Search

The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. This network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional‐level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription – quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one‐hybrid, and effector–reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL‐ASSOCIATED NAC‐DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1‐driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis. [ABSTRACT FROM AUTHOR]

Rhizoctonia solani is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. It deploys effector molecules as well as carbohydrate-active enzymes and enhances the production of reactive oxygen species for killing host tissues. Understanding R. solani ability to sustain growth under an oxidative-stress-enriched environment is important for developing disease control strategies. Here, we demonstrate that R. solani upregulates methionine biosynthetic genes, including Rs_MET13 during infection in rice, and double-stranded RNA-mediated silencing of these genes impairs the pathogen's ability to cause disease. Exogenous treatment with methionine restores the disease-causing ability of Rs_MET13-silenced R. solani and facilitates its growth on 10 mM H 2 O 2 -containing minimal-media. Notably, the Rs_MsrA gene that encodes methionine sulfoxide reductase A, an antioxidant enzyme involved in the repair of oxidative damage of methionine, is upregulated upon H 2 O 2 treatment and also during infection in rice. Rs_MsrA-silenced R. solani is unable to cause disease, suggesting that it is important for the repair of oxidative damage in methionine during host colonization. We propose that spray-induced gene silencing of Rs_MsrA and designing of antagonistic molecules that block MsrA activity can be exploited as a drug target for effective control of sheath blight disease in rice., (© 2024 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd.)

Artemisinin is the most practical medication for the treatment of malaria, but is only very minimally synthesized in Artemisia annua, significantly less than the market needs. In this study, indole-3-acetic acid (IAA) was used to investigate its effects on trichomes, artemisinin accumulation, and biosynthetic gene expression in A. anuua. The results showed that exogenous IAA could contribute to the growth and development of A. annua and increase the density of trichomes. Analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that artemisinin and dihydroartemisinic acid (DHAA) contents were increased by 1.9-fold (1.1 mg/g) and 2.1-fold (0.51 mg/g) after IAA treatment in comparison with control lines (CK), respectively. Furthermore, quantitative real-time PCR results showed that AaADS, AaCYP71AV1, AaALDH1, and AaDBR2, four critical enzyme genes for the biosynthesis of artemisinin, had relatively high transcription levels in leaves of A. annua treated with IAA. In summary, this study indicated that exogenous IAA treatment was a feasible strategy to enhance artemisinin production, which paves the way for further metabolic engineering of artemisinin biosynthesis., (© 2023 International Union of Biochemistry and Molecular Biology, Inc.)

Melatonin participates in plant growth and development and biotic and abiotic stress responses. Histone acetylation regulates many plant biological processes via transcriptional reprogramming. However, the direct relationship between melatonin and histone acetylation in plant disease resistance remains unclear. In this study, we identified cassava bacterial blight (CBB) responsive histone deacetylase 9 (HDA9), which negatively regulated disease resistance to CBB by reducing melatonin content. In addition, exogenous melatonin alleviated disease sensitivity of MeHDA9 overexpressed plants to CBB. Importantly, MeHDA9 inhibited the expression of melatonin biosynthetic genes through decreasing lysine 5 of histone 4 (H4K5) acetylation at the promoter regions of melatonin biosynthetic genes, thereby modulating melatonin accumulation in cassava. Furthermore, protein phosphatase 2C 12 (MePP2C12) interacted with MeHDA9 in vivo and in vitro, and it was involved in MeHDA9‐mediated disease resistance via melatonin biosynthetic pathway. In summary, this study highlights the direct interaction between histone deacetylation and melatonin biosynthetic genes in cassava disease resistance via histone deacetylation, providing new insights into the genetic improvement of disease resistance via epigenetic regulation of melatonin level in tropical crops. [ABSTRACT FROM AUTHOR]

Cassava bacterial blight (CBB) is one of the most serious diseases in cassava production, so it is essential to explore the underlying mechanism of immune responses. Histone acetylation is an important epigenetic modification, however, its relationship with cassava disease resistance remains unclear. Here, we identified 10 histone acetyltransferases in cassava and found that the transcript of MeHAM1 showed the highest induction to CBB. Functional analysis showed that MeHAM1 positively regulated disease resistance to CBB through modulation of salicylic acid (SA) accumulation. Further investigation revealed that MeHAM1 directly activated SA biosynthetic genes' expression via promoting lysine 9 of histone 3 (H3K9) acetylation and lysine 5 of histone 4 (H4K5) acetylation of these genes. In addition, molecular chaperone MeDNAJA2 physically interacted with MeHAM1, and MeDNAJA2 also regulated plant immune responses and SA biosynthetic genes. In conclusion, this study illustrates that MeHAM1 and MeDNAJA2 confer immune responses through transcriptional programming of SA biosynthetic genes via histone acetylation. The MeHAM1 & MeDNAJA2‐SA biosynthesis module not only constructs the direct relationship between histone acetylation and cassava disease resistance, but also provides gene network with potential value for genetic improvement of cassava disease resistance. Summary statement: Histone acetyltransferase MeHAM1 interacts with molecular chaperone MeDNAJA2, and both of them confer immune responses through promoting histone acetylation of SA biosynthetic genes and SA accumulation. [ABSTRACT FROM AUTHOR]

Rhizoctonia solani is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. It deploys effector molecules as well as carbohydrate‐active enzymes and enhances the production of reactive oxygen species for killing host tissues. Understanding R. solani ability to sustain growth under an oxidative‐stress‐enriched environment is important for developing disease control strategies. Here, we demonstrate that R. solani upregulates methionine biosynthetic genes, including Rs_MET13 during infection in rice, and double‐stranded RNA‐mediated silencing of these genes impairs the pathogen's ability to cause disease. Exogenous treatment with methionine restores the disease‐causing ability of Rs_MET13‐silenced R. solani and facilitates its growth on 10 mM H2O2‐containing minimal‐media. Notably, the Rs_MsrA gene that encodes methionine sulfoxide reductase A, an antioxidant enzyme involved in the repair of oxidative damage of methionine, is upregulated upon H2O2 treatment and also during infection in rice. Rs_MsrA‐silenced R. solani is unable to cause disease, suggesting that it is important for the repair of oxidative damage in methionine during host colonization. We propose that spray‐induced gene silencing of Rs_MsrA and designing of antagonistic molecules that block MsrA activity can be exploited as a drug target for effective control of sheath blight disease in rice. [ABSTRACT FROM AUTHOR]

