Metabolic changes in Arabidopsis thaliana expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D

Anthranilate synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 microM L-Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3 M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3 M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.     

Bacterial volatiles induce systemic resistance in Arabidopsis

Plant growth-promoting rhizobacteria, in association with plant roots, can trigger induced systemic resistance (ISR). Considering that low-molecular weight volatile hormone analogues such as methyl jasmonate and methyl salicylate can trigger defense responses in plants, we examined whether volatile organic compounds (VOCs) associated with rhizobacteria can initiate ISR. In Arabidopsis seedlings exposed to bacterial volatile blends from Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a, disease severity by the bacterial pathogen Erwinia carotovora subsp. carotovora was significantly reduced compared with seedlings not exposed to bacterial volatiles before pathogen inoculation. Exposure to VOCs from rhizobacteria for as little as 4 d was sufficient to activate ISR in Arabidopsis seedlings. Chemical analysis of the bacterial volatile emissions revealed the release of a series of low-molecular weight hydrocarbons including the growth promoting VOC (2R,3R)-(-)-butanediol. Exogenous application of racemic mixture of (RR) and (SS) isomers of 2,3-butanediol was found to trigger ISR and transgenic lines of B. subtilis that emitted reduced levels of 2,3-butanediol and acetoin conferred reduced Arabidopsis protection to pathogen infection compared with seedlings exposed to VOCs from wild-type bacterial lines. Using transgenic and mutant lines of Arabidopsis, we provide evidence that the signaling pathway activated by volatiles from GB03 is dependent on ethylene, albeit independent of the salicylic acid or jasmonic acid signaling pathways. This study provides new insight into the role of bacteria VOCs as initiators of defense responses in plants.     

Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.     

Co-regulation of indole glucosinolates and camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6 signaling pathways

Secondary plant metabolites, represented by indole glucosinolates (IGS) and camalexin, play important roles in Arabidopsis immunity. Previously, we demonstrated the importance of MPK3 and MPK6, two closely related MAPKs, in regulating Botrytis cinerea (Bc)‐induced IGS and camalexin biosynthesis. Here we report that CPK5 and CPK6, two redundant calcium‐dependent protein kinases (CPKs), are also involved in regulating the biosynthesis of these secondary metabolites. The loss‐of‐function of both CPK5 and CPK6 compromises plant resistance to Bc. Expression profiling of CPK5‐VK transgenic plants, in which a truncated constitutively active CPK5 is driven by a steroid‐inducible promoter, revealed that biosynthetic genes of both IGS and camalexin pathways are coordinately upregulated after the induction of CPK5‐VK, leading to high‐level accumulation of camalexin and 4‐methoxyindole‐3‐yl‐methylglucosinolate (4MI3G). Induction of camalexin and 4MI3G, as well as the genes in their biosynthesis pathways, is greatly compromised in cpk5 cpk6 mutant in response to Bc. In a conditional cpk5 cpk6 mpk3 mpk6 quadruple mutant, Bc resistance and induction of IGS and camalexin are further reduced in comparison to either cpk5 cpk6 or conditional mpk3 mpk6 double mutant, suggesting that both CPK5/CPK6 and MPK3/MPK6 signaling pathways contribute to promote the biosynthesis of 4MI3G and camalexin in defense against Bc.     

Oligogalacturonide-mediated induction of a gene involved in jasmonic acid synthesis in response to the cell-wall-degrading enzymes of the plant pathogen Erwinia carotovora.

Identification of Arabidopsis thaliana genes responsive to plant cell-wall-degrading enzymes of Erwinia carotovora subsp. carotovora led to the isolation of a cDNA clone with high sequence homology to the gene for allene oxide synthase, an enzyme involved in the biosynthesis of jasmonates. Expression of the corresponding gene was induced by the extracellular enzymes from this pathogen as well as by treatment with methyl jasmonate and short oligogalacturonides (OGAs). This suggests that OGAs are involved in the induction of the jasmonate pathway during plant defense response to E. carotovora subsp. carotovora attack.     

