RESUMO
Brassinosteroids (BR) and gibberellins (GA) regulate plant height and leaf angle in maize (Zea mays). Mutants with defects in BR or GA biosynthesis or signaling identify components of these pathways and enhance our knowledge about plant growth and development. In this study, we characterized three recessive mutant alleles of GRAS transcription factor 42 (gras42) in maize, a GRAS transcription factor gene orthologous to the DWARF AND LOW TILLERING (DLT) gene of rice (Oryza sativa). These maize mutants exhibited semi-dwarf stature, shorter and wider leaves, and more upright leaf angle. Transcriptome analysis revealed a role for GRAS42 as a determinant of BR signaling. Analysis of the expression consequences from loss of GRAS42 in the gras42-mu1021149 mutant indicated a weak loss of BR signaling in the mutant, consistent with its previously demonstrated role in BR signaling in rice. Loss of BR signaling was also evident by the enhancement of weak BR biosynthetic mutant alleles in double mutants of nana plant1-1 and gras42-mu1021149. The gras42-mu1021149 mutant had little effect on GA-regulated gene expression, suggesting that GRAS42 is not a regulator of core GA signaling genes in maize. Single-cell expression data identified gras42 expressed among cells in the G2/M phase of the cell cycle consistent with its previously demonstrated role in cell cycle gene expression in Arabidopsis (Arabidopsis thaliana). Cis-acting natural variation controlling GRAS42 transcript accumulation was identified by expression genome-wide association study (eGWAS) in maize. Our results demonstrate a conserved role for GRAS42/SCARECROW-LIKE 28 (SCL28)/DLT in BR signaling, clarify the role of this gene in GA signaling, and suggest mechanisms of tillering and leaf angle control by BR.
Assuntos
Brassinosteroides , Regulação da Expressão Gênica de Plantas , Mutação , Proteínas de Plantas , Transdução de Sinais , Fatores de Transcrição , Zea mays , Zea mays/genética , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Brassinosteroides/metabolismo , Mutação/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Giberelinas/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Fenótipo , Alelos , Perfilação da Expressão Gênica , Genes de PlantasRESUMO
The plant hormone gibberellin (GA) impacts plant growth and development differently depending on the developmental context. In the maize (Zea mays) tassel, application of GA alters floral development, resulting in the persistence of pistils. GA signaling is achieved by the GA-dependent turnover of DELLA domain transcription factors, encoded by dwarf8 (d8) and dwarf9 (d9) in maize. The D8-Mpl and D9-1 alleles disrupt GA signaling, resulting in short plants and normal tassel floret development in the presence of excess GA. However, D9-1 mutants are unable to block GA-induced pistil development. Gene expression in developing tassels of D8-Mpl and D9-1 mutants and their wild-type siblings was determined upon excess GA3 and mock treatments. Using GA-sensitive transcripts as reporters of GA signaling, we identified a weak loss of repression under mock conditions in both mutants, with the effect in D9-1 being greater. D9-1 was also less able to repress GA signaling in the presence of excess GA3 . We treated a diverse set of maize inbred lines with excess GA3 and measured the phenotypic consequences on multiple aspects of development (e.g., height and pistil persistence in tassel florets). Genotype affected all GA-regulated phenotypes but there was no correlation between any of the GA-affected phenotypes, indicating that the complexity of the relationship between GA and development extends beyond the two-gene epistasis previously demonstrated for GA and brassinosteroid biosynthetic mutants.
Assuntos
Proteínas de Arabidopsis , Giberelinas , Giberelinas/metabolismo , Zea mays/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Inflorescência/metabolismo , Brassinosteroides/metabolismo , Plantas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Arabidopsis/metabolismoRESUMO
Meristems house the stem cells needed for the developmental plasticity observed in adverse environmental conditions and are crucial for determining plant architecture. Meristem development is particularly sensitive to deficiencies of the micronutrient boron, yet how boron integrates into meristem development pathways is unknown. We addressed this question using the boron-deficient maize mutant, tassel-less1 (tls1). Reduced boron uptake in tls1 leads to a progressive impairment of meristem development that manifests in vegetative and reproductive defects. We show, that the tls1 tassel phenotype (male reproductive structure) was partially suppressed by mutations in the CLAVATA1 (CLV1)-ortholog, thick tassel dwarf1 (td1), but not by other mutants in the well characterized CLV-WUSCHEL pathway, which controls meristem size. The suppression of tls1 by td1 correlates with altered signaling of the phytohormone cytokinin. In contrast, mutations in the meristem maintenance gene knotted1 (kn1) enhanced both vegetative and reproductive defects in tls1. In addition, reduced transcript levels of kn1 and cell cycle genes are early defects in tls1 tassel meristems. Our results show that specific meristem maintenance and hormone pathways are affected in tls1, and suggest that reduced boron levels induced by tls1 are the underlying cause of the observed defects. We, therefore, provide new insights into the molecular mechanisms affected by boron deficiency in maize, leading to a better understanding of how genetic and environmental factors integrate during shoot meristem development.