SUMMARY: Senescence is a gradual physiological process involving the integration of numerous internal and environmental signals. Abscisic acid (ABA) is a well‐known inducer of senescence. However, the regulatory mechanisms underlying ABA‐mediated senescence remain largely unknown. Here, we report that the citrus homeodomain leucine zipper I (HD‐ZIP I) transcription factor CsHB5 functions as a regulator of ABA‐triggered senescence. CsHB5 acts as a nucleus‐localized transcriptional activator, the expression of which appeared to be closely associated with citrus senescence. Overexpression of CsHB5 in citrus calli upregulated the expression of ABA‐ and reactive oxygen species (ROS)‐related genes, and significantly increased the content of ABA and hydrogen peroxide (H2O2), whereas silencing CsHB5 in citrus calli downregulated the expression of ABA‐related genes. Additionally, heterogenous overexpression of CsHB5 in Solanum lycopersicum (tomato) and Arabidopsis thaliana (Arabidopsis) leads to early leaf yellowing under dark‐induced senescence conditions. Meanwhile, the levels of ABA and H2O2 in transgenic tomatoes increased significantly and the lycopene content decreased. Transcriptome analysis of CsHB5‐overexpressing citrus calli and tomato showed that CsHB5 was involved in multiple senescence‐associated processes, including chlorophyll degradation, nutrient compound biosynthesis and transport, as well as ABA and ROS signal transduction. The results of yeast one‐hybrid assays, electrophoretic mobility shift assays and dual luciferase assays indicated that CsHB5 directly binds to the promoters of ABA biosynthetic genes, including β‐carotene hydroxylase 1 (BCH1) and 9‐cis‐epoxycarotenoid dioxygenase 2 (NCED2), thereby activating their transcription. Our findings revealed that CsHB5 participates in senescence, at least partly, by directly controlling ABA accumulation. Our work provides insight into the regulatory mechanisms underlying ABA‐mediated senescence. Significance Statement: Abscisic acid (ABA) is a well‐defined inducer of senescence. However, the regulatory mechanisms underlying ABA‐mediated senescence remain largely unknown. Here we report that transcription factor CsHB5 directly binds to the promoters of ABA biosynthetic genes and activates their expression to promote ABA accumulation, thereby triggering senescence. This study provides insight into the regulatory mechanism underlying ABA‐mediated senescence. [ABSTRACT FROM AUTHOR]

Summary: Maize is one of the world's most widely cultivated crops. As future demands for maize will continue to rise, fields will face ever more frequent and extreme weather patterns that directly affect crop productivity. Development of environmentally resilient crops with improved standability in the field, like wheat and rice, was enabled by shifting the architecture of plants to a short stature ideotype. However, such architectural change has not been implemented in maize due to the unique interactions between gibberellin (GA) and floral morphology which limited the use of the same type of mutations as in rice and wheat. Here, we report the development of a short stature maize ideotype in commercial hybrid germplasm, which was generated by targeted suppression of the biosynthetic pathway for GA. To accomplish this, we utilized a dominant, miRNA‐based construct expressed in a hemizygous state to selectively reduce expression of the ZmGA20ox3 and ZmGA20ox5 genes that control GA biosynthesis primarily in vegetative tissues. Suppression of both genes resulted in the reduction of GA levels leading to inhibition of cell elongation in internodal tissues, which reduced plant height. Expression of the miRNA did not alter GA levels in reproductive tissues, and thus, the reproductive potential of the plants remained unchanged. As a result, we developed a dominant, short‐stature maize ideotype that is conducive for the commercial production of hybrid maize. We expect that the new maize ideotype would enable more efficient and more sustainable maize farming for a growing world population. [ABSTRACT FROM AUTHOR]

Until recently, Fusarium fujikuroi was widely described as a non‐producer or a producer of little B‐series fumonisin toxins despite having a complement of fumonisin biosynthetic genes similar to those in F. verticillioides and F. proliferatum. Although high and low fumonisin‐producing F. fujikuroi strains have been shown to induce typical disease symptoms, the relationship between fumonisin production and virulence in F. fujikuroi has not been fully established. Following recent reports on the roles of bacterial endosymbionts in influencing the biology of their fungal hosts, including virulence, reproduction and the production of secondary metabolites, this study investigated the association of two strains of bacteria of the genus Enterobacter with a F. fujikuroi strain isolated from wheat in Nebraska. Results demonstrated the intracellular localization of bacteria within the hyphae of the fungus. The association with the bacteria was also correlated with macroconidia production and higher levels of virulence in the fungus. Additionally, fumonisin production was consistently and significantly (α = 0.05) higher in the bacteria‐containing F. fujikuroi strain compared with the bacteria‐free strain. Furthermore, higher levels of fumonisin production by bacteria‐containing F. fujikuroi correlated with higher expression levels of the fumonisin biosynthetic genes fum1, fum8 and fum21 under fumonisin‐inducing and non‐inducing conditions. Expression of fum15, which encodes a P450 monooxygenase, was similarly observed to be significantly (α = 0.05) higher in the bacteria‐containing fungus. Analysis of transcripts also revealed significantly (α = 0.05) higher expression of the sexual and asexual development activator protein, VeA, in the bacteria‐containing F. fujikuroi under fumonisin‐inducing conditions. [ABSTRACT FROM AUTHOR]

Summary: Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin‐based polyacylated anthocyanins. However, recent breeding programs developed several red‐flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high‐performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin‐based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red‐flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6‐O‐glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol‐6‐O‐(6′‐O‐malonyl)‐glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3‐O‐glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell‐free protein expression system and assayed. We determined that a UDP‐glucose‐dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red‐flowered gentians via copigmentation effects. [ABSTRACT FROM AUTHOR]

BACKGROUND: Anthocyanins, a major flavonoid class, determine the color and quality of wine. Recent research revealed that basal leaf removal can increase the content of these compounds. This study determined the effects of basal leaf removal on the anthocyanin profiles of Cabernet Sauvignon grapes. RESULTS: The effects of basal leaf removal on anthocyanin composition in Cabernet Sauvignon grapes were investigated over two growing seasons. Leaf removal at 5% veraison (VB6) and at 100% veraison (VC6) was compared with a control. Reducing sugar and total anthocyanin contents in the leaf removal group were significantly higher than in the control group at harvest for both vintages. Leaf removal increased the content of individual anthocyanins and significantly improved the malvidin‐3‐O‐glucoside (Mv‐3‐glc), peonidin‐3‐O‐glucoside (Pn‐3‐glc), and malvidin‐3‐O‐(6‐acetyl)‐glucoside (Mv‐3‐acglc) content of the VB6 group. At harvest, VB6 treatment increased the transcript abundance of structural and regulator genes in the anthocyanin pathway, especially VvF3′5′H, VvLDOX, and VvDFR. CONCLUSIONS: Our results suggest that leaf removal at 5% veraison may be useful for improving the anthocyanin content in grapes. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Summary: 2‐Phenylethylglucosinolate (2PE) derived from homophenylalanine is present in plants of the Brassicales order as a defense compound. It is associated with multiple biological properties, including deterrent effects on pests and antimicrobial and health‐promoting functions, due to its hydrolysis product 2‐phenylethyl isothiocyanate, which confers 2PE as a potential application in agriculture and industry. In this study, we characterized the putative key genes for 2PE biosynthesis from Barbarea vulgaris W.T. Aiton and demonstrated the feasibility of engineering 2PE production in Nicotiana benthamiana Domin. We used different combinations of genes from B. vulgaris and Arabidopsis thaliana (L.) Heynh. to demonstrate that: (i) BvBCAT4 performed more efficiently than AtBCAT4 in biosynthesis of both homophenylalanine and dihomomethionine; (ii) MAM1 enzymes were critical for the chain‐elongated profile, while CYP79F enzymes accepted both chain‐elongated methionine and homophenylalanine; (iii) aliphatic but not aromatic core structure pathway catalyzed the 2PE biosynthesis; (iv) a chimeric pathway containing BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in a two‐fold increase in 2PE production compared with the B. vulgaris‐specific chain elongation pathway; and (v) profiles of chain‐elongated products and glucosinolates partially mirrored the profiles in the gene donor plant, but were wider in N. benthamiana than in the native plants. Our study provides a strategy to produce the important homophenylalanine and 2PE in a heterologous host. Furthermore, chimeric engineering of the complex 2PE biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes – a prerequisite for understanding biochemical evolution. The new‐to‐nature gene combinations have the potential for application in biotechnological and plant breeding. Significance Statement: In this study, we engineered the production of the multifunctional 2‐phenylethylglucosinolate by heterologous expression in Nicotiana benthamiana using genes from Barbarea vulgaris and Arabidopsis thaliana, and we infer the substrate specificities of MAM1 and CYP79F enzymes by chimeric engineering of the complex biosynthetic pathway. The work provides basic knowledge on biochemical evolution of these enzymes and constitutes an important step towards unfolding the biotechnological and plant breeding potentials of 2‐phenylethylglucosinolate. [ABSTRACT FROM AUTHOR]

