Methionine-derived glucosinolates belong to a class of plant secondary metabolites that serve as chemoprotective compounds in plant biotic defense reactions and also exhibit strong anticancerogenic ...properties beneficial to human health. In a screen for the trans-activation potential of various transcription factors toward glucosinolate biosynthetic genes, we could identify the HAG1 (HIGH ALIPHATIC GLUCOSINOLATE 1, also referred to as MYB28) gene as a positive regulator of aliphatic methionine-derived glucosinolates. The content of aliphatic glucosinolates as well as transcript levels of aliphatic glucosinolate biosynthetic genes were elevated in gain-of-function mutants and decreased in HAG1 RNAi knock-down mutants. ProHAG₁:GUS expression analysis revealed strong HAG1 promoter activity in generative organs and mature leaves of A. thaliana plants, the main sites of accumulation of aliphatic glucosinolates. Mechanical stimuli such as touch or wounding transiently induced HAG1/MYB28 expression in inflorescences of flowering plants, and HAG1/MYB28 over-expression reduced insect performance as revealed by weight gain assays with the generalist lepidopteran herbivore Spodoptera exigua. Expression of HAG1/MYB28 was significantly induced by glucose, indicating a novel transcriptional regulatory mechanism for the integration of carbohydrate availability upon biotic challenge. We hypothesize that HAG1/MYB28 is a novel regulator of aliphatic glucosinolate biosynthesis that controls the response to biotic challenges.
Summary
Glucosinolates are a class of plant secondary metabolites that serve as antiherbivore compounds in plant defence. A previously identified Arabidopsis thaliana activation‐tagged line, ...displaying altered levels of secondary metabolites, was shown here to be affected in the content of indolic and aliphatic glucosinolates. The observed chemotype was caused by activation of the R2R3‐MYB transcription factor gene HIG1 (HIGH INDOLIC GLUCOSINOLATE 1, also referred to as MYB51). HIG1/MYB51 was shown to activate promoters of indolic glucosinolate biosynthetic genes leading to increased accumulation of indolic glucosinolates. The corresponding loss‐of‐function mutant hig1‐1 contained low levels of glucosinolates. Overexpression of the related transcription factor ATR1/MYB34, which had previously been described as a regulator of indolic glucosinolate and indole‐3‐acetic acid homeostasis, in the hig1‐1 mutant background led to a partial rescue of the mutant chemotype along with a severe high‐auxin growth phenotype. Overexpression of MYB122, another close homologue of HIG1/MYB51, did not rescue the hig1‐1 chemotype, but caused a high‐auxin phenotype and increased levels of indolic glucosinolates in the wild‐type. By contrast, overexpression of HIG1/MYB51 resulted in the specific accumulation of indolic glucosinolates without affecting auxin metabolism and plant morphology. Mechanical stimuli such as touch or wounding transiently induced the expression of HIG1/MYB51 but not of ATR1/MYB34, and HIG1/MYB51 overexpression reduced insect herbivory as revealed by dual‐choice assays with the generalist lepidopteran herbivore, Spodoptera exigua. We hypothesize that HIG1/MYB51 is a regulator of indolic glucosinolate biosynthesis that also controls responses to biotic challenges.
•gad12 mutant exhibited a reduced shoot growth.•gad12 plants were oversensitive to prolonged drought stress.•gad12 mutant have bigger stomata aperture, defect in stomata closure.•gad12 oversensitive ...phenotype was reversed by functional complementation with Pop2 mutation.•gad12 mutation affected the shoot amino acid composition
A rapid accumulation of γ-aminobutyric acid (GABA) during biotic and abiotic stresses is well documented. However, the specificity of the response and the primary role of GABA under such stress conditions are hardly understood. To address these questions, we investigated the response of the GABA-depleted gad1/2 mutant to drought stress. GABA is primarily synthesized from the decarboxylation of glutamate by glutamate decarboxylase (GAD) which exists in five copies in the genome of Arabidopsis thaliana. However, only GAD1 and GAD2 are abundantly expressed, and knockout of these two copies dramatically reduced the GABA content. Phenotypic analysis revealed a reduced shoot growth of the gad1/2 mutant. Furthermore, the gad1/2 mutant was wilted earlier than the wild type following a prolonged drought stress treatment. The early-wilting phenotype was due to an increase in stomata aperture and a defect in stomata closure. The increase in stomata aperture contributed to higher stomatal conductance. The drought oversensitive phenotype of the gad1/2 mutant was reversed by functional complementation that increases GABA level in leaves. The functionally complemented gad1/2 x pop2 triple mutant contained more GABA than the wild type. Our findings suggest that GABA accumulation during drought is a stress-specific response and its accumulation induces the regulation of stomatal opening thereby prevents loss of water.