Assuntos
Meristema , Zea mays , Boro , Divisão Celular , Citocininas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Inflorescência , Mutação/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/metabolismoRESUMO
Inflorescence architecture is a key determinant of yield potential in many crops and is patterned by the organization and developmental fate of axillary meristems. In cereals, flowers and grain are borne from spikelets, which differentiate in the final iteration of axillary meristem branching. In Setaria spp, inflorescence branches terminate in either a spikelet or a sterile bristle, and these structures appear to be paired. In this work, we leverage Setaria viridis to investigate a role for the phytohormones brassinosteroids (BRs) in specifying bristle identity and maintaining spikelet meristem determinacy. We report the molecular identification and characterization of the Bristleless1 (Bsl1) locus in S. viridis, which encodes a rate-limiting enzyme in BR biosynthesis. Loss-of-function bsl1 mutants fail to initiate a bristle identity program, resulting in homeotic conversion of bristles to spikelets. In addition, spikelet meristem determinacy is altered in the mutants, which produce two florets per spikelet instead of one. Both of these phenotypes provide avenues for enhanced grain production in cereal crops. Our results indicate that the spatiotemporal restriction of BR biosynthesis at boundary domains influences meristem fate decisions during inflorescence development. The bsl1 mutants provide insight into the molecular basis underlying morphological variation in inflorescence architecture.
Assuntos
Brassinosteroides/farmacologia , Diferenciação Celular/efeitos dos fármacos , Inflorescência/citologia , Meristema/citologia , Setaria (Planta)/citologia , Alelos , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Loci Gênicos , Inflorescência/efeitos dos fármacos , Inflorescência/ultraestrutura , Meristema/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Fenótipo , Filogenia , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Setaria (Planta)/efeitos dos fármacos , Setaria (Planta)/genética , Setaria (Planta)/ultraestrutura , Transdução de Sinais/efeitos dos fármacosRESUMO
A small number of phytohormones dictate the pattern of plant form affecting fitness via reproductive architecture and the plant's ability to forage for light, water, and nutrients. Individual phytohormone contributions to plant architecture have been studied extensively, often following a single component of plant architecture, such as plant height or branching. Both brassinosteroid (BR) and gibberellin (GA) affect plant height, branching, and sexual organ development in maize (Zea mays). We identified the molecular basis of the nana plant2 (na2) phenotype as a loss-of-function mutation in one of the two maize paralogs of the Arabidopsis (Arabidopsis thaliana) BR biosynthetic gene DWARF1 (DWF1). These mutants accumulate the DWF1 substrate 24-methylenecholesterol and exhibit decreased levels of downstream BR metabolites. We utilized this mutant and known GA biosynthetic mutants to investigate the genetic interactions between BR and GA. Double mutants exhibited additivity for some phenotypes and epistasis for others with no unifying pattern, indicating that BR and GA interact to affect development but in a context-dependent manner. Similar results were observed in double mutant analyses using additional BR and GA biosynthetic mutant loci. Thus, the BR and GA interactions were neither locus nor allele specific. Exogenous application of GA3 to na2 and d5, a GA biosynthetic mutant, also resulted in a diverse pattern of growth responses, including BR-dependent GA responses. These findings demonstrate that BR and GA do not interact via a single inclusive pathway in maize but rather suggest that differential signal transduction and downstream responses are affected dependent upon the developmental context.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Brassinosteroides/metabolismo , Giberelinas/metabolismo , Proteínas de Plantas/metabolismo , Homologia de Sequência de Aminoácidos , Zea mays/crescimento & desenvolvimento , Zea mays/genética , Alelos , Vias Biossintéticas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Loci Gênicos , Modelos Biológicos , Mutação/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Brassinosteroids (BRs) are important regulators that control myriad aspects of plant growth and development, including biotic and abiotic stress responses, such that modulating BR homeostasis and signaling presents abundant opportunities for plant breeding and crop improvement. Enzymes and other proteins involved in the biosynthesis and signaling of BRs are well understood from molecular genetics and phenotypic analysis in Arabidopsis thaliana; however, knowledge of the molecular functions of these genes in other plant species, especially cereal crop plants, is minimal. In this manuscript, we comprehensively review functional studies of BR genes in Arabidopsis, maize, rice, Setaria, Brachypodium, and soybean to identify conserved and diversified functions across plant species and to highlight cases for which additional research is in order. We performed phylogenetic analysis of gene families involved in the biosynthesis and signaling of BRs and re-analyzed publicly available transcriptomic data. Gene trees coupled with expression data provide a valuable guide to supplement future research on BRs in these important crop species, enabling researchers to identify gene-editing targets for BR-related functional studies.
