b and suppressed their expression. PDF1.2a overexpression diminished the enhanced susceptibility of bes1- D to B. cinerea but did not improve resistance of bes1- D to S. exigua In response to S. exigua herbivory, BES1 inhibited biosynthesis of the JA-induced insect defense-related metabolite indolic glucosinolate by interacting with transcription factors MYB DOMAIN PROTEIN34 (MYB34), MYB51, and MYB122 and suppressing expression of genes encoding CYTOCHROME P450 FAMILY79 SUBFAMILY B POLYPEPTIDE3 (CYP79B3) and UDP-GLUCOSYL TRANSFERASE 74B1 (UGT74B1). Thus, BR contributes to the growth-defense tradeoff by suppressing expression of defensin and glucosinolate biosynthesis genes.

Differential partitioning of thiols and glucosinolates between shoot and root in Chinese cabbage upon excess zinc exposure

Zinc (Zn) is one of the important elements of plant growth, however, at elevated level it is toxic. Exposure of Chinese cabbage to elevated Zn2+ concentrations (5 and 10 μM ZnCl2) resulted in enhancement of total sulfur and organic sulfur concentration. Transcript level of APS reductase (APR) as a key enzyme in biosynthesis of primary sulfur compounds (cysteine and thiols), was up-regulated in both shoot and root upon exposure to elevated Zn2+, which was accompanied by an increase in the concentration of cysteine in both tissues. In contrast, the concentration of thiols increased only in the root by 5.5 and 15-fold at 5 and 10 μM Zn2+, respectively, which was in accompanied by an upregulation of ATP sulfurylase, an enzyme responsible for activation of sulfate. An elevated content of glucosinolates, mostly indolic glucosinolates, only in the shoot of plants exposed to excess level of Zn2+ coincided with an increase in gene expression of key biosynthetic enzymes and regulators (CYP79B3, CYP83B1, MYB34). Thus distinct acuumulation patterns of sulfur containing compounds in root and shoot of Chinese cabbage may be a strategy for Chinese cabbage to combat with exposure to excess Zn.

Molecular Characterization and Expression Analysis of MYB Transcription Factors Involved in the Glucosinolate Pathway in Chinese Cabbage (Brassica rapa ssp. pekinensis)

Chinese cabbage (Brassica rapa) is a perennial crucifer vegetable that has long been used for forage. Crucifers are rich sources of glucosinolates (GSLs), which are anti-carcinogenic in humans and involved in plant defense responses. Myeloblastosis (MYB) proteins are a large family of transcription factors (TFs) in plants and play major regulatory roles in many biological processes. We identified 14 functional R2R3-MYB genes involved in glucosinolate biosynthesis in B. rapa ssp. pekinensis. Bioinformatic analysis of their phylogeny, protein motifs, gene interaction network, and molecular characteristics showed that Chinese cabbage MYB genes are comparable to those of Arabidopsis thaliana. The expression levels of the 14 BrMYB genes under fluorescent lamp, blue, and red light were quantitated using qRT-PCR analysis. Almost all of the R2R3-BrMYBs were upregulated and expressed more under red light than under fluorescent lamp or blue light, except BrMYB34s. We also calculated the total GSLs under each light condition. The total GSL content was higher under red light than under fluorescent lamp or blue light. Furthermore, the individual glucosinolates, comprised of four aliphatic GSLs (progoitrin, sinigrin, gluconapin, and glucobrassicanapin) and one indolic GSL (glucobrassicin), were higher under red light than the other light conditions. The relationships between light quality and glucosinolate biosynthesis require further investigation.

Same Difference? Low and High Glucosinolate Brassica rapa Varieties Show Similar Responses Upon Feeding by Two Specialist Root Herbivores.