The essential micronutrient boron (B) has key roles in cell wall integrity and B deficiency inhibits plant growth. The role of jasmonic acid (JA) in plant growth inhibition under B deficiency remains unclear. Here, we report that low B elevates JA biosynthesis in Arabidopsis thaliana by inducing the expression of JA biosynthesis genes. Treatment with JA inhibited plant growth and, a JA biosynthesis inhibitor enhanced plant growth, indicating that the JA induced by B deficiency affects plant growth. Furthermore, examination of the JA signaling mutants jasmonate resistant1, coronatine insensitive1‐2, and myc2 showed that JA signaling negatively regulates plant growth under B deficiency. We identified a low‐B responsive transcription factor, ERF018, and used yeast one‐hybrid assays and transient activation assays in Nicotiana benthamiana leaf cells to demonstrate that ERF018 activates the expression of JA biosynthesis genes. ERF018 overexpression (OE) lines displayed stunted growth and up‐regulation of JA biosynthesis genes under normal B conditions, compared to Col‐0 and the difference between ERF018 OE lines and Col‐0 diminished under low B. These results suggest that ERF018 enhances JA biosynthesis and thus negatively regulates plant growth. Taken together, our results highlight the importance of JA in the effect of low B on plant growth. [ABSTRACT FROM AUTHOR]

Summary: The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1,PSY2), lycopene β‐cyclase, β‐carotene hydroxylase, zeaxanthin epoxidase and capsanthin‐capsorubin synthase (CCS) genes, in 94 pepper accessions by single‐molecule real‐time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra‐performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways. [ABSTRACT FROM AUTHOR]

Banana fruit (Musa acuminate L.) ripening is a complex genetical process affected by multiple phytohormones and expression of various genes. However, whether plant hormone brassinosteroid (BR) is involved in this process remains obscure. In this work, three genes that encode BR core signaling components brassinazole resistant (BZR) proteins, namely MaBZR1 to MaBZR3, were characterized from banana fruit. MaBZR1‐MaBZR3 exhibited both nuclear and cytoplasmic localization and behaved as transcription inhibitors. Expression analysis showed that MaBZR1/2/3 were continuously decreased as fruit ripening proceeded, indicating their negative roles in banana ripening. Moreover, gel shift and transient expression assays demonstrated that MaBZR1/2 could suppress the transcription of ethylene biosynthetic genes, including MaACS1, MaACO13 and MaACO14, which increased gradually during the banana ripening, via specifically binding to CGTGT/CG sequence in their promoters. Importantly, exogenous application of BRs promotes banana ripening, which is presumably due to the accelerated expression of MaACS1 and MaACO13/14, and consequently the ethylene production. Our study indicates that MaBZR1/2 act as transcriptional repressors of ethylene biosynthetic genes during banana fruit ripening. [ABSTRACT FROM AUTHOR]

Summary: The gaseous plant hormone ethylene induces the ripening of climacteric fruit, including apple (Malus domestica). Another phytohormone, auxin, is known to promote ethylene production in many horticultural crops, but the regulatory mechanism remains unclear.Here, we found that auxin application induces ethylene production in apple fruit before the stage of commercial harvest, when they are not otherwise capable of ripening naturally.The expression of MdARF5, a member of the auxin response factor transcription factor (TF) family involved in the auxin signaling pathway, was enhanced by treatment with the synthetic auxin naphthaleneacetic acid (NAA). Further studies revealed that MdARF5 binds to the promoter of MdERF2, encoding a TF in the ethylene signaling pathway, as well as the promoters of two 1‐aminocyclopropane‐1‐carboxylic acid synthase (ACS) genes (MdACS3a and MdACS1) and an ACC oxidase (ACO) gene, MdACO1, all of which encode key steps in ethylene biosynthesis, thereby inducing their expression. We also observed that auxin‐induced ethylene production was dependent on the methylation of the MdACS3a promoter.Our findings reveal that auxin induces ethylene biosynthesis in apple fruit through activation of MdARF5 expression. [ABSTRACT FROM AUTHOR]

BACKGROUND: Glucosinolates (GSLs) are secondary metabolites, mainly existing in Brassica vegetables. Their breakdown products have health benefits and contribute to the distinctive taste of these vegetables. Because of their high value, there is a lot of interest in developing breeding strategies to increase the content of beneficial GSLs in Brassica species. GSLs are synthesized from certain amino acids and their biological roles depend largely on the structure of their side chains. Flavin‐containing monooxygenase (FMOGS‐OX) genes are involved in the synthesis of these side chains. To better understand GSL biosynthesis, we sequenced the transcriptomes of turnip (Brassica rapa var. rapa) tubers at four developmental stages (S1–S4) and determined their GSL content. RESULTS: The total GSL content was high at the early stage (S1) of tuber development and increased up to S3, then decreased at S4. We detected 61 differentially expressed genes, including five FMOGS‐OX genes, that were related for GSL biosynthesis among the four developmental stages. Most of these genes were highly expressed at stages S1 to S3, but their expression was much lower at S4. We estimated the effect of the five FMOGS‐OX genes on GSL content by overexpressing them in turnip hairy roots and found that the amount of aliphatic GSLs increased significantly in the transgenic plants. CONCLUSION: The transcriptome data and characterization of genes involved in GSL biosynthesis, particularly the FMOGS‐OX genes, will be valuable for improving the yield of beneficial GSLs in turnip and other Brassica crops. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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

