RESUMO
Altering plant water use efficiency (WUE) is a promising approach for achieving sustainable crop production in changing climate scenarios. Here, we show that WUE can be tuned by alleles of a single gene discovered in elite maize (Zea mays) breeding material. Genetic dissection of a genomic region affecting WUE led to the identification of the gene ZmAbh4 as causative for the effect. CRISPR/Cas9-mediated ZmAbh4 inactivation increased WUE without growth reductions in well-watered conditions. ZmAbh4 encodes an enzyme that hydroxylates the phytohormone abscisic acid (ABA) and initiates its catabolism. Stomatal conductance is regulated by ABA and emerged as a major link between variation in WUE and discrimination against the heavy carbon isotope (Δ13C) during photosynthesis in the C4 crop maize. Changes in Δ13C persisted in kernel material, which offers an easy-to-screen proxy for WUE. Our results establish a direct physiological and genetic link between WUE and Δ13C through a single gene with potential applications in maize breeding.
Assuntos
Ácido Abscísico , Zea mays , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Alelos , Isótopos de Carbono , Fotossíntese/genética , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Água/metabolismo , Zea mays/metabolismoRESUMO
Salt stress induces significant loss in crop yield worldwide. Although the growth-stimulating effects of micronutrient nanoparticles (NPs) application under salinity have been studied, the molecular and biochemical mechanisms underlying these effects are poorly understood. The large size of maize leaf growth zones provides an ideal model system to sample and investigate the molecular and physiological bases of growth at subzonal resolution. Using kinematic analysis, our study indicated that salinity at 150 mM inhibited maize leaf growth by decreasing cell division and expansion in the meristem and elongation zones. Consistently, salinity downregulated cell cycle gene expression (wee1, mcm4, and cyclin-B2-4). B2 O3 NP (BNP) mitigated the stress-induced growth inhibition by reducing the decrease in cell division and expansion. BNP also enhanced the photosynthesis-related parameters. Simultaneously, chlorophyll, phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase were stimulated in the mature zone. Concomitant with growth stimulation by BNP, mineral homeostasis, particularly for B and Ca, was monitored. BNP reduced oxidative stress (e.g., lessened H2 O2 generation along the leaf zones and reduced lipid peroxidation in the mature zone) induced by salinity. This resulted from better maintenance of the redox status, that is, increased the glutathione-ascorbate cycle in the meristem and elongation zones, and flavonoids and tocopherol levels in the mature zone. Our study has important implications for assessing the salinity stress impact mitigated by BNP on maize growth, providing a basis to improve the resilience of crop species under salinity stress conditions.
Assuntos
Fotossíntese , Zea mays , Zea mays/genética , Folhas de Planta/metabolismo , Oxirredução , Estresse Salino , Minerais/metabolismo , SalinidadeRESUMO
KEY MESSAGE: Carbon isotope discrimination is a promising trait for indirect screening for improved water use efficiency of C4 crops. In the context of a changing climate, drought is one of the major factors limiting plant growth and yield. Hence, breeding efforts are directed toward improving water use efficiency (WUE) as a key factor in climate resilience and sustainability of crop production. As WUE is a complex trait and its evaluation is rather resource consuming, proxy traits, which are easier to screen and reliably reflect variation in WUE, are needed. In C3 crops, a trait established to be indicative for WUE is the carbon isotopic composition (δ13C) of plant material, which reflects the preferential assimilation of the lighter carbon isotope 12C over 13C during photosynthesis. In C4 crops, carbon fixation is more complex and δ13C thus depends on many more factors than in C3 crops. Recent physiological and genetic studies indicate a correlation between δ13C and WUE also in C4 crops, as well as a colocalization of quantitative trait loci for the two traits. Moreover, significant intraspecific variation as well as a medium to high heritability of δ13C has been shown in some of the main C4 crops, such as maize, sorghum and sugarcane, indicating its potential for indirect selection and breeding. Further research on physiological, genetic and environmental components influencing δ13C is needed to support its application in improving WUE and making C4 crops resilient to climate change.
