Maize Leaf Development Research Articles

Multiplexed in situ hybridization reveals distinct lineage identities for major and minor vein initiation during maize leaf development

Leaves of flowering plants are characterized by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyze the spatiotemporal localization of a subset of auxin-related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification and how major and minor vein ranks develop with distinct identities. The localization of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is prepatterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarized as differentiation proceeds. Collectively, our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves.

The RHW1-ZCN4 regulatory pathway confers natural variation of husk leaf width in maize.

Maize husk leaf - the outer leafy layers covering the ear - modulates kernel yield and quality. Despite its importance, however, the genetic controls underlying husk leaf development remain elusive. Our previous genome-wide association study identified a single nucleotide polymorphism located in the gene RHW1 (Regulator of Husk leaf Width) that is significantly associated with husk leaf-width diversity in maize. Here, we further demonstrate that a polymorphic 18-bp InDel (insertion/deletion) variant in the 3' untranslated region of RHW1 alters its protein abundance and accounts for husk leaf width variation. RHW1 encodes a putative MYB-like transcriptional repressor. Disruption of RHW1 altered cell proliferation and resulted in a narrower husk leaf, whereas RHW1 overexpression yielded a wider husk leaf. RHW1 positively regulated the expression of ZCN4, a well-known TFL1-like protein involved in maize ear development. Dysfunction of ZCN4 reduced husk leaf width even in the context of RHW1 overexpression. The InDel variant in RHW1 is subject to selection and is associated with maize husk leaf adaption from tropical to temperate regions. Overall, our results identify that RHW1-ZCN4 regulates a pathway conferring husk leaf width variation at a very early stage of husk leaf development in maize.

Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues.

Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Although two such methods have been developed for mammalian tissues, they have a low signal-noise ratio and/or poor resolution at the single-cell level. To overcome these drawbacks, we develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels. We conduct several applications. First, the spatial expression profiling of osa-miR156 and OsSPL12 in rice leaves reveals their specific expression in mesophyll cells. Second, studying rice and its mutant lines with our method reveals opposite expression patterns of miRNA and its target mRNA in tissues. Third, the dynamic expression profiles of ZmGRF8 and zma-miR396 during maize leaf development provide evidence that zma-miR396 regulates the preferential spatial expression of ZmGRF8 in bundle sheath cells. Finally, our method can be scaled up to simultaneously detect multiple miRNAs and mRNAs in a tissue. Thus, it is a sensitive and versatile technique for studying miRNA regulation of plant tissue development.

Regulators of early maize leaf development inferred from transcriptomes of laser capture microdissection (LCM)-isolated embryonic leaf cells

The superior photosynthetic efficiency of C4 leaves over C3 leaves is owing to their unique Kranz anatomy, in which the vein is surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. Kranz anatomy development starts from three contiguous ground meristem (GM) cells, but its regulators and underlying molecular mechanism are largely unknown. To identify the regulators, we obtained the transcriptomes of 11 maize embryonic leaf cell types from five stages of pre-Kranz cells starting from median GM cells and six stages of pre-M cells starting from undifferentiated cells. Principal component and clustering analyses of transcriptomic data revealed rapid pre-Kranz cell differentiation in the first two stages but slow differentiation in the last three stages, suggesting early Kranz cell fate determination. In contrast, pre-M cells exhibit a more prolonged transcriptional differentiation process. Differential gene expression and coexpression analyses identified gene coexpression modules, one of which included 3 auxin transporter and 18 transcription factor (TF) genes, including known regulators of Kranz anatomy and/or vascular development. In situ hybridization of 11 TF genes validated their expression in early Kranz development. We determined the binding motifs of 15 TFs, predicted TF target gene relationships among the 18 TF and 3 auxin transporter genes, and validated 67 predictions by electrophoresis mobility shift assay. From these data, we constructed a gene regulatory network for Kranz development. Our study sheds light on the regulation of early maize leaf development and provides candidate leaf development regulators for future study.

SCARECROW is deployed in distinct contexts during rice and maize leaf development.

