RESUMO
Strigol is the first identified and one of the most important strigolactones (SLs), but the biosynthetic pathway remains elusive. We functionally identified a strigol synthase (cytochrome P450 711A enzyme) in the Prunus genus through rapid gene screening in a set of SL-producing microbial consortia, and confirmed its unique catalytic activity (catalyzing multistep oxidation) through substrate feeding experiments and mutant analysis. We also reconstructed the biosynthetic pathway of strigol in Nicotiana benthamiana and reported the total biosynthesis of strigol in the Escherichia coli-yeast consortium, from the simple sugar xylose, which paves the way for large-scale production of strigol. As proof of concept, strigol and orobanchol were detected in Prunus persica root extrudes. This demonstrated a successful prediction of metabolites produced in plants through gene function identification, highlighting the importance of deciphering the sequence-function correlation of plant biosynthetic enzymes to more accurately predicate plant metabolites without metabolic analysis. This finding revealed the evolutionary and functional diversity of CYP711A (MAX1) in SL biosynthesis, which can synthesize different stereo-configurations of SLs (strigol- or orobanchol-type). This work again emphasizes the importance of microbial bioproduction platform as an efficient and handy tool to functionally identify plant metabolism.
Assuntos
Reguladores de Crescimento de Plantas , Prunus , Reguladores de Crescimento de Plantas/metabolismo , Plantas/metabolismo , Lactonas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Strigolactones (SLs) are a class of phytohormones regulating branching/tillering, and their biosynthesis has been associated with nutritional signals and plant adaptation to nutrient-limiting conditions. The enzymes in the SL biosynthetic pathway downstream of carlactone are of interest as they are responsible for structural diversity in SLs, particularly cytochrome P450 CYP711A subfamily members, such as MORE AXILLARY GROWTH1 (MAX1) in Arabidopsis. We identified 13 MAX1 homologues in wheat, clustering in four clades and five homoeologous subgroups. The utilization of RNA-sequencing data revealed a distinct expression pattern of MAX1 homologues in above- and below-ground tissues, providing insights into the distinct roles of MAX1 homologues in wheat. In addition, a transcriptional analysis showed that SL biosynthetic genes were systematically regulated by nitrogen supply. Nitrogen limitation led to larger transcriptional changes in the basal nodes than phosphorus limitation, which was consistent with the observed tillering suppression, as wheat showed higher sensitivity to nitrogen. The opposite was observed in roots, with phosphorus limitation leading to stronger induction of most SL biosynthetic genes compared with nitrogen limitation. The observed tissue-specific regulation of SL biosynthetic genes in response to nutritional signals is likely to reflect the dual role of SLs as rhizosphere signals and branching inhibitors.
Assuntos
Arabidopsis , Triticum , Triticum/genética , Triticum/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Lactonas/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Fósforo/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Flower sepals are critical for flower development and vary greatly in life span depending on their function post-pollination. Very little is known about what controls sepal longevity. Using a sepal senescence mutant screen, we identified two Arabidopsis mutants with delayed senescence directly connecting strigolactones with senescence regulation in a novel floral context that hitherto has not been explored. The mutations were in the strigolactone biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) and in the strigolactone receptor gene DWARF14 (AtD14). The mutation in AtD14 changed the catalytic Ser97 to Phe in the enzyme active site, which is the first mutation of its kind in planta. The lesion in MAX1 was in the haem-iron ligand signature of the cytochrome P450 protein, converting the highly conserved Gly469 to Arg, which was shown in a transient expression assay to substantially inhibit the activity of MAX1. The two mutations highlighted the importance of strigolactone activity for driving to completion senescence initiated both developmentally and in response to carbon-limiting stress, as has been found for the more well-known senescence-associated regulators ethylene and abscisic acid. Analysis of transcript abundance in excised inflorescences during an extended night suggested an intricate relationship among sugar starvation, senescence, and strigolactone biosynthesis and signalling.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Compostos Heterocíclicos com 3 Anéis , Lactonas , Reguladores de Crescimento de PlantasRESUMO
During neural development, growing axons express specific surface receptors in response to various environmental guidance cues. These axon guidance receptors are regulated through intracellular trafficking and degradation to enable navigating axons to reach their targets. In Caenorhabditis elegans, the UNC-5 receptor is necessary for dorsal migration of developing motor axons. We previously found that MAX-1 is required for UNC-5-mediated axon repulsion, but its mechanism of action remained unclear. Here, we demonstrate that UNC-5-mediated axon repulsion in C. elegans motor axons requires both max-1 SUMOylation and the AP-3 complex ß subunit gene, apb-3 Genetic interaction studies show that max-1 is SUMOylated by gei-17/PIAS1 and acts upstream of apb-3 Biochemical analysis suggests that constitutive interaction of MAX-1 and UNC-5 receptor is weakened by MAX-1 SUMOylation and by the presence of APB-3, a competitive interactor with UNC-5. Overexpression of APB-3 reroutes the trafficking of UNC-5 receptor into the lysosome for protein degradation. In vivo fluorescence recovery after photobleaching experiments shows that MAX-1 SUMOylation and APB-3 are required for proper trafficking of UNC-5 receptor in the axon. Our results demonstrate that SUMOylation of MAX-1 plays an important role in regulating AP-3-mediated trafficking and degradation of UNC-5 receptors during axon guidance.