BACKGROUND: The pinewood nematode (Bursaphelenchus xylophilus) causes severe damage to pine trees. The nematophagous fungus, Esteya vermicola, exhibits considerable promise in the biological control of Bursaphelenchus xylophilus due to its infectivity. Notably, the lunate conidia produced by E. vermicola can infect Bursaphelenchus xylophilus. In the study, we aim to investigate the genes involved in the formation of the lunate conidia of E. vermicola CBS115803. RESULTS: Esteya vermicola CBS115803 yielded 95% lunate conidia on the complete medium (CM) and 86% bacilloid conidia on the minimal medium (MM). Transcriptomic analysis of conidia from both media revealed a significant enrichment of differentially expressed genes in the pathway related to 'cellular amino acid biosynthesis and metabolism'. Functional assessment showed that the knockout of two arginine biosynthesis genes (EV232 and EV289) resulted in defects in conidia germination, mycelial growth, lunate conidia formation, and virulence of E. vermicola CBS115803 in Bursaphelenchus xylophilus. Remarkably, the addition of arginine to the MM improved mycelial growth, conidiation and lunate conidia formation in the mutants and notably increased conidia yield and the lunate conidia ratio in the wild‐type E. vermicola CBS115803. CONCLUSION: This investigation confirms the essential role of two arginine biosynthesis genes in lunate conidia formation in E. vermicola CBS115803. The findings also suggest that the supplementation of arginine to the culture medium can enhance the lunate conidia yield. These insights contribute significantly to the application of E. vermicola CBS115803 in managing Bursaphelenchus xylophilus infections. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Summary: Phosphatidylcholine (PC) plays an important role in maintaining membrane integrity and functionality. In this study, two key genes (Mrpct and Mrpem) putatively involved in the cytidine diphosphate (CDP)‐choline and phosphatidylethanolamine N‐methyltransferase (PEMT) pathways for PC biosynthesis were characterized in the insect pathogenic fungus Metarhizium robertsii. The results indicated that disruption of Mrpct did not lead to any reduction of total PC content but impaired fungal virulence and increased cellular accumulation of triacylglycerol. Deletion of Mrpem reduced PC content and impaired fungal conidiation and infection structure differentiation but did not result in virulence defects. Lipidomic analysis revealed that deletion of Mrpct and Mrpem resulted in dissimilar effects on increase and decrease of PC moieties and other phospholipid species accumulations. Interestingly, we found that these two genes played opposite roles in activation of cell autophagy when the fungi were grown in a nutrient‐rich medium. The connection between PC metabolism and autophagy was confirmed because PC content was drastically reduced in Mratg8Δ and that the addition of PC could rescue null mutant sporulation defect. The results of this study facilitate the understanding of PC metabolism on fungal physiology. [ABSTRACT FROM AUTHOR]

Specific transcription factors have been identified in various heterotrophic bacterial species that regulate the sets of genes required for fatty acid metabolism. Here, we report that expression of the fab genes, encoding fatty acid biosynthetic enzymes, is regulated by the global regulator LexA in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803. Sll1626, an ortholog of the well-known LexA repressor involved in the SOS response in heterotrophic bacteria, was isolated from crude extracts of Synechocystis by DNA affinity chromatography, reflecting its binding to the upstream region of the acpP-fabF and fabI genes. An electrophoresis mobility shift assay revealed that the recombinant LexA protein can bind to the upstream region of each fab gene tested ( fabD, fabH, fabF, fabG, fabZ and fabI). Quantitative RT- PCR analysis of the wild type and a lexA-disrupted mutant strain suggested that LexA acts as a repressor of the fab genes involved in initiation of fatty acid biosynthesis ( fabD, fabH and fabF) and the first reductive step in the subsequent elongation cycle ( fabG) under normal growth conditions. Under nitrogen-depleted conditions, downregulation of fab gene expression is partly achieved through an increase in LexA-repressing activity. In contrast, under phosphate-depleted conditions, fab gene expression is upregulated, probably due to the loss of repression by LexA. We further demonstrate that elimination of LexA largely increases the production of fatty acids in strains modified to secrete free fatty acids. [ABSTRACT FROM AUTHOR]

The lipid membrane is one of the most characteristic traits distinguishing the three domains of life. Membrane lipids of Bacteria and Eukarya are composed of fatty acids linked to glycerol-3-phosphate (G3P) via ester bonds, while those of Archaea possess isoprene-based alkyl chains linked by ether linkages to glycerol-1-phosphate (G1P), resulting in the opposite stereochemistry of the glycerol phosphate backbone. This 'lipid divide' has raised questions on the evolution of microbial life since eukaryotes are thought to have evolved from the Archaea, requiring a radical change in membrane composition. Here, we searched for homologs of enzymes involved in membrane lipid and fatty acid synthesis in a wide variety of archaeal genomes and performed phylogenomic analyses. We found that two uncultured archaeal groups, i.e. marine euryarchaeota group II/III and 'Lokiarchaeota', recently discovered descendants of the archaeal ancestor leading to eukaryotes, lack the gene to synthesize G1P and, consequently, the capacity to synthesize archaeal membrane lipids. However, our analyses reveal their genetic capacity to synthesize G3P-based 'chimeric lipids' with either two ether-bound isoprenoidal chains or with an ester-bound fatty acid instead of an ether-bound isoprenoid. These archaea may reflect the 'archaea-to-eukaryote' membrane transition stage which have led to the current 'lipid divide'. [ABSTRACT FROM AUTHOR]

BACKGROUND In addition to their high carbohydrate content, potatoes are also an important dietary source of vitamin C and bioactive secondary metabolites, including phenolic compounds and carotenoids, which have been suggested to play a role in human health. The expression of genes encoding key enzymes involved in the synthesis of these compounds was assessed by reverse transcription-quantitative polymerase chain reaction and compared to the accumulation of the corresponding product in seven potato varieties showing contrasting levels of metabolite accumulation. RESULTS Strong positive correlations were found between phenolic content in the flesh of tubers and transcript levels of phenylalanine ammonia lyase ( PAL) and chalcone synthase ( CHS) genes. The expression of PAL and CHS was also related to that of AN1, a transcription factor involved in the synthesis of anthocyanins, suggesting that these genes are regulated in a coordinated manner. No clear relationship was found between transcript levels of phytoene synthase ( PSY) or l-galactono-1,4-lactone dehydrogenase ( GLDH) genes and total carotenoid or vitamin C accumulation, respectively. CONCLUSION Data indicate that levels of total phenolic and flavonoid compounds in potato are controlled primarily by PAL and CHS gene expression. Transcript levels of PSY and GLDH did not control accumulation of carotenoids or vitamin C. © 2015 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Summary Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific β-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 ( cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava ( Manihot esculenta) and sorghum ( Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario. [ABSTRACT FROM AUTHOR]

Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture-dependent and -independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR-43 exhibited cytotoxic activity against cancer cell lines. JBIR-34 and -35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains. [ABSTRACT FROM AUTHOR]

With 6 figures and 2 tables To elucidate the regulatory mechanisms of carotenoid accumulation in tepals of Asiatic hybrid lily ( Lilium spp.), the content and composition of carotenoids and transcriptional changes of carotenoid biosynthetic genes were compared between tepals of yellow, red, pink and white flowers. Yellow and red tepal cultivars accumulated high amounts of carotenoids, and cultivars with pink and white tepals accumulated small amounts of carotenoids. Most of the carotenoids in yellow petals were antheraxanthin, (9 Z)-violaxanthin, cis-lutein and violaxanthin. Capsanthin was accumulated in red tepals. Transcription of phytoene synthase, phytoene desaturase, ζ-carotene desaturase, carotenoid isomerase and β-ring hydroxylase in tepals increased as tepals grew. Among yellow and red tepal cultivars, the amounts of carotenoids were well associated with the transcription levels of biosynthetic genes, indicating that transcription levels regulate carotenoid accumulation in these cultivars. However, in pink and white tepals, the transcription levels of biosynthetic genes were similar to those in red tepals, indicating that small amounts of carotenoids are not explained by down-regulation of biosynthetic genes. Another factor should reduce carotenoid amounts in these tepals. [ABSTRACT FROM AUTHOR]