Classic myrosinase‐dependent degradation of indole glucosinolate attenuates fumonisin B1‐induced programmed cell death in Arabidopsis

The mycotoxin fumonisin B1 (FB1) causes the accumulation of reactive oxygen species (ROS) which then leads to programmed cell death (PCD) in Arabidopsis. In the process of studying FB1‐induced biosynthesis of glucosinolates, we found that indole glucosinolate (IGS) is involved in attenuating FB1‐induced PCD. Treatment with FB1 elevates the expression of genes related to the biosynthesis of camalexin and IGS. Mutants deficient in aliphatic glucosinolate (AGS) or camalexin biosynthesis display similar lesions to Col‐0 upon FB1 infiltration; however, the cyp79B2 cyp79B3 double mutant, which lacks induction of both IGS and camalexin, displays more severe lesions. Based on the fact that the classic myrosinase β‐thioglucoside glucohydrolase (TGG)‐deficient double mutant tgg1 tgg2, rather than atypical myrosinase‐deficient mutant pen2‐2, is more sensitive to FB1 than Col‐0, and the elevated expression of TGG1, but not of PEN2, correlates with the decrease in IGS, we conclude that TGG‐dependent IGS hydrolysis is involved in FB1‐induced PCD. Indole‐3‐acetonitrile (IAN) and indole‐3‐carbinol (I3C), the common derivatives of IGS, were used in feeding experiments, and this rescued the severe cell death phenotype, which is associated with reduced accumulation of ROS as well as increased activity of antioxidant enzymes and ROS‐scavenging ability. Despite the involvement of indole‐3‐acetic acid (IAA) in restricting FB1‐induced PCD, feeding of IAN and I3C attenuated FB1‐induced PCD in the IAA receptor mutant tir1‐1 just as in Col‐0. Taken together, our results indicate that TGG‐catalyzed breakdown products of IGS decrease the accumulation of ROS by their antioxidant behavior, and attenuate FB1 induced PCD in an IAA‐independent way.     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

Erwinia carotovora subsp. carotovora and Erwinia-derived elicitors HrpN and PehA trigger distinct but interacting defense responses and cell death in Arabidopsis

We have used an hrp-positive strain of the soft rot pathogen Erwinia carotovora subsp. carotovora to elucidate plant responses to this bacterial necrotroph. Purified virulence determinants, harpin (HrpN) and polygalacturonase (PehA), were used as tools to facilitate this analysis. We show that HrpN elicits lesion formation in Arabidopsis and tobacco and triggers systemic resistance in Arabidopsis. Establishment of resistance is accompanied by the expression of salicylic acid (SA)-dependent, but also jasmonate/ethylene (JA/ET)-dependent, marker genes PR1 and PDF1.2, respectively, suggesting that both SA-dependent and JA/ET-dependent defense pathways are activated. Use of pathway-specific mutants and transgenic NahG plants show that both pathways are required for the induction of resistance. Arabidopsis plants treated simultaneously with both elictors PehA, known to trigger only JA/ET-dependent defense signaling, and HrpN react with accelerated and enhanced induction of the marker genes PR1 and PDF1.2 both locally and systemically. This mutual amplification of defense gene expression involves both SA-dependent and JA/ET-dependent defense signaling. The two elicitors produced by E. carotovora subsp. carotovora also cooperate in triggering increased production of superoxide and lesion formation.     

Coordinate regulation of the tryptophan biosynthetic pathway and indolic phytoalexin accumulation in Arabidopsis.

Little is known about the mechanisms that couple regulation of secondary metabolic pathways to the synthesis of primary metabolic precursors. Camalexin, an indolic secondary metabolite, appears to be the major phytoalexin in Arabidopsis. It was previously shown that camalexin accumulation is caused by infection with plant pathogens, by abiotic elicitors, and in spontaneous lesions in the accelerated cell death mutant acd2. We demonstrate that the accumulation of this phytoalexin is accompanied by the induction of the mRNAs and proteins for all of the tryptophan biosynthetic enzymes tested. A strong correlation was observed between the magnitude of camalexin accumulation and the induction of tryptophan biosynthetic proteins, indicating coordinate regulation of these processes. Production of disease symptoms is not sufficient for the response because systemic infection with cauliflower mosaic virus or cucumber mosaic virus did not induce the tryptophan pathway enzymes or camalexin accumulation. Salicylic acid appears to be required, but unlike other documented pathogenesis-related proteins, it is not sufficient for the coordinate induction. Results with trp mutants suggest that the tryptophan pathway is not rate limiting for camalexin accumulation. Taken together, these results are consistent with the hypothesis that the regulation of the tryptophan pathway in plants responds to needs for biosynthesis of secondary metabolites.     