A comparative transcriptome analysis for successive stages of Arabidopsis (Arabidopsis thaliana) developmental leaf senescence (NS), darkening-induced senescence of individual leaves attached to the ...plant (DIS), and senescence in dark-incubated detached leaves (DET) revealed many novel senescence-associated genes with distinct expression profiles. The three senescence processes share a high number of regulated genes, although the overall number of regulated genes during DIS and DET is about 2 times lower than during NS. Consequently, the number of NS-specific genes is much higher than the number of DIS- or DET-specific genes. The expression profiles of transporters (TPs), receptor-like kinases, autophagy genes, and hormone pathways were analyzed in detail. The Arabidopsis TPs and other integral membrane proteins were systematically reclassified based on the Transporter Classification system. Coordinate activation or inactivation of several genes is observed in some TP families in all three or only in individual senescence types, indicating differences in the genetic programs for remobilization of catabolites. Characteristic senescence type-specific differences were also apparent in the expression profiles of (putative) signaling kinases. For eight hormones, the expression of biosynthesis, metabolism, signaling, and (partially) response genes was investigated. In most pathways, novel senescence-associated genes were identified. The expression profiles of hormone homeostasis and signaling genes reveal additional players in the senescence regulatory network.
In a previous transactivation screen, two Arabidopsis thaliana R2R3-MYB transcription factors, HAG2/MYB76 and HAG3/MYB29, along with the already characterized HAG1/MYB28, were identified as putative ...regulators of aliphatic glucosinolate biosynthesis. Molecular and biochemical characterization of HAG2/MYB76 and HAG3/MYB29 functions was performed using transformants with increased or repressed transcript levels. Real-time PCR assays, cotransformation assays and measurements of glucosinolate contents were used to assess the impact of both MYB factors on the steady-state level of glucosinolate biosynthetic genes and accumulation of aliphatic glucosinolates. Both HAG2/MYB76 and HAG3/MYB29 were shown to be positive regulators of aliphatic glucosinolate biosynthesis. Expression of promoter-β-glucuronidase (GUS) fusions indicated GUS activities in both vegetative and generative organs, with distinct characteristics for each MYB factor. HAG1/MYB28, HAG2/MYB76 and HAG3/ MYB29 reciprocally transactivated each other in the control of aliphatic glucosinolate biosynthesis and downregulated the expression of genes involved in the control of indolic glucosinolate biosynthesis, pointing to a reciprocal negative regulation of these two pathways. All three HAG transcription factors exert a coordinated control on aliphatic glucosinolate biosynthesis.
Photosynthesis is central to all life on earth, providing not only oxygen but also organic compounds that are synthesized from atmospheric CO
2 and water using light energy as the driving force. The ...still-increasing world population poses a serious challenge to further enhance biomass production of crop plants. Crop yield is determined by various parameters,
inter alia by the light energy conversion efficiency of the photosynthetic machinery. Photosynthesis can be looked at from different perspectives: (i) light reactions and carbon assimilation, (ii) leaves and canopy structure, and (ii) source-sink relationships. In this review, we discuss opportunities and prospects to increase photosynthetic performance at the different layers, taking into account the recent progress made in the respective fields.
Arabidopsis (Arabidopsis thaliana) overexpressing glycolate oxidase (GO) in chloroplasts accumulates both hydrogen peroxide (H₂O₂) and glyoxylate. GO-overexpressing lines (GO plants) grown at 75 μmol ...quanta m⁻² s⁻¹ show retarded development, yellowish rosettes, and impaired photosynthetic performance, while at 30 μmol quanta m⁻² s⁻¹, this phenotype virtually disappears. The GO plants develop oxidative stress lesions under photorespiratory conditions but grow like wild-type plants under nonphotorespiratory conditions. GO plants coexpressing enzymes that further metabolize glyoxylate but still accumulate H₂O₂ show all features of the GO phenotype, indicating that H₂O₂ is responsible for the GO phenotype. The GO plants can complete their life cycle, showing that they are able to adapt to the stress conditions imposed by the accumulation of H₂O₂ during the light period. Moreover, the data demonstrate that a response to oxidative stress is installed, with increased expression and/or activity of known oxidative stress-responsive components. Hence, the GO plants are an ideal noninvasive model system in which to study the effects of H₂O₂ directly in the chloroplasts, because H₂O₂ accumulation is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions.