Trienoic fatty acids (TAs), the major constituents in plant membrane lipids, play essential roles in stress signalling as precursors of the phytohormone jasmonic acid (JA). Arabidopsis FAD7 encodes a plastidial ω-3 fatty acid desaturase, which catalyses the production of TAs. In coordination with other JA-biosynthetic genes, expression of FAD7 is induced locally by wounding. This provides a feedforward mechanism for the rapid and sustainable accumulation of JA. To identify molecular components involved in this mechanism, a transgenic Arabidopsis line carrying the FAD7 promoter ( pFAD7) fused to the firefly luciferase gene ( LUC) was constructed. Reciprocal crossing experiments revealed that the induction of FAD7 expression depends largely on JA biosynthesis and the SCFCOI1-mediated signalling mechanism, whereas JA alone is insufficient for its maximal induction. Full induction required synergistic interactions between JA-dependent and -independent wound signalling mechanisms. A genetic screen for aberrant pFAD7::LUC expression yielded a recessive mutant showing enhanced wound-induced LUC bioluminescence. The mutation was associated with the cpl1 locus encoding an RNA polymerase II C-terminal domain (CTD) phosphatase, and conferred wound hyper-responsiveness on the promoters of several JA-biosynthetic genes. The picture of signalling mechanisms underlying the wound-regulated FAD7 expression, and potential roles of CPL proteins as attenuators of wound-induced JA biosynthesis, are discussed. [ABSTRACT FROM AUTHOR]

The Streptomyces produce a plethora of secondary metabolites including antibiotics and undergo a complex developmental cycle. As a means of establishing the pathways that regulate secondary metabolite production by this important bacterial genus, the model species Streptomyces coelicolor and its relatives have been the subject of several genetic screens. However, despite the identification and characterization of numerous genes that affect antibiotic production, there is still no overall understanding of the network that integrates the various environmental and growth signals to bring about changes in the expression of biosynthetic genes . To establish new links, we are taking a biochemical approach to identify transcription factors that regulate antibiotic production in S. coelicolor. Here we describe the identification and characterization of a transcription factor, designated AtrA, that regulates transcription of actII-ORF4, the pathway-specific activator of the actinorhodin biosynthetic gene cluster in S. coelicolor. Disruption of the corresponding atrA gene, which is not associated with any antibiotic gene cluster, reduced the production of actinorhodin, but had no detectable effect on the production of undecylprodigiosin or the calcium-dependent antibiotic. These results indicate that atrA has specificity with regard to the biosynthetic genes it influences. An orthologue of atrA is present in the genome of Streptomyces avermitilis, the only other streptomycete for which there is a publicly available complete sequence. We also show that S. coelicolor AtrA can bind in vitro to the promoter of strR, a transcriptional activator unrelated to actII-ORF4 that is the final regulator of streptomycin production in Streptomyces griseus. These findings provide further evidence that the path leading to the expression of pathway-specific activators of antibiotic biosynthesis genes in disparate Streptomyces may share evolutionarily conserved components in at least some cases, even though the final activators are not related, and suggests that the regulation of streptomycin production, which serves an important paradigm, may be more complex than represented by current models. [ABSTRACT FROM AUTHOR]

Myelination of peripheral nerves by Schwann cells requires a large amount of lipid and cholesterol biosynthesis. To understand the transcriptional coordination of the myelination process, we have investigated the developmental relationship between early growth response 2 (Egr2)/Krox20– a pivotal regulator of peripheral nerve myelination– and the sterol regulatory element binding protein (SREBP) pathway, which controls expression of cholesterol/lipid biosynthetic genes. During myelination of sciatic nerve, there is a very significant induction of SREBP1 and SREBP2, as well as their target genes, suggesting that the SREBP transactivators are important regulators in the myelination process. Egr2/Krox20 does not appear to directly regulate the levels of SREBP pathway components, but rather, we found that Egr2/Krox20 and SREBP transactivators can synergistically activate promoters of several SREBP target genes, indicating that direct induction of cholesterol/lipid biosynthetic genes by Egr2/Krox20 is a part of the myelination program regulated by this transactivator. [ABSTRACT FROM AUTHOR]

Vitrella brassicaformis is the second discovered species in the Chromerida, and first in the family Vitrellaceae. Chromera velia, the first discovered species, forms an independent photosynthetic lineage with V. brassicaformis, and both are closely related to peridinin-containing dinoflagellates and nonphotosynthetic apicomplexans; both also show phylogenetic closeness with red algal plastids. We have utilized gas chromatography/mass spectrometry to identify two free sterols, 24-ethylcholest-5-en-3β-ol, and a minor unknown sterol which appeared to be a C28:4 compound. We have also used RNA Seq analysis to identify seven genes found in the nonmevalonate/methylerythritol pathway ( MEP) for sterol biosynthesis. Subsequent genome analysis of V. brassicaformis showed the presence of two mevalonate ( MVA) pathway genes, though the genes were not observed in the transcriptome analysis. Transcripts from four genes ( dxr, ispf, ispd, and idi) were selected and translated into proteins to study the phylogenetic relationship of sterol biosynthesis in V. brassicaformis and C. velia to other groups of algae and apicomplexans. On the basis of our genomic and transcriptomic analyses, we hypothesize that the MEP pathway was the primary pathway that apicomplexans used for sterol biosynthesis before they lost their sterol biosynthesis ability, although contribution of the MVA pathway cannot be discounted. [ABSTRACT FROM AUTHOR]

Zn[2]-Cys[6] binuclear transcription factors Upc2p and Ecm22p regulate the expression of genes involved in ergosterol biosynthesis and exogenous sterol uptake in Saccharomyces cerevisiae. We identified two UPC2/ ECM22 homologues in the pathogenic fungus Candida glabrata which we designated CgUPC2A and CgUPC2B. The contribution of these two genes to sterol homeostasis was investigated. Cells that lack CgUPC2A ( upc2AΔ) exhibited enhanced susceptibility to the sterol biosynthesis inhibitors, fluconazole and lovastatin, whereas upc2BΔ-mutant cells were as susceptible to the drugs as wild-type cells. The growth of upc2AΔ cells was also severely attenuated under anaerobic conditions. Lovastatin treatment enhanced the expression of ergosterol biosynthetic genes, ERG2 and ERG3 in wild-type and upc2BΔ but not in upc2AΔ cells. Similarly, serum-induced expression of ERG2 and ERG3 was completely impaired in upc2AΔ cells but was unaffected in upc2BΔ cells, whereas serum-induced expression of the sterol transporter gene CgAUS1 was impaired in both upc2AΔ and upc2BΔ cells. These results suggest that in C. glabrata CgUPC2A but not in CgUPC2B is the main transcriptional regulator of the genes responsible for maintaining sterol homeostasis as well as susceptibility to sterol inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: Volatile terpenoids released from different plant parts play crucial roles in pollinator attraction, plant defense, and interaction with the surrounding environment. Two distinct pathways localized in different subcellular compartments are responsible for the biosynthesis of these compounds. Recent advances in the characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have revealed new aspects of volatile terpenoid biosynthesis. This review summarizes recent progress in the characterization of volatile terpenoid biosynthetic genes, their spatio-temporal expression patterns and subcellular localization of corresponding proteins. In addition, recent information obtained from metabolic engineering is discussed. [Copyright &y& Elsevier]

