Regulation Of Phenylpropanoid Metabolism Research Articles

Comparative protein analysis of two maize genotypes with contrasting tolerance to low temperature

BackgroundLow temperature (LT) stress is one of the major environmental stress factors affecting the growth and yield of maize (Zea mays L.). Hence, it is important to unravel the molecular mechanisms behind LT stress tolerance to improve molecular breeding in LT tolerant genotypes. In the present study, two maize genotypes viz. Gurez local from Kashmir Himalaya and tropical grown GM6, were dissected for their LT stress response in terms of accumulation of differentially regulated proteins (DRPs). Leaf proteome analysis at three-leaf stage of maize seedlings subjected to LT stress of 6 °C for a total of 12 h duration was performed using two dimensional gel electrophoresis (2D-PAGE) followed by subsequent identification of the proteins involved.ResultsAfter MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) and bioinformatics analysis, 19 proteins were successfully identified in Gurez local, while as 10 proteins were found to get successful identification in GM6. The interesting observations from the present investigation is the identification of three novel proteins viz. threonine dehydratase biosynthetic chloroplastic, thylakoidal processing peptidase 1 chloroplastic, and nodulin-like protein, whose role in abiotic stress tolerance, in general, and LT stress, in particular, has not been reported so far. It is important to highlight here that most of LT responsive proteins including the three novel proteins were identified from Gurez local only, owing to its exceptional LT tolerance. From the protein profiles, obtained in both genotypes immediately after LT stress perception, it was inferred that stress responsive protein accumulation and their expression fashion help the Gurez local in seedling establishment and withstand unfavorable conditions as compared to GM6. This was inferred from the findings of pathway enrichment analysis like regulation of seed growth, timing of floral transition, lipid glycosylation, and aspartate family amino acid catabolic processes, besides other key stress defense mechanisms. However, in GM6, metabolic pathways enriched were found to be involved in more general processes including cell cycle DNA replication and regulation of phenylpropanoid metabolism. Furthermore, majority of the qRT-PCR results of the selected proteins demonstrated positive correlation between protein levels and transcript abundance, thereby strengthening our findings.ConclusionsIn conclusion, our findings reported majority of the identified proteins in Gurez local exhibiting up-regulated pattern under LT stress as compared to GM6. Furthermore, three novel proteins induced by LT stress were found in Gurez local, requiring further functional validation. Therefore, our results offer more insights for elucidating the molecular networks mediating LT stress tolerance in maize.

Regulation of phenylpropanoid metabolism during moderate freezing and post-freezing recovery in Dendrobium officinale

ABSTRACT Freezing injury is a major environmental limiting factor affecting plant yield and geographical distribution. Little is known about the regulation of phenylpropanoid metabolism during freezing (FT) and post-freezing recovery (FR). Dendrobium officinale is a valuable medicinal plant and hypersensitive to low temperature, especially freezing in winter. Here, we used metabolomics and transcriptomics analyses to reveal the regulation of of phenylpropanoid metabolism during FT and FR in D. officinale. Most anthocyanin and flavonol significantly increased during FR, including quercetin 3-O-glucoside (Q3G) and cyanidin 3-O-sophoroside (C3S) increased 104.6-fold and 66.8-fold in FR vs. CK. Transcriptomics analyses revealed the expression levels of flavonoid 3′- hydroxylase (F3`H) and flavonol synthase (FLS), which were related to an increase in quercetin derivatives. Six phytohormone-related GO terms were enriched during FT and FR. Four genes were significantly affected by FT and FR, including MYB97, MYB39, fructosyltransferase, and zinc finger transcript factor. Four GA response-related genes, 2-oxoglutarate-dependent dioxygenases, were specially induced during freezing stress. Moreover, jasmonic acid (JA) and salicylic acid (SA) were significantly increased during FT, and only SA decreased during FR. This study provided understanding of phenylpropanoid metabolism under freezing stress and will be useful for enhancing freezing tolerance via molecular breeding in D. officinale.