Assuntos
Axônios/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sumoilação/fisiologia , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Proteínas do Tecido Nervoso/genética , Transporte Proteico/fisiologia , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: More than 70 cytoplasmic male sterility (CMS) types have been identified in Helianthus, but only for less than half of them, research of mitochondrial organization has been conducted. Moreover, complete mitochondrion sequences have only been published for two CMS sources - PET1 and PET2. It has been demonstrated that other sunflower CMS sources like MAX1, significantly differ from the PET1 and PET2 types. However, possible molecular causes for the CMS induction by MAX1 have not yet been proposed. In the present study, we have investigated structural changes in the mitochondrial genome of HA89 (MAX1) CMS sunflower line in comparison to the fertile mitochondrial genome. RESULTS: Eight significant major reorganization events have been determined in HA89 (MAX1) mtDNA: one 110 kb inverted region, four deletions of 439 bp, 978 bp, 3183 bp and 14,296 bp, respectively, and three insertions of 1999 bp, 5272 bp and 6583 bp. The rearrangements have led to functional changes in the mitochondrial genome of HA89 (MAX1) resulting in the complete elimination of orf777 and the appearance of new ORFs - orf306, orf480, orf645 and orf1287. Aligning the mtDNA of the CMS sources PET1 and PET2 with MAX1 we found some common reorganization features in their mitochondrial genome sequences. CONCLUSION: The new open reading frame orf1287, representing a chimeric atp6 gene, may play a key role in MAX1 CMS phenotype formation in sunflower, while the contribution of other mitochondrial reorganizations seems to appear negligible for the CMS development.
Assuntos
Genoma Mitocondrial/genética , Helianthus/genética , Helianthus/fisiologia , Infertilidade das Plantas/fisiologia , Proteínas de Plantas/metabolismo , Infertilidade das Plantas/genética , Proteínas de Plantas/genéticaRESUMO
Strigolactones (SLs) are rhizosphere signalling molecules exuded by plants that induce seed germination of root parasitic weeds and hyphal branching of arbuscular mycorrhiza. They are also phytohormones regulating plant architecture. MORE AXILLARY GROWTH 1 (MAX1) and its homologs encode cytochrome P450 (CYP) enzymes that catalyse the conversion of the strigolactone precursor carlactone to canonical strigolactones in rice (Oryza sativa), and to an SL-like compound in Arabidopsis. Here, we characterized the tomato (Solanum lycopersicum) MAX1 homolog, SlMAX1. The targeting induced local lesions in genomes method was used to obtain Slmax1 mutants that exhibit strongly reduced production of orobanchol, solanacol and didehydro-orobanchol (DDH) isomers. This results in a severe strigolactone mutant phenotype in vegetative and reproductive development. Transient expression of SlMAX1 - together with SlD27, SlCCD7 and SlCCD8 - in Nicotiana benthamiana showed that SlMAX1 catalyses the formation of carlactonoic acid from carlactone. Plant feeding assays showed that carlactone, but not 4-deoxy-orobanchol, is the precursor of orobanchol, which in turn is the precursor of solanacol and two of the three DDH isomers. Inhibitor studies suggest that a 2-oxoglutarate-dependent dioxygenase is involved in orobanchol biosynthesis from carlactone and that the formation of solanacol and DDH isomers from orobanchol is catalysed by CYPs.
