RESUMEN
The development of efficient pipelines for the bioconversion of grass lignocellulosic feedstocks is challenging due to the limited understanding of the molecular mechanisms controlling the synthesis, deposition, and degradation of the varying polymers unique to grass cell walls. Here, we describe a large-scale forward genetic approach resulting in the identification of a collection of chemically mutagenized maize mutants with diverse alterations in their cell wall attributes such as crystalline cellulose content or hemicellulose composition. Saccharification yield, i.e. the amount of lignocellulosic glucose (Glc) released by means of enzymatic hydrolysis, is increased in two of the mutants and decreased in the remaining six. These mutants, termed candy-leaf (cal), show no obvious plant growth or developmental defects despite associated differences in their lignocellulosic composition. The identified cal mutants are a valuable tool not only to understand recalcitrance of grass lignocellulosics to enzymatic deconstruction but also to decipher grass-specific aspects of cell wall biology once the genetic basis, i.e. the location of the mutation, has been identified.
RESUMEN
Narrow odd dwarf (nod) and Liguleless narrow (Lgn) are pleiotropic maize mutants that both encode plasma membrane proteins, cause similar developmental patterning defects, and constitutively induce stress signaling pathways. To investigate how these mutants coordinate maize development and physiology, we screened for protein interactors of NOD by affinity purification. LGN was identified by this screen as a strong candidate interactor, and we confirmed the NOD-LGN molecular interaction through orthogonal experiments. We further demonstrated that LGN, a receptor-like kinase, can phosphorylate NOD in vitro, hinting that they could act in intersecting signal transduction pathways. To test this hypothesis, we generated Lgn-R;nod mutants in two backgrounds (B73 and A619), and found that these mutations enhance each other, causing more severe developmental defects than either single mutation on its own, with phenotypes including very narrow leaves, increased tillering, and failure of the main shoot. Transcriptomic and metabolomic analyses of the single and double mutants in the two genetic backgrounds revealed widespread induction of pathogen defense genes and a shift in resource allocation away from primary metabolism in favor of specialized metabolism. These effects were similar in each single mutant and heightened in the double mutant, leading us to conclude that NOD and LGN act cumulatively in overlapping signaling pathways to coordinate growth-defense tradeoffs in maize.
Asunto(s)
Proteínas de Plantas , Zea mays , Zea mays/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Hojas de la Planta/metabolismo , Fenotipo , Mutación , Regulación de la Expresión Génica de las PlantasRESUMEN
The presence of mixed-linkage (1,3;1,4)-ß-d-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals, the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. In this study, we performed a large-scale forward genetic screen for maize (Zea mays) mutants with altered cell wall polysaccharide structural properties. As a result, we identified a maize mutant with increased MLG content in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. The causative mutation was found in the GRMZM2G137535 gene, encoding a GH17 licheninase as demonstrated by an in vitro activity assay of the heterologously expressed protein. In addition, maize plants overexpressing GRMZM2G137535 exhibit a 90% reduction in MLG content, indicating that the protein is not only required, but its expression is sufficient to degrade MLG. Accordingly, the mutant was named MLG hydrolase 1 (mlgh1). mlgh1 plants show increased saccharification yields upon enzymatic digestion. Stacking mlgh1 with lignin-deficient mutations results in synergistic increases in saccharification. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles, inversely associated with MLGH1 transcript accumulation. This cycling is absent in the mlgh1 mutant, suggesting that the mechanism involved requires MLG degradation, which may in turn regulate MLGH1 gene expression.
Asunto(s)
Pared Celular/metabolismo , Oscuridad , Glucanos/metabolismo , Hidrolasas/metabolismo , Hojas de la Planta/metabolismo , Polisacáridos/metabolismo , Zea mays/genética , Zea mays/metabolismo , Pared Celular/genética , Productos Agrícolas/genética , Productos Agrícolas/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , Genotipo , Glucanos/genética , Hidrolasas/genética , Mutación , Hojas de la Planta/genética , Polisacáridos/genéticaRESUMEN
Plant meristems are self-renewing groups of pluripotent stem cells that produce lateral organs in a stereotypical pattern. Of interest is how the radially symmetrical meristem produces laminar lateral organs. Both the male and female inflorescence meristems of the dominant Fascicled ear (Fas1) mutant fail to grow as a single point and instead show deep branching. Positional cloning of two independent Fas1 alleles identified an â¼160 kb region containing two floral genes, the MADS-box gene, zmm8, and the YABBY gene, drooping leaf2 (drl2). Both genes are duplicated within the Fas1 locus and spatiotemporally misexpressed in the mutant inflorescence meristems. Increased zmm8 expression alone does not affect inflorescence development; however, combined misexpression of zmm8, drl2, and their syntenic paralogs zmm14 and drl1, perturbs meristem organization. We hypothesize that misexpression of the floral genes in the inflorescence and their potential interaction cause ectopic activation of a laminar program, thereby disrupting signaling necessary for maintenance of radially symmetrical inflorescence meristems. Consistent with this hypothesis, RNA sequencing and in situ analysis reveal altered expression patterns of genes that define distinct zones of the meristem and developing leaf. Our findings highlight the importance of strict spatiotemporal patterns of expression for both zmm8 and drl2 and provide an example of phenotypes arising from tandem gene duplications.