Assuntos
Isótopos de Carbono/análise , Mudança Climática , Produtos Agrícolas/genética , Melhoramento Vegetal , Secas , Locos de Características Quantitativas , Saccharum/genética , Sorghum/genética , Zea mays/genéticaRESUMO
To understand the growth response to drought, we performed a proteomics study in the leaf growth zone of maize (Zea mays L.) seedlings and functionally characterized the role of starch biosynthesis in the regulation of growth, photosynthesis and antioxidant capacity, using the shrunken-2 mutant (sh2), defective in ADP-glucose pyrophosphorylase. Drought altered the abundance of 284 proteins overrepresented for photosynthesis, amino acid, sugar and starch metabolism, and redox-regulation. Changes in protein levels correlated with enzyme activities (increased ATP synthase, cysteine synthase, starch synthase, RuBisCo, peroxiredoxin, glutaredoxin, thioredoxin and decreased triosephosphate isomerase, ferredoxin, cellulose synthase activities, respectively) and metabolite concentrations (increased ATP, cysteine, glycine, serine, starch, proline and decreased cellulose levels). The sh2 mutant showed a reduced increase of starch levels under drought conditions, leading to soluble sugar starvation at the end of the night and correlating with an inhibition of leaf growth rates. Increased RuBisCo activity and pigment concentrations observed in WT, in response to drought, were lacking in the mutant, which suffered more oxidative damage and recovered more slowly after re-watering. These results demonstrate that starch biosynthesis contributes to maintaining leaf growth under drought stress and facilitates enhanced carbon acquisition upon recovery.
Assuntos
Secas , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Amido/metabolismo , Zea mays/fisiologia , Aminoácidos/metabolismo , Antioxidantes/metabolismo , Divisão Celular , Desidratação , Regulação da Expressão Gênica de Plantas , Mutação , Fotossíntese/fisiologia , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Estômatos de Plantas/fisiologia , Amido/biossíntese , Zea mays/citologiaRESUMO
KEY MESSAGE: A genomic segment on maize chromosome 7 influences carbon isotope composition, water use efficiency, and leaf growth sensitivity to drought, possibly by affecting stomatal properties. Climate change is expected to decrease water availability in many agricultural production areas around the globe. Therefore, plants with improved ability to grow under water deficit are urgently needed. We combined genetic, phenomic, and physiological approaches to understand the relationship between growth, stomatal conductance, water use efficiency, and carbon isotope composition in maize (Zea mays L.). Using near-isogenic lines derived from a maize introgression library, we analysed the effects of a genomic region previously identified as affecting carbon isotope composition. We show stability of trait expression over several years of field trials and demonstrate in the phenotyping platform Phenodyn that the same genomic region also influences the sensitivity of leaf growth to evaporative demand and soil water potential. Our results suggest that the studied genomic region affecting carbon isotope discrimination also harbours quantitative trait loci playing a role in maize drought sensitivity possibly via stomatal behaviour and development. We propose that the observed phenotypes collectively originate from altered stomatal conductance, presumably via abscisic acid.
Assuntos
Isótopos de Carbono/análise , Secas , Água/fisiologia , Zea mays/genética , Zea mays/fisiologia , Cromossomos de Plantas/genética , Fenótipo , Folhas de Planta/fisiologia , Estômatos de Plantas/fisiologia , Locos de Características Quantitativas , Estresse FisiológicoRESUMO
The plant hormone abscisic acid (ABA) regulates many processes, including response to drought, seed dormancy and abscission of leaves and fruits. For maintenance of ABA homeostasis, catabolism of ABA by 8'-hydroxylation and subsequent cyclisation to phaseic acid (PA) is crucial. However, detection of ABA 8'-hydroxylation activity is tedious. We present a simple and rapid method for detection of ABA 8'-hydroxylase activity by cloning cDNAs of interest and expressing the respective protein in yeast. Upon addition of ABA, PA is formed and subsequently quantified in the yeast cell culture supernatant by heart cutting 2D-HPLC or GC-MS.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Saccharomyces cerevisiae/enzimologia , CiclizaçãoRESUMO
Drought is the most important crop yield-limiting factor, and detailed knowledge of its impact on plant growth regulation is crucial. The maize (Zea mays) leaf growth zone offers unique possibilities for studying the spatiotemporal regulation of developmental processes by transcriptional analyses and methods that require more material, such as metabolite and enzyme activity measurements. By means of a kinematic analysis, we show that drought inhibits maize leaf growth by inhibiting cell division in the meristem and cell expansion in the elongation zone. Through a microarray study, we observed the down-regulation of 32 of the 54 cell cycle genes, providing a basis for the inhibited cell division. We also found evidence for an up-regulation of the photosynthetic machinery and the antioxidant and redox systems. This was confirmed by increased chlorophyll content in mature cells and increased activity of antioxidant enzymes and metabolite levels across the growth zone, respectively. We demonstrate the functional significance of the identified transcriptional reprogramming by showing that increasing the antioxidant capacity in the proliferation zone, by overexpression of the Arabidopsis (Arabidopsis thaliana) iron-superoxide dismutase gene, increases leaf growth rate by stimulating cell division. We also show that the increased photosynthetic capacity leads to enhanced photosynthesis upon rewatering, facilitating the often-observed growth compensation.