Role of the 4-phosphopantetheinyl transferase of Trichoderma virens in secondary metabolism and induction of plant defense responses

Trichoderma virens is a ubiquitous soil fungus successfully used in biological control due to its efficient colonization of plant roots. In fungi, 4-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary and secondary metabolism. Therefore, we cloned the PPTase gene ppt1 from T. virens and generated PPTase-deficient (?ppt1) and overexpressing strains to investigate the role of this enzyme in biocontrol and induction of plant defense responses. The ?ppt1 mutants were auxotrophic for lysine, produced nonpigmented conidia, and were unable to synthesize nonribosomal peptides. Although spore germination was severely compromised under both low and high iron availability, mycelial growth occurred faster than the wild type, and the mutants were able to efficiently colonize plant roots. The ?ppt1 mutants were unable of inhibiting growth of phytopathogenic fungi in vitro. Arabidopsis thaliana seedlings co-cultivated with wild-type T. virens showed increased expression of pPr1a:uidA and pLox2:uidA markers, which correlated with enhanced accumulation of salicylic acid (SA), jasmonic acid, camalexin, and resistance to Botrytis cinerea. Co-cultivation of A. thaliana seedlings with ?ppt1 mutants compromised the SA and camalexin responses, resulting in decreased protection against the pathogen. Our data reveal an important role of T. virens PPT1 in antibiosis and induction of SA and camalexin-dependent plant defense responses.     

B1-phytoprostanes trigger plant defense and detoxification responses

Phytoprostanes are prostaglandin/jasmonate-like products of nonenzymatic lipid peroxidation that not only occur ubiquitously in healthy plants but also increase in response to oxidative stress. In this work, we show that the two naturally occurring B(1)-phytoprostanes (PPB(1)) regioisomers I and II (each comprising two enantiomers) are short-lived stress metabolites that display a broad spectrum of biological activities. Gene expression analysis of Arabidopsis (Arabidopsis thaliana) cell cultures treated with PPB(1)-I or -II revealed that both regioisomers triggered a massive detoxification and defense response. Interestingly, expression of several glutathione S-transferases, glycosyl transferases, and putative ATP-binding cassette transporters was found to be increased by one or both PPB(1) regioisomers, and hence, may enhance the plant's capacity to inactivate and sequester reactive products of lipid peroxidation. Moreover, pretreatment of tobacco (Nicotiana tabacum) suspension cells with PPB(1) considerably prevented cell death caused by severe CuSO(4) poisoning. Several Arabidopsis genes induced by PPB(1), such as those coding for adenylylsulfate reductase, tryptophan synthase beta-chain, and PAD3 pointed to an activation of the camalexin biosynthesis pathway that indeed led to the accumulation of camalexin in PPB(1) treated leaves of Arabidopsis. Stimulation of secondary metabolism appears to be a common plant reaction in response to PPB(1). In three different plant species, PPB(1)-II induced a concentration dependent accumulation of phytoalexins that was comparable to that induced by methyl jasmonate. PPB(1)-I was much weaker active or almost inactive. No differences were found between the enantiomers of each regioisomer. Thus, results suggest that PPB(1) represent stress signals that improve plants capacity to cope better with a variety of stresses.     