Asunto(s)
Duplicación de Gen , Meristema/crecimiento & desarrollo , Zea mays/genética , Flores/genética , Flores/crecimiento & desarrollo , Meristema/citología , Meristema/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Zea mays/crecimiento & desarrolloRESUMEN
Maize is monecious, with separate male and female inflorescences. Maize flowers are initially bisexual but achieve separate sexual identities through organ arrest. Loss-of-function mutants in the jasmonic acid (JA) pathway have only female flowers due to failure to abort silks in the tassel. Tasselseed5 (Ts5) shares this phenotype but is dominant. Positional cloning and transcriptomics of tassels identified an ectopically expressed gene in the CYP94B subfamily, Ts5 (ZmCYP94B1). CYP94B enzymes are wound inducible and inactivate bioactive jasmonoyl-L-isoleucine (JA-Ile). Consistent with this result, tassels and wounded leaves of Ts5 mutants displayed lower JA and JA-lle precursors and higher 12OH-JA-lle product than the wild type. Furthermore, many wounding and jasmonate pathway genes were differentially expressed in Ts5 tassels. We propose that the Ts5 phenotype results from the interruption of JA signaling during sexual differentiation via the upregulation of ZmCYP94B1 and that its proper expression maintains maize monoecy.
Asunto(s)
Ciclopentanos/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Regulación de la Expresión Génica de las Plantas , Oxilipinas/metabolismo , Proteínas de Plantas/genética , Zea mays/genética , Zea mays/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Inducción Enzimática , Flores/genética , Flores/metabolismo , Mutación , Fenotipo , Filogenia , Proteínas de Plantas/metabolismo , Transducción de Señal , Zea mays/clasificaciónRESUMEN
Plant architecture results from a balance of indeterminate and determinate cell fates. Cells with indeterminate fates are located in meristems, comprising groups of pluripotent cells that produce lateral organs. Meristematic cells are also found in intercalary stem tissue, which provides cells for internodes, and at leaf margins to contribute to leaf width. We identified a maize (Zea mays) mutant that has a defect in balancing determinacy and indeterminacy. The mutant has narrow leaves and short internodes, suggesting a reduction in indeterminate cells in the leaf and stem. In contrast, the mutants fail to control indeterminacy in shoot meristems. Inflorescence meristems are fasciated, and determinate axillary meristems become indeterminate. Positional cloning identified growth regulating factor-interacting factor1 (gif1) as the responsible gene. gif1 mRNA accumulates in distinct domains of shoot meristems, consistent with tissues affected by the mutation. We determined which GROWTH REGULATING FACTORs interact with GIF1 and performed RNA-seq analysis. Many genes known to play roles in inflorescence architecture were differentially expressed in gif1 Chromatin immunoprecipitation identified some differentially expressed genes as direct targets of GIF1. The interactions with these diverse direct and indirect targets help explain the paradoxical phenotypes of maize GIF1. These results provide insights into the biological functions of gif1.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Zea mays/genética , Meristema/genética , Meristema/crecimiento & desarrollo , Meristema/fisiología , Mutación , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/fisiología , Zea mays/crecimiento & desarrollo , Zea mays/fisiologíaRESUMEN
Plant architecture is dictated by precise control of meristematic activity. In the shoot, an imbalance in positive or negative maintenance signals can result in a fasciated or enlarged meristem phenotype. fasciated ear4 (fea4) is a semidwarfed mutant with fasciated ears and tassels as well as greatly enlarged vegetative and inflorescence meristems. We identified FEA4 as a bZIP transcription factor, orthologous to Arabidopsis thaliana PERIANTHIA. FEA4 was expressed in the peripheral zone of the vegetative shoot apical meristem and in the vasculature of immature leaves and conspicuously excluded from the stem cell niche at the tip of the shoot apical meristem and from incipient leaf primordia. Following the transition to reproductive fate, FEA4 was expressed throughout the entire inflorescence and floral meristems. Native expression of a functional YFP:FEA4 fusion recapitulated this pattern of expression. We used chromatin immunoprecipitation-sequencing to identify 4060 genes proximal to FEA4 binding sites, including ones that were potentially bound and modulated by FEA4 based on transcriptional changes in fea4 mutant ears. Our results suggest that FEA4 promotes differentiation in the meristem periphery by regulating auxin-based responses and genes associated with leaf differentiation and polarity, potentially in opposition to factors such as KNOTTED1 and WUSCHEL.
Asunto(s)
Meristema/metabolismo , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Zea mays/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Hojas de la Planta/metabolismoRESUMEN
In Arabidopsis, SHOOT MERISTEMLESS (STM) and CLAVATA1 (CLV1) competitively regulate meristem homeostasis. Here, we explore the interaction of their maize homologs knotted1 (kn1) and thick tassel dwarf1 (td1). kn1 mutants form fewer lateral organs and td1 inflorescences are fasciated with additional floral organs. Double mutants show kn1 epistatic to td1 in seedling and ear development but dose-sensitivity exists later to promote leaf initiation. Thus kn1 and td1 function in a pathway to maintain meristem homeostasis but their products may interact with different partners during development.