ABSTRACTThe flexible deployment of developmental regulators is an increasingly appreciated aspect of plant development and evolution. The GRAS transcription factor SCARECROW (SCR) regulates the development of the endodermis in Arabidopsis and maize roots, but during leaf development it regulates the development of distinct cell types; bundle-sheath in Arabidopsis and mesophyll in maize. In rice, SCR is implicated in stomatal patterning, but it is unknown whether this function is additional to a role in inner leaf patterning. Here, we demonstrate that two duplicated SCR genes function redundantly in rice. Contrary to previous reports, we show that these genes are necessary for stomatal development, with stomata virtually absent from leaves that are initiated after germination of mutants. The stomatal regulator OsMUTE is downregulated in Osscr1;Osscr2 mutants, indicating that OsSCR acts early in stomatal development. Notably, Osscr1;Osscr2 mutants do not exhibit the inner leaf patterning perturbations seen in Zmscr1;Zmscr1h mutants, and Zmscr1;Zmscr1h mutants do not exhibit major perturbations in stomatal patterning. Taken together, these results indicate that SCR was deployed in different developmental contexts after the divergence of rice and maize around 50 million years ago.

Functional Identification and Molecular Mechanism Analysis of BHLH Transcription Factor ABP7-like in Maize Leaf and Grain Development

Functional Identification and Molecular Mechanism Analysis of BHLH Transcription Factor ABP7-like in Maize Leaf and Grain Development

A forward genetics approach integrating genome-wide association study and expression quantitative trait locus mapping to dissect leaf development in maize (Zea mays).

SUMMARYThe characterization of the genetic basis of maize (Zea mays) leaf development may support breeding efforts to obtain plants with higher vigor and productivity. In this study, a mapping panel of 197 biparental and multiparental maize recombinant inbred lines (RILs) was analyzed for multiple leaf traits at the seedling stage. RNA sequencing was used to estimate the transcription levels of 29 573 gene models in RILs and to derive 373 769 single nucleotide polymorphisms (SNPs), and a forward genetics approach combining these data was used to pinpoint candidate genes involved in leaf development. First, leaf traits were correlated with gene expression levels to identify transcript–trait correlations. Then, leaf traits were associated with SNPs in a genome‐wide association (GWA) study. An expression quantitative trait locus mapping approach was followed to associate SNPs with gene expression levels, prioritizing candidate genes identified based on transcript–trait correlations and GWAs. Finally, a network analysis was conducted to cluster all transcripts in 38 co‐expression modules. By integrating forward genetics approaches, we identified 25 candidate genes highly enriched for specific functional categories, providing evidence supporting the role of vacuolar proton pumps, cell wall effectors, and vesicular traffic controllers in leaf growth. These results tackle the complexity of leaf trait determination and may support precision breeding in maize.

Identification and meta-analysis of QTLs for four leaf shape traits in two maize populations under contracting watering environments

Maize leaf length, leaf width, leaf angle, and leaf orientation value are determinant traits influencing leaf shape, yield potential, and can sense water stress signals. However, little is known regarding their genetic controls under drought stress. Using two F3 populations, we identified 72 QTLs under contrasting environments at V18 and R1 stages by single-environment mapping, 58.3% QTLs were identified under drought-stressed environments. Thirty-three joint QTLs were detected by joint analysis of all environments, 7 QTLs involved in QTL-by-environment interactions, 11 pair epistasis exhibited additive-by-additive effects. Twelve constitutive QTLs were dissected that will be potentially useful for genetic improvement of maize leaf development through QTL pyramiding. Additionally, 13 meta-QTLs were identified via meta-analysis, 15 candidate genes were identified in these meta-QTLs. These results provide valuable information for QTLs fine mapping, revealing genetic mechanisms of leaf shape under contrasting watering environments, and alleles provide useful targets for marker-assisted selection breeding.