Many of the genes for lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae are phase variable. The mechanism of this variable expression involves slippage of tetranucleotide repeats located within the reading frame of these genes. Based on this, we hypothesized that tetranucleotide repeat sequences might be used to identify as yet unrecognized LPS biosynthetic genes. Synthetic oligonucleotides (20 bases), representing all previously reported LPS-related tetranucleotide repeat sequences in H. influenzae, were used to probe a collection of 25 genetically and epidemiologically diverse strains of non-typeable H. influenzae. A novel gene identified through this strategy was a homologue of oafA, a putative O-antigen LPS acetylase of Salmonella typhimurium, that was present in all 25 non-typeable H. influenzae, 19 of which contained multiple copies of the tetranucleotide 5′-GCAA. Using lacZ fusions, we showed that these tetranucleotide repeats could mediate phase variation of this gene. Structural analysis of LPS showed that a major site of acetylation was the distal heptose (HepIII) of the LPS inner-core. An oafA deletion mutant showed absence of O-acetylation of HepIII. When compared with wild type, oafA mutants displayed increased susceptibility to complement-mediated killing by human serum, evidence that O-acetylation of LPS facilitates resistance to host immune clearance mechanisms. These results provide genetic and structural evidence that H. influenzae oafA is required for phase variable O-acetylation of LPS and functional evidence to support the role of O-acetylation of LPS in pathogenesis. [ABSTRACT FROM AUTHOR]

The Saccharomyces cerevisiae MOT3 gene encodes a nuclear protein implicated in both repression and activation of transcription. However, a mot3δ mutation causes only mild phenotypes under normal growth conditions. To learn more about Mot3 function, we have performed a synthetic lethal screen. This screen identified PAN1, a gene required for normal endocytosis, and VPS41, a gene required for vacuotar fusion and protein targeting, suggesting a role for Mot3 in the regulation of membrane-related genes. Transcriptional analyses show that Mot3 represses transcription of ERG2, ERG6 and ERG9, genes required for ergosterol biosynthesis, during both aerobic and hypoxic growth. Chromatin immuno- precipitation experiments suggest that this repression is direct. Ergosterol has been shown to be required for endocytosis and homotypic vacuole fusion, providing a link between Mot3 and these processes. Consistent with these results, mot3δ mutants have a number of related defects, including impaired homotypic vacuole fusion and increased sterol levels. Taken together, our data suggest that proper transcriptional regulation of ergosterol biosynthetic genes by Mot3 is important for normal vacuolar function and probably for the endocytic membrane transport system. [ABSTRACT FROM AUTHOR]

Ent-kaurenoic acid oxidase ( KAO), a class of cytochrome P450 monooxygenases of the subfamily CYP88A, catalyzes the conversion of ent-kaurenoic acid ( KA) to gibberellin ( GA) GA12, the precursor of all GAs, thereby playing an important role in determining GA concentration in plants. Past work has demonstrated the importance of KAO activity for growth in various plant species. In Arabidopsis , this enzyme is encoded by two genes designated KAO1 and KAO2. In this study, we used various approaches to determine the physiological roles of KAO1 and KAO2 throughout plant development. Analysis of gene expression pattern reveals that both genes are mainly expressed in germinating seeds and young developing organs, thus suggesting functional redundancy. Consistent with this, kao1 and kao2 single mutants are indistinguishable from wild-type plants. By contrast, the kao1 kao2 double mutant exhibits typical non-germinating GA-dwarf phenotypes, similar to those observed in the severely GA-deficient ga1-3 mutant. Phenotypic characterization and quantitative analysis of endogenous GA contents of single and double kao mutants further confirm an overlapping role of KAO1 and KAO2 throughout Arabidopsis development. [ABSTRACT FROM AUTHOR]

The antifungal activity of bacteria from the genus C ollimonas has been well documented, but the chemistry and gene functions that underlie this phenotype are still poorly understood. Screening of a random plasposon insertion library of C ollimonas fungivorans Ter331 for loss-of-function mutants revealed the importance of gene cluster K, which is annotated to code for the biosynthesis of a secondary metabolite and which features genes for fatty acid desaturases and polyketide synthases. Mutants in gene cluster K had lost the ability to inhibit hyphal growth of the fungus A spergillus niger and were no longer able to produce and secrete several metabolites that after extraction and partial purification from wildtype strain Ter331 were shown to share a putative ene-triyne moiety. Some but not all of these metabolites were able to inhibit growth of A . niger, indicating functional variation within this group of C ollimonas-produced polyyne-like 'collimomycins'. Polymerase chain reaction analysis of isolates representing different C ollimonas species indicated that the possession of cluster K genes correlated positively with antifungal ability, further strengthening the notion that this cluster is involved in collimomycin production. We discuss our findings in the context of other bacterially produced polyynes and the potential use of collimomycins for the control of harmful fungi. [ABSTRACT FROM AUTHOR]

Streptomyces cinnamonensis DSM 1042 produces two types of isoprenoid secondary metabolites: the prenylated naphthalene derivative furanonaphthoquinone I (FNQ I), and isoprenylated phenazines which are termed endophenazines. Previously, a 55 kb gene cluster was identified which contained genes for both FNQ I and endophenazine biosynthesis. However, several genes required for the biosynthesis of these metabolites were not present in this cluster. We now re-screened the cosmid library for genes of the mevalonate pathway and identified a separate genomic locus which contains the previously missing genes. This locus (15 kb) comprised orthologues of four phenazine biosynthesis genes known from Pseudomonas strains. Furthermore, the locus contained a putative operon of six genes of the mevalonate pathway, as well as the gene epzP which showed sequence similarity to a recently discovered class of prenyltransferases. Inactivation and complementation experiments proved the involvement of epzP in the prenylation reaction in endophenazine biosynthesis. This newly identified genomic locus is more than 40 kb distant from the previously identified cluster. The protein EpzP was expressed in Escherichia coli in form of a his-tag fusion protein and purified. The enzyme catalysed the prenylation of 5,10-dihydrophenazine-1-carboxylic acid (dihydro-PCA) using dimethylallyl diphosphate (DMAPP) as isoprenoid substrate. K(m) values were determined as 108 µM for dihydro-PCA and 25 µM for DMAPP., (© 2010 The Authors. Journal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Chromera velia is a recently discovered, photosynthetic, marine alveolate closely related to apicomplexan parasites, and more distantly to perkinsids and dinoflagellates. To date, there are no published studies on the sterols of C. velia. Because apicomplexans and perkinsids are not known to synthesize sterols de novo, but rather obtain them from their host organisms, our objective was to examine the composition of the sterols of C. velia to assess whether or not there is any commonality with dinoflagellates as the closest taxonomic group capable of synthesizing sterols de novo. Furthermore, knowledge of the sterols of C. velia may provide insight into the sterol biosynthetic capabilities of apicomplexans prior to loss of sterol biosynthesis. We have found that C. velia possesses two primary sterols, 24-ethylcholesta-5,22E-dien-3β-ol, and 24-ethylcholest-5-en-3β-ol, not common to dinoflagellates, but rather commonly found in other classes of algae and plants. In addition, we have identified computationally three genes, SMT1 (sterol-24C-methyltransferase), FDFT1 (farnesyl diphosphate farnesyl transferase, squalene synthase), and IDI1 (isopentenyl diphosphate Δ-isomerase), predicted to be involved in sterol biosynthesis by their similarity to analogous genes in other sterol-producing eukaryotes, including a number of algae. [ABSTRACT FROM AUTHOR]