Asunto(s)
Epistasis Genética/fisiología , Proteínas de Homeodominio/genética , Meristema/crecimiento & desarrollo , Proteínas de Plantas/genética , Zea mays/crecimiento & desarrollo , Zea mays/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Meristema/genética , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Reproducción/genética , Zea mays/fisiologíaRESUMEN
Organogenesis in plants is controlled by meristems. Axillary meristems, which give rise to branches and flowers, play a critical role in plant architecture and reproduction. Maize (Zea mays) and rice (Oryza sativa) have additional types of axillary meristems in the inflorescence compared to Arabidopsis (Arabidopsis thaliana) and thus provide an excellent model system to study axillary meristem initiation. Previously, we characterized the barren inflorescence2 (bif2) mutant in maize and showed that bif2 plays a key role in axillary meristem and lateral primordia initiation in the inflorescence. In this article, we cloned bif2 by transposon tagging. Isolation of bif2-like genes from seven other grasses, along with phylogenetic analysis, showed that bif2 is a co-ortholog of PINOID (PID), which regulates auxin transport in Arabidopsis. Expression analysis showed that bif2 is expressed in all axillary meristems and lateral primordia during inflorescence and vegetative development in maize and rice. Further phenotypic analysis of bif2 mutants in maize illustrates additional roles of bif2 during vegetative development. We propose that bif2/PID sequence and expression are conserved between grasses and Arabidopsis, attesting to the important role they play in development. We provide further support that bif2, and by analogy PID, is required for initiation of both axillary meristems and lateral primordia.
Asunto(s)
Copas de Floración/crecimiento & desarrollo , Organogénesis/genética , Poaceae/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Elementos Transponibles de ADN , Copas de Floración/metabolismo , Expresión Génica , Ácidos Indolacéticos/metabolismo , Meristema/crecimiento & desarrollo , Datos de Secuencia Molecular , Poaceae/enzimología , Poaceae/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Development in higher plants depends on the activity of meristems, formative regions that continuously initiate new organs at their flanks. Meristems must maintain a balance between stem cell renewal and organ initiation. In fasciated mutants, organ initiation fails to keep pace with meristem proliferation. The thick tassel dwarf1 (td1) mutation of maize affects both male and female inflorescence development. The female inflorescence, which results in the ear, is fasciated, with extra rows of kernels. The male inflorescence, or tassel, shows an increase in spikelet density. Floral meristems are also affected in td1 mutants; for example, male florets have an increase in stamen number. These results suggest that td1 functions in the inflorescence to limit meristem size. In addition, td1 mutants are slightly shorter than normal siblings, indicating that td1 also plays a role in vegetative development. td1 encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) that is a putative ortholog of the Arabidopsis CLAVATA1 protein. These results complement previous work showing that fasciated ear2 encodes a CLAVATA2-like protein, and suggest that the CLAVATA signaling pathway is conserved in monocots. td1 maps in the vicinity of quantitative trait loci that affect seed row number, spikelet density and plant height. We discuss the possible selection pressures on td1 during maize domestication.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Copas de Floración/genética , Proteínas de Plantas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Zea mays/genética , Secuencia de Aminoácidos , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Copas de Floración/crecimiento & desarrollo , Datos de Secuencia Molecular , Mutación , Fenotipo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Zea mays/metabolismoRESUMEN
The Maize Gene Discovery Project (MGDP) is a 5-year NSF-funded plant genome initiative that began in 1998. The MGDP collaboration involves researchers at six universities from diverse disciplines with the common goal of discovering new maize genes and developing tools for the phenotypic characterization of maize mutants. The project utilizes several approaches: EST sequencing, cDNA microarray production, and the discovery of gene function and genomic sequence through the use of a recombinant Mu1 transposon ( RescueMu). Current achievements of the MGDP (NSF 98-72657) include the sequencing of over 120,000 maize ESTs from diverse cDNA libraries, and over 70,000 RescueMu flanking sequences, as well as the cataloguing of mutant seed and cob phenotypes of 23,000 maize ears, 6,200 families of maize seedlings, and 4,000 families of adult maize plants carrying MuDR/Mu and RescueMu insertion alleles. A consolidation of over 24,000 unique sequences from 19 libraries has been made into the first two of the planned set of four "Unigene" microarray slides. In addition, slides for four EST libraries have been produced. These microarray slides, EST clones, library plates of immortalized RescueMu bacterial cultures, and seed are all available online (http://www.zmdb.iastate.edu). The ZmDB website posts periodic assemblies of all maize EST and genomic sequences available from GenBank. ZmDB is also a portal for sequence analysis software designed to aid in gene discovery: MuSeqBox, GeneSeqer, and SplicePredictor. In addition, ZmDB contains links to other plant and genetics websites.