Assuntos
Secas , Folhas de Planta/crescimento & desenvolvimento , Zea mays/fisiologia , Arabidopsis/genética , Ciclo Celular/genética , Regulação da Expressão Gênica de Plantas , Estresse Oxidativo/genética , Fotossíntese/genética , Folhas de Planta/citologia , Folhas de Planta/fisiologia , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
We studied the drought response of eight commercial hybrid maize lines with contrasting drought sensitivity together with the reference inbred line B73 using a non-invasive platform for root and shoot phenotyping and a kinematics approach to quantify cell level responses in the leaf. Drought treatments strongly reduced leaf growth parameters including projected leaf area, elongation rate, final length and width of the fourth and fifth leaf. Physiological measurements including water use efficiency, chlorophyll fluorescence and photosynthesis were also significantly affected. By performing a kinematic analysis, we show that leaf growth reduction in response to drought is mainly due to a decrease in cell division rate, whereas a marked reduction in cell expansion rate is compensated by increased duration of cell expansion. Detailed analysis of root growth in rhizotrons under drought conditions revealed a strong reduction in total root length as well as rooting depth and width. This was reflected by corresponding decreases in fresh and dry weight of the root system. We show that phenotypic differences between lines differing in geographic origin (African vs. European) and in drought tolerance under field conditions can already be identified at the seedling stage by measurements of total root length and shoot dry weight of the plants. Moreover, we propose a list of candidate traits that could potentially serve as traits for future screening strategies.
Assuntos
Adaptação Fisiológica , Secas , Hibridização Genética , Raízes de Plantas/fisiologia , Brotos de Planta/fisiologia , Plântula/fisiologia , Zea mays/fisiologia , Análise de Variância , Fenômenos Biomecânicos , Fenótipo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Análise de Componente Principal , Característica Quantitativa Herdável , Zea mays/genéticaRESUMO
We studied the impact of drought on growth regulation in leaves of 13 maize varieties with different drought sensitivity and geographic origins (Western Europe, Egypt, South Africa) and the inbred line B73. Combining kinematic analysis of the maize leaf growth zone with biochemical measurements at a high spatial resolution allowed us to examine the correlation between the regulation of the cellular processes cell division and elongation, and the molecular redox-regulation in response to drought. Moreover, we demonstrated differences in the response of the maize lines to mild and severe levels of water deficit. Kinematic analysis indicated that drought tolerant lines experienced less impact on leaf elongation rate due to a smaller reduction of cell production, which, in turn, was due to a smaller decrease of meristem size and number of cells in the leaf meristem. Clear differences in growth responses between the groups of lines with different geographic origin were observed in response to drought. The difference in drought tolerance between the Egyptian hybrids was significantly larger than between the European and South-African hybrids. Through biochemical analyses, we investigated whether antioxidant activity in the growth zone, contributes to the drought sensitivity differences. We used a hierarchical clustering to visualize the patterns of lipid peroxidation, H2O2 and antioxidant concentrations, and enzyme activities throughout the growth zone, in response to stress. The results showed that the lines with different geographic region used different molecular strategies to cope with the stress, with the Egyptian hybrids responding more at the metabolite level and African and the European hybrids at the enzyme level. However, drought tolerance correlated with both, higher antioxidant levels throughout the growth zone and higher activities of the redox-regulating enzymes CAT, POX, APX, and GR specifically in leaf meristems. These findings provide evidence for a link between antioxidant regulation in the leaf meristem, cell division, and drought tolerance.
RESUMO
Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.
Assuntos
Microscopia de Interferência , Células Vegetais , Folhas de Planta/crescimento & desenvolvimento , Zea mays/citologia , Fenômenos Biomecânicos , Diferenciação Celular , Divisão Celular , Folhas de Planta/citologiaRESUMO
The Arabidopsis thaliana root tip has been a key experimental system to study organ growth regulation. It has clear advantages for genetic, transcriptomic, and cell biological studies that focus on the control of cell division and expansion along its longitudinal axis. However, the system shows some limitations for methods that currently require too much tissue to perform them at subzonal resolution, including quantification of proteins, enzyme activity, hormone, and metabolite levels and cell wall extensibility. By contrast, the larger size of the maize leaf does allow such analyses. Here we highlight exciting new possibilities to advance mechanistic understanding of plant growth regulation by using the maize leaf as a complimentary system to the Arabidopsis root tip.