AtMYB32 regulates the ABA response by targeting ABI3, ABI4 and ABI5 and the drought response by targeting CBF4 in Arabidopsis

The Arabidopsis thaliana R2R3-MYB transcription factor AtMYB32 and its homologs AtMYB4 and AtMYB7 play crucial roles in the regulation of phenylpropanoid metabolism. In addition, AtMYB4 and AtMYB7 are involved in the response to abiotic stress. However, the function of AtMYB32 remains unclear. In this study, we found that AtMYB32 is induced by abscisic acid (ABA) and repressed by drought stress. AtMYB32 positively regulates ABA-mediated seed germination and early seedling development. The expression of ABSCISIC ACID-INSENSITIVE 3 (ABI3), ABI4 and ABI5, which encode key positive regulators of ABA signaling, was upregulated in response to ABA in AtMYB32-overexpressing plants and downregulated in the atmyb32-1 mutant. In addition, we found that the atmyb32-1 mutant was drought resistant. Consistent with the drought-resistant phenotype, the transcript levels of C-repeat binding factor 4 (CBF4) were higher in the atmyb32-1 mutant in response to drought stress. Electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays revealed that AtMYB32 binds directly to the ABI3, ABI4, ABI5 and CBF4 promoters both in vitro and in vivo. Genetically, ABI4 was found to be epistatic to AtMYB32 for ABA-induced inhibition of seed germination and early seedling development. Taken together, our findings revealed that AtMYB32 regulates the ABA response by directly promoting ABI3, ABI4 and ABI5 expression and that the drought stress response likely occurs because of repression of CBF4 expression.

Contribution of phenylpropanoid metabolism to plant development and plant-environment interactions.

Phenylpropanoid metabolism is one of the most important metabolisms in plants, yielding more than 8,000 metabolites contributing to plant development and plant-environment interplay. Phenylpropanoid metabolism materialized during the evolution of early freshwater algae that were initiating terrestrialization and land plants have evolved multiple branches of this pathway, which give rise to metabolites including lignin, flavonoids, lignans, phenylpropanoid esters, hydroxycinnamic acid amides, and sporopollenin. Recent studies have revealed that many factors participate in the regulation of phenylpropanoid metabolism, and modulate phenylpropanoid homeostasis when plants undergo successive developmental processes and are subjected to stressful environments. In this review, we summarize recent progress on elucidating the contribution of phenylpropanoid metabolism to the coordination of plant development and plant-environment interaction, and metabolic flux redirection among diverse metabolic routes. In addition, our review focuses on the regulation of phenylpropanoid metabolism at the transcriptional, post-transcriptional, post-translational, and epigenetic levels, and in response to phytohormones and biotic and abiotic stresses.

MYB Repressors as Regulators of Phenylpropanoid Metabolism in Plants.

The phenylpropanoid pathway gives rise to lignin, flavonoids, and other metabolites and is regulated by MYB transcription factors. Many R2R3-MYB transcriptional activators are known, but the prevalence of MYB repressors has only recently become recognized. This review article summarizes recent progress on function and mechanism of these MYB repressors. The characterized phenylpropanoid R2R3-MYB repressors comprise two phylogenetic clades that act on the lignin and general phenylpropanoid genes, or the flavonoid genes, respectively; anthocyanin R3-MYB repressors form a separate clade. While some flavonoid MYBs repressors can bind basic-helix-loop-helix factors and disrupt the MBW complex, for the lignin repressor MYBs interactions with promoter cis-elements have been demonstrated. The role of the conserved repression motifs that define the MYB repressors is not yet known, however.

Transcription factors, sucrose, and sucrose metabolic genes interact to regulate potato phenylpropanoid metabolism