Glucosinolates (GSLs) evolved in Brassicaceae as chemical defenses against herbivores. The GSL content in plants is affected by both abiotic and biotic factors, but also depends on the genetic background of the plant. Since the bitter taste of GSLs can be unfavorable for both livestock and human consumption, several plant varieties with low GSL seed or leaf content have been bred. Due to their lower GSL levels, such varieties can be more susceptible to herbivore pests. However, low GSL varieties may quickly increase GSL levels upon herbivore feeding by activating GSL biosynthesis, hydrolysis, or transporter genes. To analyze differences in herbivore-induced GSL responses in relation to constitutive GSL levels, we selected four Brassica rapa varieties, containing either low or high root GSL levels. Plants were infested either with Delia radicum or Delia floralis larvae. The larvae of both root flies are specialists on Brassica plants. Root samples were collected after 3, 5, and 7 days. We compared the effect of root herbivore damage on the expression of GSL biosynthesis (CYP79A1, CYP83B2), transporter (GTR1A2, GTR2A2), and GSL hydrolysis genes (PEN2, TGG2) in roots of low and high GSL varieties in conjugation with their GSL levels. We found that roots of high GSL varieties contained higher levels of aliphatic, indole, and benzyl GSLs than low GSL varieties. Infestation with D. radicum larvae led to upregulation of indole GSL synthesis genes in low and high GSL varieties. High GSL varieties showed no or later responses than low varieties to D. floralis herbivory. Low GSL varieties additionally upregulated the GSL transporter gene expression. Low GSL varieties did not show a stronger herbivore-induced response than high GSL varieties, which indicates that there is no trade-off between constitutive and induced GSLs.

Growth and glucosinolate profiles of a common Asian green leafy vegetable, Brassica rapa subsp. chinensis var. parachinensis (choy sum), under LED lighting

Technological advancements in light-emitting diode (LED) technology have led to the production, under controlled indoor conditions, of value-added crops that are high in nutritional or nutraceutical contents. In this study, the growth and glucosinolate (GSL) profiles of a common Asian vegetable, Brassica rapa subsp. chinensis var. parachinensis (choy sum or cai xin) were determined under LED lighting. The growth of choy sum was influenced by both the LED light intensity and spectrum, with plants grown under 160 μmol m−2 s−1 red-blue light (160RB) producing the highest shoot biomass (∼100–300 % increment in adult plants). GSL content was found to vary across growth stages regardless of LED treatment. Total GSLs per gram fresh weight were four times higher in one-leafed seedlings than in adult plants. A shift from a high proportion of aliphatic GSLs in one-leafed seedlings to indolic and aromatic GSLs in three-leafed seedlings and adult plants was attributed to an increase in the proportion of glucobrassicin and/or 4-hydroxy-glucobrassicin, and gluconasturtiin which are known to have anti-cancer properties. Our results will be useful for the production of GSL-enhanced vegetables through urban farming.

Modulation of Glucosinolate Composition in Brassicaceae Seeds by Germination and Fungal Elicitation.

Glucosinolates (GSLs) are of interest for potential antimicrobial activity of their degradation products and exclusive presence in Brassicaceae. Compositional changes of aliphatic, benzenic, and indolic GSLs of Sinapis alba, Brassica napus, and B. juncea seeds by germination and fungal elicitation were studied. Rhizopus oryzae (nonpathogenic), Fusarium graminearum (nonpathogenic), and F. oxysporum (pathogenic) were employed. Thirty-one GSLs were detected by reversed-phase ultrahigh-performance liquid chromatography photodiode array with in-line electrospray ionization mass spectrometry (RP-UHPLC-PDA-ESI-MSn). Aromatic-acylated derivatives of 3-butenyl GSL, p-hydroxybenzyl GSL, and indol-3-ylmethyl GSL were for the first time tentatively annotated and confirmed to be not artifacts. For S. alba, germination, Rhizopus elicitation, and F. graminearum elicitation increased total GSL content, mainly consisting of p-hydroxybenzyl GSL, by 2–3 fold. For B. napus and B. juncea, total GSL content was unaffected by germination or elicitation. In all treatments, aliphatic GSL content was decreased (≥50%) in B. napus and remained unchanged in B. juncea. Indolic GSLs were induced in all species by germination and nonpathogenic elicitation.

Selenium Application During Radish (Raphanus sativus) Plant Development Alters Glucosinolate Metabolic Gene Expression and Results in the Production of 4-(methylseleno)but-3-enyl glucosinolate.