Plants assimilate carbon dioxide during photosynthesis in chloroplasts. Assimilated carbon is subsequently allocated throughout the plant. Generally, two types of organs can be distinguished, mature ...green source leaves as net photoassimilate exporters, and net importers, the sinks, e.g., roots, flowers, small leaves, and storage organs like tubers. Within these organs, different tissue types developed according to their respective function, and cells of either tissue type are highly compartmentalized. Photoassimilates are allocated to distinct compartments of these tissues in all organs, requiring a set of metabolite transporters mediating this intercompartmental transfer. The general route of photoassimilates can be briefly described as follows. Upon fixation of carbon dioxide in chloroplasts of mesophyll cells, triose phosphates either enter the cytosol for mainly sucrose formation or remain in the stroma to form transiently stored starch which is degraded during the night and enters the cytosol as maltose or glucose to be further metabolized to sucrose. In both cases, sucrose enters the phloem for long distance transport or is transiently stored in the vacuole, or can be degraded to hexoses which also can be stored in the vacuole. In the majority of plant species, sucrose is actively loaded into the phloem via the apoplast. Following long distance transport, it is released into sink organs, where it enters cells as source of carbon and energy. In storage organs, sucrose can be stored, or carbon derived from sucrose can be stored as starch in plastids, or as oil in oil bodies, or - in combination with nitrogen - as protein in protein storage vacuoles and protein bodies. Here, we focus on transport proteins known for either of these steps, and discuss the implications for yield increase in plants upon genetic engineering of respective transporters.
Aliphatic glucosinolate biosynthesis is highly compartmentalized, requiring import of 2-keto acids or amino acids into chloroplasts for side chain elongation and export of the resulting compounds ...into the cytosol for conversion into glucosinolate. Aliphatic glucosinolate biosynthesis in Arabidopsis thaliana is regulated by three R2R3-MYB transcription factors, the major player being High Aliphatic Glucosinolate 1 (HAG1/MYB28). Here, we show that BAT5, which belongs to the putative bile acid transporter family, is the only member of this family that is transactivated by HAG1/MYB28, HAG2/MYB76, and HAG3/MYB29. Furthermore, two isopropylmalate isomerases genes, IPMI1 and IPMI2, and the isopropylmalate dehydrogenase gene, IPMDH1, were identified as targets of HAG1/MYB28 and the corresponding proteins localized to plastids, suggesting a role in plastidic chain elongation reactions. The BAT proteins also localized to plastids; however, only mutants defective in BAT5 function contained strongly reduced levels of aliphatic glucosinolates. The bat5 mutant chemotype was rescued by induced overexpression of BAT5. Feeding experiments using 2-keto acids and amino acids of different chain length suggest that BAT5 is a plastidic transporter of (chain-elongated) 2-keto acids. Mechanical stimuli and methyl jasmonate transiently induced BAT5 expression in inflorescences and leaves. Thus, BAT5 was identified as the first transporter component of the aliphatic glucosinolate biosynthetic pathway.
Plants can metabolize sulfate by two pathways, which branch at the level of adenosine 5'-phosphosulfate (APS). APS can be reduced to sulfide and incorporated into Cys in the primary sulfate ...assimilation pathway or phosphorylated by APS kinase to 3'-phosphoadenosine 5'-phosphosulfate, which is the activated sulfate form for sulfation reactions. To assess to what extent APS kinase regulates accumulation of sulfated compounds, we analyzed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, and the sulfated 12-hydroxyjasmonate were reduced approximately fivefold in apk1 apk2 plants. Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. It also led to great alterations in sulfur metabolism: the levels of sulfate and thiols increased in the apk1 apk2 plants. The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in the synthesis of secondary sulfated metabolites and are required for normal growth rates.
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