Much remains unknown about how transcription factors and sugars regulate phenylpropanoid metabolism in tuber crops like potato (Solanum tuberosum). Based on phylogeny and protein similarity to known regulators of phenylpropanoid metabolism, 15 transcription factors were selected and their expression was compared in white, yellow, red, and purple genotypes with contrasting phenolic and anthocyanin profiles. Red and purple genotypes had increased phenylalanine ammonia lyase (PAL) enzyme activity, markedly higher levels of phenylpropanoids, and elevated expression of most phenylpropanoid structural genes, including a novel anthocyanin O-methyltransferase. The transcription factors Anthocyanin1 (StAN1), basic Helix Loop Helix1 (StbHLH1), and StWD40 were more strongly expressed in red and purple potatoes. Expression of 12 other transcription factors was not associated with phenylpropanoid content, except for StMYB12B, which showed a negative relationship. Increased expression of AN1, bHLH1, and WD40 was also associated with environmentally mediated increases in tuber phenylpropanoids. Treatment of potato plantlets with sucrose induced hydroxycinnamic acids, flavonols, anthocyanins, structural genes, AN1, bHLH1, WD40, and genes encoding the sucrose-hydrolysing enzymes SUSY1, SUSY4, and INV2. Transient expression of StAN1 in tobacco leaves induced bHLH1, structural genes, SUSY1, SUSY4, and INV1, and increased phenylpropanoid amounts. StAN1 infiltration into tobacco leaves decreased sucrose and glucose concentrations. In silico promoter analysis revealed the presence of MYB and bHLH regulatory elements on sucrolytic gene promoters and sucrose-responsive elements on the AN1 promoter. These findings reveal an interesting dynamic between AN1, sucrose, and sucrose metabolic genes in modulating potato phenylpropanoids.

Comprehensive Analysis of Glucan Elicitor-Regulated Gene Expression in Tobacco BY-2 Cells Reveals a Novel MYB Transcription Factor Involved in the Regulation of Phenylpropanoid Metabolism

We previously demonstrated that a beta-1,3-, 1,6-oligoglucan (AaGlucan) from the fungus Alternaria alternata 102 shows strong elicitor activity in tobacco BY-2 cells. We have used cDNA microarray analysis to monitor global changes in gene expression in tobacco cells treated with this A. alternata fraction or with laminarin. In total, we identified 265 genes that were induced 1 h after treatment with an AaGlucan-enriched fraction or laminarin. Among them, we characterized in detail a novel tobacco R2R3 MYB-type transcription factor homolog (NtMYBGR1) and two DC1 domain-containing genes (NtDC1A and NtDC1B). Microarray data, together with overexpression and metabolic analyses, indicated that NtMYBGR1, but not the NtDC1 proteins, primarily targets the phenylpropanoid synthesis-related genes PAL and 4CL. These results suggest that NtMYBGR1 specifically regulates defense responses in BY-2 cells by enhancing phenylpropanoid metabolism in response to AaGlucan and laminarin elicitors.

Transcription factor AtDOF4;2 affects phenylpropanoid metabolism in Arabidopsis thaliana

In a phenotypic screen of plants constitutively overexpressing DOF (DNA-binding-with-one-finger) transcription factors under the control of the Cauliflower mosaic virus 35S promoter, AtDOF4;2 was identified as a gene inducing a bushy plant phenotype and potentially being involved in the regulation of phenylpropanoid metabolism in Arabidopsis. Further molecular and biochemical characterization was performed in parallel using transgenic plants with enhanced and reduced AtDOF4;2 expression. The expression pattern of AtDOF4;2 was determined by quantitative real-time polymerase chain reaction (Q-RTPCR) and through promoter-beta-glucuronidase (GUS) fusions, indicating preferential transcriptional activity in axillary buds of the flower stalk, the hypocotyls periderm and in tapetum cells. Constitutive overexpression and RNAi-mediated silencing of AtDOF4;2 caused reciprocal changes in the expression of flavonoid biosynthetic genes and the accumulation of flavonoids under cold and high-light conditions. Moreover, tapetum-specific overexpression of AtDOF4;2 led to pollen grains devoid of flavonols. In contrast to its negative influence on flavonoid biosynthesis and coincident with high expression in the periderm and tapetum, AtDOF4;2 positively influences the production of hydroxycinnamic acids in the hypocotyl and flower buds, implicating its possible importance for suberin and sporopollenin production. These data provide evidence that AtDOF4;2, influences phenylpropanoid metabolism in an environmental and tissue-specific manner.