Bacterial production of long-chain fatty acids via a polyketide synthase-related mechanism has thus far only been investigated in isolate-based studies. Here, the genetic capacity for production of long-chain fatty acids was investigated using a culture-independent approach. PCR primers targeting the keto-acyl synthase (KS) domain of the pfaA gene involved in omega-3 polyunsaturated fatty acid (PUFA) biosynthesis were used to construct clone libraries to investigate KS sequence diversity in disparate marine habitats. Of the 446 sequences recovered, 123 (27.6%) clustered with KS sequences involved in the synthesis of eicosapentaenoic acid (EPA, C20:5 n-3), docosahexaenoic acid (DHA, C22:6 n-3) and arachidonic acid (AA, C20:4 n-6). The remaining 72.4% of clones formed environmental-only groups or grouped with the KS domains of pfaA homologues from organisms producing unidentified products. In total, 17 groups were recovered - four known and 13 newly identified. A query of metagenomic data sets revealed sequences related to EPA KS domains, as well as sequences related to four environmental-only groups discovered in the clone libraries. The phylogenetic affiliation and end product of these environmental-only KS clusters is unknown. These findings reveal a widespread capacity for long-chain fatty acid production in marine microorganisms, including biosynthetic pathways not yet characterized. [ABSTRACT FROM AUTHOR]

The WRINKLED1 (WRI1) protein is an important regulator of oil accumulation in maturing Arabidopsis seeds. WRI1 is a member of a plant-specific family of transcription factors (AP2/EREBP) that share either one or two copies of a DNA-binding domain called the AP2 domain. Here, it is shown that WRI1 acts as a transcriptional enhancer of genes involved in carbon metabolism in transgenic seeds overexpressing this transcription factor. PKp-β1 and BCCP2, two genes encoding enzymes of the glycolysis and fatty acid biosynthetic pathway, respectively, have been chosen to investigate the regulatory action exerted by WRI1 over these pathways. Using the reporter gene uidA, it was possible to demonstrate in planta that WRI1 regulates the activity of both PKp-β1 and BCCP2 promoters. Electrophoretic mobility-shift assays and yeast one-hybrid experiments showed that WRI1 was able to interact with the BCCP2 promoter. To further elucidate the regulatory mechanism controlling the transcription of these genes, functional dissections of PKp-β1 and BCCP2 promoters were performed. Two enhancers, of 54 and 79 bp, respectively, have thus been isolated that are essential to direct the activity of these promoters in oil-accumulating tissues of the embryo. A consensus site is present in these enhancers as well as in other putative target promoters of WRI1. Loss of this consensus sequence in the BCPP2 promoter decreases both the strength of the interaction between WRI1 and this promoter in yeast and the activity of the promoter in planta. [ABSTRACT FROM AUTHOR]

This article discusses a study which identified the bicistronic wce-II and monocistronic wce-III loci in Pantoea stewartii ssp. stewartii (P. stewartii), a Gram-negative bacterium. The study extended the inventory of stewartan exopolysaccharide (EPS) biosynthetic in P. stewartii. It revealed the function of the gene systems to encode a putative glycosyl-transferase (GT) with functional roles in the priming or completion of the stewartan repeat units. It also showed the control of the gene systems by Esal/EsaR QS and the Rcs signal transduction system.

Overexpression of the proline biosynthetic gene P5CS1 results in early flowering in Arabidopsis. However, the p5cs1 loss-of-function mutant exhibits a modest delay in flowering, suggesting that P5CS2, a duplicated P5CS1 gene present in the Arabidopsis, may also play a role in flower transition. In situ mRNA hybridizations and quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that P5CS1 and P5CS2 are expressed at similar levels and with the same pattern of expression in vegetative and floral shoot apical meristems as well as in axillary meristems. Arabidopsis lines homozygous for the p5cs1 mutant and simultaneously heterozygous for the p5cs2 mutation showed a stronger late-flowering phenotype than p5cs1 single mutants, confirming that also P5CS2 plays a role in flower transition and supporting the notion of overlapping functions of the two P5CS genes in this developmental process. P5CS1 and P5CS2 have identical messenger RNA (mRNA) distributions also in embryos, but only p5cs2 mutant embryos exhibit alterations of the cellular division planes and consequently stop developing. This suggests a specific role of P5CS2 in embryogenesis and an involvement of proline in cell division. Accordingly, exogenous proline accelerated organ growth and meristem formation, and stimulated expression of the cell cycle-related protein CYCB1;1. [ABSTRACT FROM AUTHOR]

• Anthocyanins are secondary metabolites, which play an important role in the physiology of plants. Both sucrose and hormones regulate anthocyanin synthesis. Here, the interplay between sucrose and plant hormones was investigated in the expression of sucrose-regulated genes coding for anthocyanin biosynthetic enzymes in Arabidopsis seedlings. • The expression pattern of 14 genes involved in the anthocyanin biosynthetic pathway, including two transcription factors ( PAP1, PAP2), was analysed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in Arabidopsis seedlings treated with sucrose and plant hormones. • Sucrose-induction of the anthocyanin synthesis pathway was repressed by the addition of gibberellic acid (GA) whereas jasmonate (JA) and abscisic acid (ABA) had a synergic effect with sucrose. The gai mutant was less sensitive to GA-dependent repression of dihydroflavonol reductase. This would seem to prove that GAI signalling is involved in the crosstalk between sucrose and GA in wild-type Arabidopsis seedlings. Conversely, the inductive effect of sucrose was not strictly ABA mediated. Sucrose induction of anthocyanin genes required the COI1 gene, but not JAR1, which suggests a possible convergence of the jasmonate- and sucrose-signalling pathways. • The results suggest the existence of a crosstalk between the sucrose and hormone signalling pathways in the regulation of the anthocyanin biosynthetic pathway. [ABSTRACT FROM AUTHOR]