RESUMO
Forward genetics is used to identify the genetic basis for a phenotype. The approach involves identifying a mutant organism exhibiting a phenotype of interest and then mapping the causative locus or gene. Bulked-segregant analysis (BSA) is a quick and effective approach to map mutants using pools of mutants and wild-type plants from a segregating population to identify linkage of the mutant phenotype, and this approach has been successfully used in plants. Traditional linkage mapping approaches are outdated and time intensive, and can be very difficult. With the highly evolved development and reduction in cost of high-throughput sequencing, this new approach combined with BSA has become extremely effective in multiple plant species, including Zea mays (maize). While the approach is incredibly powerful, careful experimental design, bioinformatic mapping techniques, and interpretation of results are important to obtain the desired results in an effective and timely manner. Poor design of a mapping population, limitations in bioinformatic experience, and inadequate understanding of sequence data are limitations of these approaches for the researcher. Here, we describe a straightforward protocol for mapping mutations responsible for a phenotype of interest in maize, using high-throughput sequencing and BSA. Specifically, we discuss relevant aspects of developing a mutant mapping population. This is followed by a detailed protocol for DNA preparation and analysis of short-read sequences to map and identify candidate causative mutations responsible for the mutant phenotype of interest. We provide command-line and perl scripts to complete the bioinformatic analysis of the mutant sequence data. This protocol lays out the design of the BSA, bioinformatic approaches, and interpreting the sequencing data. These methods are very adaptable to any forward genetics experiment and provide a step-by-step approach to identifying the genetic basis of a maize mutant phenotype. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Bulked-segregant analysis and high-throughput sequencing to map maize mutants.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Zea mays , Zea mays/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mapeamento Cromossômico/métodos , Ligação Genética , FenótipoRESUMO
The nuclear pore complex (NPC) regulates the movement of macromolecules between the nucleus and cytoplasm. Dysfunction of many components of the NPC results in human genetic diseases, including triple A syndrome (AAAS) as a result of mutations in ALADIN. Here, we report a nonsense mutation in the maize ortholog, aladin1 (ali1-1), at the orthologous amino acid residue of an AAAS allele from humans, alters plant stature, tassel architecture, and asymmetric divisions of subsidiary mother cells (SMCs). Crosses with the stronger nonsense allele ali1-2 identified complex allele interactions for plant height and aberrant SMC division. RNA-seq analysis of the ali1-1 mutant identified compensatory transcript accumulation for other NPC components as well as gene expression consequences consistent with conservation of ALADIN1 functions between humans and maize. These findings demonstrate that ALADIN1 is necessary for normal plant development, shoot architecture, and asymmetric cell division in maize.