Selenium (Se) is an essential micronutrient for human health, entering the diet mainly through the consumption of plant material. Members of the Brassicaceae are Se-accumulators that can accumulate up to 1g Se kg−1 dry weight (DW) from the environment without apparent ill effect. The Brassicaceae also produce glucosinolates (GSLs), sulfur (S)-rich compounds that benefit human health. Radish (Raphanus sativus) has a unique GSL profile and is a Se-accumulating species that is part of the human diet as sprouts, greens and roots. In this report we describe the effects of Se-fertilisation on GSL production in radish during five stages of early development (from seed to mature salad greens) and on the transcript abundance of eight genes encoding enzymes involved in GSL metabolism. We tentatively identified (by tandem mass spectrometry) the selenium-containing glucosinolate, 4-(methylseleno)but-3-enyl glucosinolate, with the double bond geometry not resolved. Two related isothiocyanates were tentatively identified by Gas Chromatography-Mass Spectrometry as (E/Z?) isomers of 4-(methylseleno)but-3-enyl isothiocyanate. Se fertilisation of mature radish led to the presence of selenoglucosinolates in the seed. While GSL concentration generally reduced during radish development, GSL content was generally not affected by Se fertilisation, aside from the indole GSL, indol-3-ylmethyl glucosinolate, which increased on Se treatment, and the Se-GSLs, which significantly increased during development. The transcript abundance of genes involved in aliphatic GSL biosynthesis declined with Se treatment while that of genes involved in indole GSL biosynthesis tended to increase. APS kinase transcript abundance increased significantly in three of the four developmental stages following Se treatment. The remaining genes investigated were not significantly changed following Se treatment. We hypothesise that increased APS kinase expression in response to Se treatment is part of a general protection mechanism controlling the uptake of S and the production of S-containing compounds such as GSLs. The upregulation of genes encoding enzymes involved in indole GSL biosynthesis and a decrease in those involved in aliphatic GSL biosynthesis may be part of a similar mechanism protecting the plant’s GSL complement whilst limiting the amount of Se-GSLs produced.

Profiling glucosinolate metabolites in human urine and plasma after broccoli consumption using non-targeted and targeted metabolomic analyses

Broccoli is a popular brassica vegetable and its consumption may decrease the occurrence of cancer in certain populations. To gain insight into the metabolites that may induce physiological responses to broccoli intake, a non-targeted metabolomic approach and a targeted approach for analysis of glucosinolate metabolites were developed using high resolution accurate mass spectrometry. A human study was conducted in which 6 subjects consumed a single meal of 200 g of uncooked broccoli florets. The metabolomic analysis revealed changes in endogenous metabolites and a decrease in hippuric acid after broccoli consumption. Targeted analysis using high-resolution, accurate mass-mass spectrometry (HRAM-MS) enabled detection of low concentrations (nM) of glucosinolate metabolites in human urine and plasma. Glucosinolate metabolites were found in human urine (13) and plasma (8), respectively. Metabolites from methoxyl-indole glucosinolates, arising from broccoli consumption, are reported for the first time. Most glucosinolate metabolites reached their peak concentration in urine 2–4 h after consumption while, in plasma, peak maxima were achieved 2 h after intake. The results suggest that glucoraphanin metabolites (sulforaphane, sulforaphane cysteine, sulforaphane N-acetyl cysteine) and indole metabolites (ascorbigen and methoxyl ascorbigen from indole glucosinolates) may serve as marker compounds for the intake of broccoli.

Negative Regulation of Age-Related Developmental Leaf Senescence by the IAOx Pathway, PEN1, and PEN3.

Early age-related developmental senescence was observed in Arabidopsis cyp79B2/cyp79B3 double mutants that cannot produce indole-3-acetaldoxime (IAOx), the precursor to indole glucosinolates (IGs), camalexin and auxin. The early senescence phenotype was not observed when senescence was induced by darkness. The cyp79B2/cyp79B3 mutants had lower auxin levels, but did not display auxin-deficient phenotypes. Camalexin biosynthesis mutants senesced normally; however, IG transport and exosome-related pen1/pen3 double mutants displayed early senescence. The early senescence in pen1/pen3 mutants depended on salicylic acid and was not observed in pen1 or pen3 single mutants. Quantitation of IGs showed reduced levels in cyp79B2/cyp79B3 mutants, but unchanged levels in pen1/pen3, even though both of these double mutants display early senescence. We discuss how these genetic data provide evidence that IAOx metabolites are playing a protective role in leaf senescence that is dependent on proper trafficking by PEN1 and PEN3, perhaps via the formation of exosomes.