Targeting novel chemical and constitutive primed metabolites against Plectosphaerella cucumerina

Priming is a physiological state for protection of plants against a broad range of pathogens, and is achieved through stimulation of the plant immune system. Various stimuli, such as beneficial microbes and chemical induction, activate defense priming. In the present study, we demonstrate that impairment of the high‐affinity nitrate transporter 2.1 (encoded by NRT2.1) enables Arabidopsis to respond more quickly and strongly to Plectosphaerella cucumerina attack, leading to enhanced resistance. The Arabidopsis thaliana mutant lin1 (affected in NRT2.1) is a priming mutant that displays constitutive resistance to this necrotroph, with no associated developmental or growth costs. Chemically induced priming by β–aminobutyric acid treatment, the constitutive priming mutant ocp3 and the constitutive priming present in the lin1 mutant result in a common metabolic profile within the same plant–pathogen interactions. The defense priming significantly affects sugar metabolism, cell‐wall remodeling and shikimic acid derivatives levels, and results in specific changes in the amino acid profile and three specific branches of Trp metabolism, particularly accumulation of indole acetic acid, indole‐3–carboxaldehyde and camalexin, but not the indolic glucosinolates. Metabolomic analysis facilitated identification of three metabolites in the priming fingerprint: galacturonic acid, indole‐3–carboxylic acid and hypoxanthine. Treatment of plants with the latter two metabolites by soil drenching induced resistance against P. cucumerina, demonstrating that these compounds are key components of defense priming against this necrotrophic fungus. Here we demonstrate that indole‐3–carboxylic acid induces resistance by promoting papillae deposition and H₂O₂ production, and that this is independent of PR1, VSP2 and PDF1.2 priming.     

UVR8 mediates UV-B-induced Arabidopsis defense responses against Botrytis cinerea by controlling sinapate accumulation

Light is emerging as a central regulator of plant immune responses against herbivores and pathogens. Solar UV-B radiation plays an important role as a positive modulator of plant defense. However, since UV-B photons can interact with a wide spectrum of molecular targets in plant tissues, the mechanisms that mediate their effects on plant defense have remained elusive. Here, we show that ecologically meaningful doses of UV-B radiation increase Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea and that this effect is mediated by the photoreceptor UVR8. The UV-B effect on plant resistance was conserved in mutants impaired in jasmonate (JA) signaling (jar1-1 and P35S:JAZ10.4) or metabolism of tryptophan-derived defense compounds (pen2-1, pad3-1, pen2 pad3), suggesting that neither regulation of the JA pathway nor changes in levels of indolic glucosinolates (iGS) or camalexin are involved in this response. UV-B radiation, acting through UVR8, increased the levels of flavonoids and sinapates in leaf tissue. The UV-B effect on pathogen resistance was still detectable in tt4-1, a mutant deficient in chalcone synthase and therefore impaired in the synthesis of flavonoids, but was absent in fah1-7, a mutant deficient in ferulic acid 5-hydroxylase, which is essential for sinapate biosynthesis. Collectively, these results indicate that UVR8 plays an important role in mediating the effects of UV-B radiation on pathogen resistance by controlling the expression of the sinapate biosynthetic pathway.     

Cytosolic γ-glutamyl peptidases process glutathione conjugates in the biosynthesis of glucosinolates and camalexin in Arabidopsis

The defense-related plant metabolites known as glucosinolates play important roles in agriculture, ecology, and human health. Despite an advanced biochemical understanding of the glucosinolate pathway, the source of the reduced sulfur atom in the core glucosinolate structure remains unknown. Recent evidence has pointed toward GSH, which would require further involvement of a GSH conjugate processing enzyme. In this article, we show that an Arabidopsis thaliana mutant impaired in the production of the γ-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. We also show that the double mutant is impaired in the production of camalexin and accumulates high amounts of the camalexin intermediate GS-IAN upon induction. In addition, we demonstrate that the cellular and subcellular localization of GGP1 and GGP3 matches that of known glucosinolate and camalexin enzymes. Finally, we show that the purified recombinant GGPs can metabolize at least nine of the 10 glucosinolate-related GSH conjugates as well as GS-IAN. Our results demonstrate that GSH is the sulfur donor in the biosynthesis of glucosinolates and establish an in vivo function for the only known cytosolic plant γ-glutamyl peptidases, namely, the processing of GSH conjugates in the glucosinolate and camalexin pathways.     