Discovery of leaf region and time point related modules and genes in maize (Zea mays L.) leaves by Weighted Gene Co-expression Network analysis (WGCNA) of gene expression profiles of carbon metabolism

Maize (Zea mays L.) yield depends not only on the conversion and accumulation of carbohydrates in kernels, but also on the supply of carbohydrates by leaves. However, the carbon metabolism process in leaves can vary across different leaf regions and during the day and night. Hence, we used Weighted Gene Co-expression Network analysis (WGCNA) with the gene expression profiles of carbon metabolism to identify the modules and genes that may associate with particular regions in a leaf and time of day. There were a total of 45 samples of maize leaves that were taken from three different regions of a growing maize leaf at five time points. Robust Multi-array Average analysis was used to pre-process the raw data of GSE85963 (accession number), and quality control of data was based on Pearson correlation coefficients. We obtained eight co-expression network modules. The modules with the highest significance of association with LeafRegion and TimePoint were selected. Functional enrichment and gene-gene interaction analyses were conducted to acquire the hub genes and pathways in these significant modules. These results can support the findings of similar studies by providing evidence of potential module genes and enriched pathways associated with leaf development in maize.

Effect of temperature under different evaporative demand conditions on maize leaf expansion

Leaf area affects light interception, carbon assimilation and plant growth. How maize leaf development and expansion is affected by temperature under different evaporative demand conditions is not well understood. Effects of three temperatures (21, 27 and 32 °C) under high and low evaporative demand conditions were tested in growth chambers. Leaf expansion rate (LER), leaf appearance rate (LAR), leaf number, plant height, photosynthesis (A), net assimilation rate (NAR), stomatal conductance (gs), and transpiration (E) increased with increasing temperature under low evaporative demand environments. Under high evaporative demand, while there was an increase with temperature until 27 °C, at 32 °C there was a decrease in LER, plant height, A, NAR, gs, and E. However, the variation in the rate of leaf appearance appears to be linked to temperature but not VPD. The development of stomata and epidermal cells were affected by VPD with large size stomata and more epidermal cells under low VPD. The differential responses of LER to low and high evaporative demands could be attributed to changes in expression of Expansin A2, A4, and B1 genes and XTH23. The significant effect of temperature and VPD on the expression of vacuolar H + pyrophosphatase and teosinte branched gene was detected. Overall, we found a negative effect of high VPD due to high evaporative demand for maize leaf expansion and development.

The trans and cis zeatin isomers play different roles in regulating growth inhibition induced by high nitrate concentrations in maize

Abscisic acid (ABA), auxins, and cytokinins (CKs) are known to be closely linked to nitrogen signaling. In particular, CKs control the effects of nitrate availability on plant growth. Our group has shown that treatment with high nitrate concentrations limits root growth and leaf development in maize, and conditions the development of younger roots and leaves. CKs also affect source-sink relationships in plants. Based on these results, we hypothesized that CKs regulate the source-sink relationship in maize via a mechanism involving complex crosstalk with the main auxin indole-3-acetic acid (IAA) and ABA. To evaluate this hypothesis, various CK metabolites, IAA, and ABA were quantified in the roots and in source and sink leaves of maize plants treated with high and normal nitrate concentrations. The data obtained suggest that the cis and trans isomers of zeatin play completely distinct roles in maize growth regulation by a complex crosstalk with IAA and ABA. We demonstrate that while trans-zeatin (tZ) and isopentenyladenine (iP) regulate nitrate uptake and thus control final leaf sizes, cis-zeatin (cZ) regulates source and sink strength, and thus controls leaf development. The implications of these findings relating to the roles of ABA and IAA in plants’ responses to varying nitrate concentrations are also discussed.

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C3 or C4 photosynthesis. As part of a multinational effort to introduce C4 traits into rice to boost crop yield, candidate regulators of C4 leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C4-like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C4 plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C3 plants.