Transcriptome-wide identification of indole glucosinolate dependent flg22-response genes in Arabidopsis

Indole glucosinolates are known to play essential and diverse roles in Arabidopsis immunity to pathogens. However, a complete understanding of the function of these compounds in plant immunity remains unclear. In this study, we investigated the transcriptome profile in loss-of-function mutant of MYB51, the key transcription factor that controls the biosynthesis of indole glucosinolates. Upon treatment with flg22 (a 22-amino acid peptide derived from bacterial flagellin), the genes that responded in a MYB51-dependent manner were analyzed. The results suggested that MYB51 was possibly implicated in most resistance processes, including pathogen recognition, signal transduction and PR protein activation. Of note, several genes in the ethylene pathway and the WRKY family, including WRKY33, were induced by flg22 in a MYB51-dependent manner. WRKY33 and ethylene were demonstrated to be crucial regulators in plant immunity defense and are functionally upstream of MYB51 during MAMP triggered immunity (MTI). This result suggested a “positive feedback loop” between MYB51 and its upstream regulators.

Quantification of Total Glucosinolates and Isothiocyanates for Common Brassicaceous Vegetables Consumed in the US Market Using Cyclocondensation and Thiocyanate Ion Measurement Methods

Glucosinolates are a group of important sulfur and nitrogen containing plant secondary metabolites found in Brassicaceous vegetables that are associated with many health benefits. There is a growing interest in assessing dietary glucosinolates and their degradation products’ isothiocyanates exposure to humans. Determination of glucosinolates in plants is a very challenging task. In this study, the total glucosinolates from 22 commonly consumed Brassicaceous vegetables in the US market were determined by employing 1,2-benzenedithiol cyclocondensation for the determination of isothiocyanates (ITCs) from non-indole glucosinolates and a colorimetric method for the determination of thiocyanate ion (SCN−) from indole glucosinolates. The total glucosinolates were determined from the sum of these two methods. In the 22 investigated vegetables, the highest level of total ITCs was found in horse radish (5189.0 ± 544.8 nmol/g dry weight) and the highest level of total SCN− level was found in brussel sprouts (1294.0 ± 141.6 nmol/g dry weight). The total glucosinolates ranged from 18.6 ± 3.2 to 2271.2 ± 215.3 nmol/g dry weight. The total glucosinolate and ITC data are necessary for the USDA glucosinolates database and important for establishing the relationship of glucosinolates to human nutrition.

The role of CYP71A12 monooxygenase in pathogen-triggered tryptophan metabolism and Arabidopsis immunity.

Effective defense of Arabidopsis against filamentous pathogens requires two mechanisms, both of which involve biosynthesis of tryptophan (Trp)-derived metabolites. Extracellular resistance involves products of PEN2-dependent metabolism of indole glucosinolates (IGs). Restriction of further fungal growth requires PAD3-dependent camalexin and other, as yet uncharacterized, indolics. This study focuses on the function of CYP71A12 monooxygenase in pathogen-triggered Trp metabolism, including the biosynthesis of indole-3-carboxylic acid (ICA). Moreover, to investigate the contribution of CYP71A12 and its products to Arabidopsis immunity, we analyzed infection phenotypes of multiple mutant lines combining pen2 with pad3, cyp71A12, cyp71A13 or cyp82C2. Metabolite profiling of cyp71A12 lines revealed a reduction in ICA accumulation. Additionally, analysis of mutant plants showed that low amounts of ICA can form during an immune response by CYP71B6/AAO1-dependent metabolism of indole acetonitrile, but not via IG hydrolysis. Infection assays with Plectosphaerella cucumerina and Colletotrichum tropicale, two pathogens with different lifestyles, revealed cyp71A12-, cyp71A13- and cyp82C2-associated defects associated with Arabidopsis immunity. Our results indicate that CYP71A12, but not CYP71A13, is the major enzyme responsible for the accumulation of ICA in Arabidopsis in response to pathogen ingression. We also show that both enzymes are key players in the resistance of Arabidopsis against selected filamentous pathogens after they invade.

CIRCADIAN CLOCK-ASSOCIATED1 Controls Resistance to Aphids by Altering Indole Glucosinolate Production.

CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), a well-known central circadian clock regulator, coordinates plant responses to environmental challenges. Its daily rhythmic expression in Arabidopsis (Arabidopsis thaliana) confers host resistance to the caterpillar Trichoplusia ni However, it is unclear whether CCA1 plays a role in defense against phloem sap-feeding aphids. In this study, we showed that green peach aphid (Myzus persicae) displayed an intrinsic circadian feeding rhythm. Under constant light, wild-type Columbia-0 (Col-0) Arabidopsis plants coentrained with aphids in the same light/dark cycles exhibited greater antixenotic activity than plants preentrained in the opposite cycle from the aphids. Consistently, circadian mutants cca1-1, cca1-11, lhy-21, ztl-1, ztl-4, and lux-2 suffered more severe damage than Col-0 plants when infested by aphids, suggesting that the Arabidopsis circadian clock plays a defensive role. However, the arrhythmic CCA1 overexpression line (CCA1-OX) displayed strong antixenotic and antibiotic activities despite its loss of circadian regulation. Aphids feeding on CCA1-OX plants exhibited lower reproduction and smaller body size and weight than those on Col-0. Apparently, CCA1 regulates both clock-dependent and -independent defense responses. Systematic investigation based on bioinformatics analyses indicated that resistance to aphids in CCA1-OX plants was due primarily to heightened basal indole glucosinolate levels. Interestingly, aphid feeding induced alternatively spliced intron-retaining CCA1a/b transcripts, which are normally expressed at low levels, whereas expression of the major fully spliced CCA1 transcript remained largely unchanged. We hypothesize that posttranscriptional modulation of CCA1 expression upon aphid infestation maximizes the potential of circadian-mediated defense and stress tolerance while ensuring normal plant development.

Copper toxicity affects indolic glucosinolates and gene expression of key enzymes for their biosynthesis in Chinese cabbage

ABSTRACT Excessive levels of Cu2+ are phytotoxic and exposure of Chinese cabbage to elevated Cu2+ concentrations led to reduction of the plant biomass. To get more insight into the role of glucosinolates upon copper stress, the impact of elevated Cu2+ levels on glucosinolates biosynthesis were studied in Chinese cabbage. The content of total glucosinolates was only elevated in the roots, mostly due to indolic and aromatic glucosinolates. The results showed a higher contribution of indolic glucosinolates, notably glucobrassicin, a 2- and 4-fold increase in Chinese cabbage exposed to 5 and 10 µM Cu2+, respectively. Furthermore, the increase in the indolic glucosinolates was accompanied by enhanced transcript levels of CYP79B2 and CYP83B1, two genes involved in biosynthesis of indolic glucosinolates, and that of the MYB51, a transcription factor involved in regulation of indolic glucosinolate biosynthesis pathway, at elevated Cu2+ concentrations. In addition, total sulfur and nitrogen remained unaffected in the root, but total glucosinolate was significantly enhanced upon exposure to elevated Cu2+. This result may show that relatively more sulfur and nitrogen was channeled into glucosinolates in the root. In conclusion, accumulation of indolic glucosinolates in the root can be considered as a strategy for Chinese cabbage to combat elevated Cu2+ concentrations.

A Gain-of-Function Screen Reveals Redundant ERF Transcription Factors Providing Opportunities for Resistance Breeding Toward the Vascular Fungal Pathogen Verticillium longisporum.

Verticillium longisporum is a vascular fungal pathogen leading to severe crop loss, particular in oilseed rape. Transcription factors (TF) are highly suited for genetic engineering of pathogen-resistant crops, as they control sets of functionally associated genes. Applying the AtTORF-Ex (Arabidopsis thaliana transcription factor open reading frame expression) collection, a simple and robust screen of TF-overexpressing plants was established displaying reduced fungal colonization. Distinct members of the large ethylene response factor (ERF) family, namely ERF96 and the six highly related subgroup IXb members ERF102 to ERF107, were identified. Whereas overexpression of these ERF significantly reduces fungal propagation, single loss-of-function approaches did not reveal altered susceptibility. Hence, this gain-of-function approach is particularly suited to identify redundant family members. Expression analyses disclosed distinct ERF gene activation patterns in roots and leaves, suggesting functional differences. Transcriptome studies performed on chemically induced ERF106 expression revealed an enrichment of genes involved in the biosynthesis of antimicrobial indole glucosinolates (IG), such as CYP81F2 (CYTOCHROME P450-MONOOXYGENASE 81F2), which is directly regulated by IXb-ERF via two GCC-like cis-elements. The impact of IG in restricting fungal propagation was further supported as the cyp81f2 mutant displayed significantly enhanced susceptibility. Taken together, this proof-of-concept approach provides a novel strategy to identify candidate TF that are valuable genetic resources for engineering or breeding pathogen-resistant crop plants.