A potato gene encoding a WRKY-like transcription factor is induced in interactions with Erwinia carotovora subsp. atroseptica and Phytophthora infestans and is coregulated with class I endochitinase expression

A potato gene encoding a putative WRKY protein was isolated from a cDNA library enriched by suppression subtractive hybridization for sequences upregulated 1 h postinoculation with Erwinia carotovora subsp. atroseptica. The cDNA encodes a putative polypeptide of 172 amino acids, containing a single WRKY domain with a zinc finger motif and preceded by a potential nuclear localization site. St-WRKY1 was strongly upregulated in compatible, but only weakly in incompatible, interactions with Phytophthora infestans where, in all cases, it was coregulated with class I endochitinase, associating its expression with a known defense response. Whereas St-WRKY1 was strongly induced by E. carotovora culture filtrate (CF), confirming it to be an elicitor-induced gene, no such induction was detected after treatment with salicylic acid, methyl jasmonate, ethylene, or wounding. St-WRKY1 was upregulated by treatment of potato leaves with CFs from recombinant Escherichia coli containing plasmids expressing E. carotovora pectate lyase genes pelB and pelD, suggesting that either proteins encoded by these genes, or oligogalacturonides generated by their activity, elicit a potato defense pathway associated with St-WRKY1.     

Yeast increases resistance in Arabidopsis against Pseudomonas syringae and Botrytis cinerea by salicylic acid-dependent as well as -independent mechanisms

Cell-wall and glucopeptide components of yeast have been reported to exhibit elicitor activity. The mode of action of defense activation by yeast is not known so far. In this study, we used the model plant Arabidopsis to investigate the activation of defense responses by yeast, the effect on resistance against different pathogens, and the mode of action. Treatment of Arabidopsis plants with an autoclaved yeast suspension induced the expression of systemic acquired resistance-related genes and accumulation of the phytoalexin camalexin. Symptom development and bacterial growth after infection with a virulent strain of the pathogen Pseudomonas syringae was reduced in yeast-pretreated plants. No protection was detectable in mutants affected in the salicylate pathway, while mutants in the jasmonate or camalexin pathway were protected by yeast, indicating that the salicylate pathway is necessary for the yeast-induced resistance against P. syringae. Yeast also reduced symptom development after challenge with Botrytis cinerea. This protection was detectable in all mutants tested, indicating that it is independent of the salicylate, jasmonate, and camalexin pathway.     

WRKY33-mediated indolic glucosinolate metabolic pathway confers resistance against Alternaria brassicicola in Arabidopsis and Brassica crops

The tryptophan (Trp)-derived plant secondary metabolites, including camalexin, 4-hydroxy-indole-3-carbonylnitrile, and indolic glucosinolate (IGS), show broad-spectrum antifungal activity. However, the distinct regulations of these metabolic pathways among different plant species in response to fungus infection are rarely studied. In this study, our results revealed that WRKY33 directly regulates IGS biosynthesis, notably the production of 4-methoxyindole-3-ylmethyl glucosinolate (4MI3G), conferring resistance to Alternaria brassicicola, an important pathogen which causes black spot in Brassica crops. WRKY33 directly activates the expression of CYP81F2, IGMT1, and IGMT2 to drive side-chain modification of indole-3-ylmethyl glucosinolate (I3G) to 4MI3G, in both Arabidopsis and Chinese kale (Brassica oleracea var. alboglabra Bailey). However, Chinese kale showed a more severe symptom than Arabidopsis when infected by Alternaria brassicicola. Comparative analyses of the origin and evolution of Trp metabolism indicate that the loss of camalexin biosynthesis in Brassica crops during evolution might attenuate the resistance of crops to Alternaria brassicicola. As a result, the IGS metabolic pathway mediated by WRKY33 becomes essential for Chinese kale to deter Alternaria brassicicola. Our results highlight the differential regulation of Trp-derived camalexin and IGS biosynthetic pathways in plant immunity between Arabidopsis and Brassica crops.