Two regulatory genes, thnI and thnU, were identified in the thienamycin ( thn) gene cluster from Streptomyces cattleya. ThnI resembles LysR-type transcriptional activators and ThnU belongs to the SARP family of transcriptional activators. Their functional role was established after independent inactivation by gene replacement together with transcriptional analysis involving reverse transcription polymerase chain reaction (RT-PCR). Deletion of thnI abolished thienamycin production showing its involvement in thienamycin biosynthesis. Gene expression analysis applied to the thn gene cluster demonstrated that ThnI is a transcriptional activator essential for thienamycin biosynthesis that regulates the expression of nine genes involved in thienamycin assembly and export ( thnH, thnJ, thnK, thnL, thnM, thnN, thnO, thnP and thnQ). Unexpectedly, the thnU disrupted mutant was not affected in thienamycin production but turned out to be essential for cephamycin C biosynthesis. Transcript analysis applied to early and late structural genes for cephamycin C biosynthesis ( pcbAB and cmcI), revealed that ThnU is the transcriptional activator of these cephamycin C genes although they are not physically linked to the thn cluster. In addition, it was shown that deletion of thnI has an upregulatory effect on pcbAB and cmcI transcription consistent with a significant increase in cephamycin C biosynthesis in this mutant. [ABSTRACT FROM AUTHOR]

Differential screening by PCR-select subtraction was carried out for cDNAs from leaves of red and green perilla, two chemovarietal forms of Perilla frutescens regarding anthocyanin accumulation. One hundred and twenty cDNA fragments were selected as the clones preferentially expressed in anthocyanin-accumulating red perilla over the nonaccumulating green perilla. About half of them were the cDNAs encoding the proteins related presumably to phenylpropanoid-derived metabolism. The cDNAs encoding glutathione S-transferase (GST), PfGST1, and chalcone isomerase (CHI), PfCHI1, were further characterized. The expression of PfGST1 in an Arabidopsis thaliana tt19 mutant lacking the GST-like gene involved in vacuole transport of anthocyanin rescued the lesion of anthocyanin accumulation in tt19, indicating a function of PfGST1 in vacuole sequestration of anthocyanin in perilla. The recombinant PfCHI1 could stereospecifically convert naringenin chalcone to (2 S)-naringenin. PfGST1 and PfCHI1 were preferentially expressed in the leaves of red perilla, agreeing with the accumulation of anthocyanin and expression of other previously identified genes for anthocyanin biosynthesis. These results suggest that the genes of the whole anthocyanin biosynthetic pathway are regulated in a coordinated manner in perilla. [ABSTRACT FROM AUTHOR]

Three transposon mutants of Rhizobium tropici CIAT899 affected in lipopolysaccharide (LPS) biosynthesis were characterized and their maize rhizosphere and endophytic root colonization abilities were evaluated. The disrupted genes coded for the following putative products: the ATPase component of an O antigen ABC-2 type transporter ( wzt), a nucleotide-sugar dehydratase ( lpsβ2) and a bifunctional enzyme producing GDP-mannose ( noeJ). Electrophoretic analysis of affinity purified LPS showed that all mutants lacked the smooth LPS bands indicating an O antigen minus phenotype. In the noeJ mutant, the rough LPS band migrated faster than the parental band, suggesting a truncated LPS core. When inoculated individually, the wzt and noeJ mutants colonize the rhizosphere and root to a lower extent than the parental strain while no differences were observed between the lpsβ2 mutant and the parental strain. All mutants were impaired in competitive rhizosphere and root colonization. Pleiotropic effects of the mutations on known colonization traits such as motility and growth rate were observed, but they were not sufficient to explain the colonization behaviours. It was found that the LPS mutants were sensitive to the maize antimicrobial 6-methoxy-2-benzoxazolinone (MBOA). Only the combined effects of altered growth rate and susceptibility to maize antimicrobials could account for all the observed colonization phenotypes. The results suggest an involvement of the LPS in protecting R. tropici against maize defence response during rhizosphere and root colonization. [ABSTRACT FROM AUTHOR]

The activity of the gibberellin (GA) biosynthetic enzymes GA 20-oxidases (GA20ox) is of particular importance in determining GA concentration in many plant species. In Arabidopsis these enzymes are encoded by a family of five genes: AtGA20ox1– AtGA20ox5. Transcript analysis indicated that they have different expression patterns and may thus participate differentially in GA-regulated developmental processes. We have used reverse genetics to determine the physiological roles of AtGA20ox1 and AtGA20ox2, the most highly expressed GA20ox genes during vegetative and early reproductive development. AtGA20ox1 and AtGA20ox2 act redundantly to promote hypocotyl and internode elongation, flowering time, elongation of anther filaments, the number of seeds that develop per silique and elongation of siliques, with AtGA20ox1 making the greater contribution to internode and filament elongation, and AtGA20ox2 making the greater contribution to flowering time and silique length. Pollination of the double mutant with wild-type pollen indicated that the GA promoting silique elongation is of maternal origin. The ga20ox2 phenotype revealed that GA promotes the number of stem internodes that elongate upon bolting, and does so independently of its effect on internode elongation. Comparison of the phenotype of the double mutant with that of the highly GA-deficient ga1-3 mutant indicates that other GA20ox genes contribute to all the developmental processes examined, and, in some cases such as root growth and leaf expansion, make major contributions, as these processes were unaffected in the double mutant. In addition, the effects of the mutations are mitigated by the homeostatic mechanism that acts on expression of other GA dioxygenase and GID1 receptor genes. [ABSTRACT FROM AUTHOR]

Regulated expression of structural genes involved in yeast phospholipid biosynthesis is mediated bynositol/holine-esponsivelement (ICRE) upstream motifs, bound by the heterodimeric activator complex Ino2 + Ino4. Gene repression occurs in the presence of sufficient inositol and choline, requiring an intact Opi1 repressor which binds to Ino2. For a better understanding of interactions among regulators, we mapped an 18 aa repressor interaction domain (RID, aa 118–135) within Ino2 necessary and sufficient for binding by Opi1. By alanine scanning mutagenesis of the entire RID we were able to identify nine residues critical for Opi1-dependent repression of Ino2 function. Consequently, the corresponding dominant Ino2 variants conferred constitutive expression of an ICRE-dependent reporter gene and were no longer inhibited even by overproduction of Opi1. Interestingly, Ino2 RID partially overlaps with transcriptional activation domain TAD2. As certain mutations exclusively affect repression while others affect both repression and activation, both functions of Ino2 can be functionally uncoupled. Correspondingly, we mapped the RID–binding activator interaction domain (AID, aa 321–380) at the C-terminus of Opi1 and introduced missense mutations at selected positions. An Opi1 variant simultaneously mutated at three highly conserved positions showed complete loss of repressor function, confirming RID–AID interaction as the crucial step of regulated expression of ICRE-dependent genes. [ABSTRACT FROM AUTHOR]

The expression of anthocyanin biosynthesis genes during flower colour development inAnthurium andraeanum(anthurium) was studied. A cDNA library was constructed from mRNA from the anthurium spathe, and full-length cDNA clones identified for the flavonoid biosynthetic enzymes chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). These were used to measure transcript levels in the spathe during flower development, in cultivars with different flower colours, over the diurnal cycle, and in the spadix.CHS,F3HandANSwere expressed at all stages of spathe and spadix development. However,DFRtranscript levels varied significantly between stages, and DFR may represent a key point of regulation. A diurnal rhythm ofDFRtranscript abundance in the spathe was also observed, with transcript levels high at dawn and dusk and low at noon. Control of anthocyanin biosynthesis in anthurium spathe differs from that described for flowers of other species, withDFRa key regulatory point and a complex mix of developmental and environmental control signals. [ABSTRACT FROM AUTHOR]