Assuntos
Poro Nuclear , Zea mays , Humanos , Zea mays/fisiologia , Poro Nuclear/genética , Poro Nuclear/metabolismo , Divisão Celular Assimétrica , Divisão Celular/genética , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
The diversity of plant architecture is determined by axillary meristems (AMs). AMs are produced from small groups of stem cells in the axils of leaf primordia and generate vegetative branches and reproductive inflorescences. Previous studies identified genes critical for AM development that function in auxin biosynthesis, transport, and signaling. barren stalk1 (ba1), a basic helix-loop-helix transcription factor, acts downstream of auxin to control AM formation. Here, we report the cloning and characterization of barren stalk2 (ba2), a mutant that fails to produce ears and has fewer branches and spikelets in the tassel, indicating that ba2 functions in reproductive AM development. Furthermore, the ba2 mutation suppresses tiller growth in the teosinte branched1 mutant, indicating that ba2 also plays an essential role in vegetative AM development. The ba2 gene encodes a protein that co-localizes and heterodimerizes with BA1 in the nucleus. Characterization of the genetic interaction between ba2 and ba1 demonstrates that ba1 shows a gene dosage effect in ba2 mutants, providing further evidence that BA1 and BA2 act together in the same pathway. Characterization of the molecular and genetic interaction between ba2 and additional genes required for the regulation of ba1 further supports this finding. The ba1 and ba2 genes are orthologs of rice genes, LAX PANICLE1 (LAX1) and LAX2, respectively, hence providing insights into pathways controlling AMs development in grasses.
Assuntos
Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/metabolismo , Inflorescência/genética , Inflorescência/crescimento & desenvolvimento , Inflorescência/metabolismo , Meristema/genética , Mutação , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Zea mays/genética , Zea mays/crescimento & desenvolvimentoRESUMO
Phytohormone biosynthesis produces metabolites with profound effects on plant growth and development. Modulation of hormone levels during developmental events, in response to the environment, by genetic polymorphism, or by chemical application, can reveal the plant processes most responsive to a phytohormone. Applications of chemical inhibitors and subsequent measurements of specific phytohormones can determine whether, and which, phytohormone is affected by a molecule. In many cases, the sensitivity of biochemical testing has determined multiple pathways affected by a single inhibitor. Genetic studies are not subject to this problem, and a wealth of data about the morphological impacts of hormone biosynthetic inhibition have accumulated through the study of enzyme mutants. In this work, we sought to assess the specificity of three triazole inhibitors of cytochrome P450s by determining their abilities to recapitulate the phenotypes of single and double mutants affected in the production of brassinosteroid (BR) and gibberellin (GA) biosynthesis. The GA biosynthetic inhibitors uniconazole (UCZ) and paclobutrazol (PAC) were applied to the BR biosynthetic mutant nana plant2 (na2), and all double-mutant phenotypes were recovered in the UCZ treatment. PAC was unable to suppress the retention of pistils in the tassels of na2 mutant plants. The BR biosynthetic inhibitor propiconazole (PCZ) suppressed tiller outgrowth in the GA biosynthetic mutant dwarf5 (d5). All treatments were additive with genetic mutants for effects on plant height. Due to additional measurements performed here but not in previous studies of the double mutants, we detected new interactions between GA and BR biosynthesis affecting the days to tassel emergence and tassel branching. These experiments, a refinement of our previous model, and a discussion of the extension of this type of work are presented.
RESUMO
Plant growth regulators, such as hormones and their respective biosynthesis inhibitors, are effective tools to elucidate the physiological function of phytohormones in plants. A problem of chemical treatments, however, is the potential for interaction of the active compound with the growth media substrate. We studied the interaction and efficacy of propiconazole, a potent and specific inhibitor of brassinosteroid biosynthesis, with common soilless greenhouse growth media for rice, sorghum, and maize. Many of the tested growth media interacted with propiconazole reducing its efficacy up to a hundred fold. To determine the molecular interaction of inhibitors with media substrates, Fourier Transform Infrared Spectroscopy and sorption isotherm analysis was applied. While mica clay substrates absorbed up to 1.3 mg of propiconazole per g substrate, calcined clays bound up to 12 mg of propiconazole per g substrate. The efficacy of the gibberellic acid biosynthesis inhibitor, uniconazole, and the most active brassinosteroid, brassinolide, was impacted similarly by the respective substrates. Conversely, gibberellic acid showed no distinct growth response in different media. Our results suggest that the reduction in efficacy of propiconazole, uniconazole, and brassinolide in bioassays when grown in calcined clay is caused by hydrophobic interactions between the plant growth regulators and the growth media. This was further confirmed by experiments using methanol-water solvent mixes with higher hydrophobicity values, which reduce the interaction of propiconazole and calcined clay.
Assuntos
Meios de Cultura , Reguladores de Crescimento de Plantas/farmacologia , Plantas/efeitos dos fármacos , Adsorção , Meios de Cultura/química , Fenótipo , Reguladores de Crescimento de Plantas/química , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimentoRESUMO
Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA(3) co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.