The AmMYB308 and AmMYB330 transcription factors from antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco

MYB-related transcription factors are known to regulate different branches of flavonoid metabolism in plants and are believed to play wider roles in the regulation of phenylpropanoid metabolism in general. Here, we demonstrate that overexpression of two MYB genes from Antirrhinum represses phenolic acid metabolism and lignin biosynthesis in transgenic tobacco plants. The inhibition of this branch of phenylpropanoid metabolism appears to be specific to AmMYB308 and AmMYB330, suggesting that they recognize their normal target genes in these transgenic plants. Experiments with yeast indicate that AmMYB308 can act as a very weak transcriptional activator so that overexpression may competitively inhibit the activity of stronger activators recognizing the same target motifs. The effects of the transcription factors on inhibition of phenolic acid metabolism resulted in complex modifications of the growth and development of the transgenic plants. The inhibition of monolignol production resulted in plants with at least 17% less lignin in their vascular tissue. This reduction is of importance when designing strategies for the genetic modification of woody crops.

Regulation of phenylpropanoid metabolism by exogenous precursors in axenic cultures of Sphagnum fallax

Regulation of phenylpropanoid metabolism by exogenous precursors in axenic cultures of Sphagnum fallax

Regulation of phenylpropanoid metabolism by exogenous precursors in axenic cultures of Sphagnum fallax

Sphagnum plantlets, cultivated in continuous‐feed bioreactors, are characterised by high levels of free endogenous phenolics and a pronounced excretion of some phenolics into the effluent culture medium. The transfer of Sphagnum fallax, precultivated in continuous‐feed bioreactors, to batch cultures resulted in an increased flux through phenylpropanoid metabolism and an accumulation of p‐coumaric acid to 0.1 μM and of trans‐sphagnum acid up to 0.5 μM in the external medium [3H]‐labelled L‐phenylalanine (7.7 GBq mol−1) was rapidly taken up, resulting in an enhanced synthesis and excretion of p‐coumaric and trans‐sphagnum acid. Specific activities were 6.9 and 5.4 GBq mol−1, respectively, for these cinnamic acids excreted into the external medium. Endogenous pools of trans‐cinnamic and p‐coumaric acid did not increase and no labelling could be detected in these compounds. Cell wall‐bound activity amounted to ca 14% of the applied activity after 48 h of incubation, 59% of which was recovered in dioxane/2 M HCl extracts of the cell wall. Exogenously applied trans‐cinnamic acid (0.1 mM) was taken up to 46% and resulted in a transient endogenous accumulation of trans‐cinnamic acid, the level of free endogenous p‐coumaric and trans‐sphagnum acid was found to have decreased. The concentrations of p‐coumaric and trans‐sphagnum acid in the culture medium rose to 17 and 2.4 μM, respectively, after 48 h of incubation in 0.1 mMtrans‐cinnamic acid. Exogenously applied p‐coumaric acid (0.1 mM) was taken up to 79% from the incubation solution but not stored endogenously, as metabolic products trans‐sphagnum acid and an unknown p‐coumaric acid‐conjugate accumulated in the external medium and endogenously. These results give evidence for the biosynthetical route from phenylalanine to sphagnum acid and a channelling of pathway intermediates by the enzymes L‐phenylalanine ammonia‐lyase (EC 4.3.1.5) and cinnamic acid 4‐hydroxylase (EC 1.14.13.11).

Immunochemical localization of phenylalanine ammonia-lyase and chalcone synthase in anthers

Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) from anthers of the garden tulip "Apeldoorn" have been purified to apparent homogeneity as revealed by sodium dodecyl sulfate disc-gel electrophoresis. Phenylalanine ammonia-lyase was either purified by successive chromatography on Sephacryl S 300 Superfine, HA Ultrogel and on diethylaminoethyl Sephacel or by immunoaffinity chromatography in a single step. Purification of CHS was achieved by chromatography on Sephadex G 200 and on HA Ultrogel followed by chromatofocusing. The purified enzymes were used for the immunization of rabbits. The specificity of the antisera against both PAL and CHS was tested by diverse methods. Antisera against PAL and CHS were employed to detect the localization of the enzymes in cross sections of tulip anthers using an indirect immunofluorometric method. The results show that PAL and CHS are located predominantly in the tapetum cells. These observations strengthen the view that the tapetum plays an important role in the regulation of phenylpropanoid metabolism within the loculus of anthers.