Assuntos
Lactonas/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Mutação , Fosfatos/metabolismo , Filogenia , Reguladores de Crescimento de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Nicotiana/genéticaRESUMO
Tree architecture develops over time through the collective activity of apical and axillary meristems. Although the capacity of both meristems to form buds is crucial for perennial life, a comparative analysis is lacking. As shown here for hybrid aspen, axillary meristems engage in an elaborate process of axillary bud (AXB) formation, while apical dominance prevents outgrowth of branches. Development ceased when AXBs had formed an embryonic shoot (ES) with a predictable number of embryonic leaves at the bud maturation point (BMP). Under short days, terminal buds (TBs) formed an ES similar to that of AXBs, and both the TB and young AXBs above the BMP established dormancy. Quantitative PCR and in situ hybridizations showed that this shared ability and structural similarity was reflected at the molecular level. TBs and AXBs similarly regulated expression of meristem-specific and bud/branching-related genes, including CENTRORADIALIS-LIKE1 (CENL1), BRANCHED1 (BRC1), BRC2, and the strigolactone biosynthesis gene MORE AXILLARY BRANCHES1 (MAX1). Below the BMP, AXBs maintained high CENL1 expression at the rib meristem, suggesting that it serves to maintain poise for growth. In support of this, decapitation initiated outgrowth of CENL1-expressing AXBs, but not of dormant AXBs that had switched CENL1 off. This singles out CENL1 as a rib meristem marker for para-dormancy. BRC1 and MAX1 genes, which may counterbalance CENL1, were down-regulated in decapitation-activated AXBs. The results showed that removal of apical dominance shifted AXB gene expression toward that of apices, while developing TBs adopted the expression pattern of para-dormant AXBs. Bud development thus follows a shared developmental pattern at terminal and axillary positions, despite being triggered by short days and apical dominance, respectively.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Populus/genética , Regulação da Expressão Gênica no Desenvolvimento , Meristema/genética , Meristema/crescimento & desenvolvimento , Fotoperíodo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Populus/crescimento & desenvolvimento , Populus/metabolismo , Análise de Sequência de DNARESUMO
Heat stress caused by global warming requires the development of thermotolerant crops to sustain yield. It is necessary to understand the molecular mechanisms that underlie heat tolerance in plants. Strigolactones (SLs) are a class of carotenoid-derived phytohormones that regulate plant development and responses to abiotic or biotic stresses. Although SL biosynthesis and signaling processes are well established, genes that directly regulate SL biosynthesis have rarely been reported. Here, we report that the MYB-like transcription factor AtMYBS1/AtMYBL, whose gene expression is repressed by heat stress, functions as a negative regulator of heat tolerance by directly inhibiting SL biosynthesis in Arabidopsis. Overexpression of AtMYBS1 led to heat hypersensitivity, whereas atmybs1 mutants displayed increased heat tolerance. Expression of MAX1, a critical enzyme in SL biosynthesis, was induced by heat stress and downregulated in AtMYBS1-overexpression (OE) plants but upregulated in atmybs1 mutants. Overexpression of MAX1 in the AtMYBS1-OE background reversed the heat hypersensitivity of AtMYBS1-OE plants. Loss of MAX1 function in the atmyb1 background reversed the heat-tolerant phenotypes of atmyb1 mutants. Yeast one-hybrid assays, chromatin immunoprecipitationâqPCR, and transgenic analyses demonstrated that AtMYBS1 directly represses MAX1 expression through the MYB binding site in the MAX1 promoter in vivo. The atmybs1d14 double mutant, like d14 mutants, exhibited hypersensitivity to heat stress, indicating the necessary role of SL signaling in AtMYBS1-regulated heat tolerance. Our findings provide new insights into the regulatory network of SL biosynthesis, facilitating the breeding of heat-tolerant crops to improve crop production in a warming world.
Assuntos
Arabidopsis , Termotolerância , Arabidopsis/metabolismo , Compostos Heterocíclicos com 3 Anéis/metabolismo , Lactonas/metabolismo , Plantas/metabolismo , Termotolerância/genéticaRESUMO
UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of Caenorhabditis elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. Alternately, MAX-1 might inhibit UNC-5 independent of this known mechanism. We also show that the effects of MAX-1 in growth cone protrusion are independent of UNC-40/DCC, UNC-33/CRMP, and UNC-34/Enabled. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Axônios/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Movimento Celular/fisiologia , Cones de Crescimento/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Netrinas/genética , Netrinas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Strigolactones (SLs) are a group of plant apocarotenoids that act as rhizosphere signaling molecules for both arbuscular mycorrhizal fungi and root parasitic plants. They also regulate plant architecture as phytohormones. The model legume Lotus japonicus (synonym of Lotus corniculatus) produces canonical 5-deoxystrigol (5DS) and non-canonical lotuslactone (LL). The biosynthesis pathways of the two SLs remain elusive. In this study, we characterized the L. japonicus MAX1 homolog, LjMAX1, found in the Lotus japonicus genome assembly build 2.5. The L. japonicus max1 LORE1 insertion mutant was deficient in 5DS and LL production. A recombinant LjMAX1 protein expressed in yeast microsomes converted carlactone (CL) to 18-hydroxycarlactonoic acid (18-OH-CLA) via carlactonoic acid (CLA). Identity of 18-OH-CLA was confirmed by comparison of the methyl ester derivative of the MAX1 product with chemically synthesized methyl 18-hydroycarlactonoate (18-OH-MeCLA) using LC-MS/MS. (11R)-CL was detected as an endogenous compound in the root of L. japonicus.13C-labeled CL, CLA, and 18-OH-MeCLA were converted to [13C]-5DS and LL in plant feeding experiments using L. japonicus WT. These results showed that LjMAX1 is the crucial enzyme in the biosynthesis of Lotus SLs and that 18-hydroxylated carlactonoates are possible precursors for SL biosynthesis in L. japonicus.