Transcriptome dynamics of developing maize leaves and genomewide prediction of cis elements and their cognate transcription factors

Maize is a major crop and a model plant for studying C4 photosynthesis and leaf development. However, a genomewide regulatory network of leaf development is not yet available. This knowledge is useful for developing C3 crops to perform C4 photosynthesis for enhanced yields. Here, using 22 transcriptomes of developing maize leaves from dry seeds to 192 h post imbibition, we studied gene up- and down-regulation and functional transition during leaf development and inferred sets of strongly coexpressed genes. More significantly, we developed a method to predict transcription factor binding sites (TFBSs) and their cognate transcription factors (TFs) using genomic sequence and transcriptomic data. The method requires not only evolutionary conservation of candidate TFBSs and sets of strongly coexpressed genes but also that the genes in a gene set share the same Gene Ontology term so that they are involved in the same biological function. In addition, we developed another method to predict maize TF-TFBS pairs using known TF-TFBS pairs in Arabidopsis or rice. From these efforts, we predicted 1,340 novel TFBSs and 253 new TF-TFBS pairs in the maize genome, far exceeding the 30 TF-TFBS pairs currently known in maize. In most cases studied by both methods, the two methods gave similar predictions. In vitro tests of 12 predicted TF-TFBS interactions showed that our methods perform well. Our study has significantly expanded our knowledge on the regulatory network involved in maize leaf development.

Transcriptome analysis of maize leaf systemic symptom infected by Bipolaris zeicola.

Bipolaris zeicola is a fungal pathogen that causes Northern corn leaf spot (NCLS), which is a serious foliar disease in maize and one of the most significant pathogens affecting global food security. Here, we report a genome-wide transcriptional profile analysis using next-generation sequencing (NGS) of maize leaf development after inoculation with B. zeicola. We performed High-Throughput Digital Gene Expression analysis to identify differentially expressed genes (DEGs) in resistant inbred Mo17 lines after infection with B. zeicola at four successive disease development stages—CP (contact period), PP (penetration period), IP (incubation period), and DP (disease period); the expression of the genes was compared with those in a CK (mock-treatment) control. In addition, a sensitive maize line (Zheng58) was used for the comparisons with the Mo17. Among all tested genes, 466 differentially expressed genes were identified in all libraries, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these genes suggested that they are involved in many biological processes related to systemic symptom development, such as plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis and photosynthesis. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in fungal-infected plants. This information will help in furthering our understanding of the detailed mechanisms of plant responses to fungal infection.

Maize (Zea maysL.) seedling leaf nuclear proteome and differentially expressed proteins between a hybrid and its parental lines

To better understand the underlying molecular basis of leaf development in maize, a reference map of nuclear proteins in basal region of seedling leaf was established using a combination of 2DE and MALDI-TOF-MS. In total, 441 reproducible protein spots in nuclear proteome of maize leaf basal region were detected with silver staining in a pH range of 3-10, among which 203 spots corresponding to 163 different proteins were identified. As expected, proteins implicated in RNA and protein-associated functions were overrepresented in nuclear proteome. Remarkably, a high percentage (10%) of proteins was identified to be involved in cell division and growth. In addition, comparative nuclear proteomic analysis in leaf basal region of highly heterotic hybrid Mo17/B73 and its parental lines was also performed and 52 of 445 (11.69%) detected protein spots were differentially expressed between the hybrid and its parental lines, among which 16 protein spots displayed nonadditively expressed pattern. These results indicated that hybridization between two parental lines can cause changes in the expression of a variety of nuclear proteins, which may be responsible for the observed leaf size heterosis.

Parallel Proteomic and Phosphoproteomic Analyses of Successive Stages of Maize Leaf Development

We performed large-scale, quantitative analyses of the maize (Zea mays) leaf proteome and phosphoproteome at four developmental stages. Exploiting the developmental gradient of maize leaves, we analyzed protein and phosphoprotein abundance as maize leaves transition from proliferative cell division to differentiation to cell expansion and compared these developing zones to one another and the mature leaf blade. Comparison of the proteomes and phosphoproteomes suggests a key role for posttranslational regulation in developmental transitions. Analysis of proteins with cell wall- and hormone-related functions illustrates the utility of the data set and provides further insight into maize leaf development. We compare phosphorylation sites identified here to those previously identified in Arabidopsis thaliana. We also discuss instances where comparison of phosphorylated and unmodified peptides from a particular protein indicates tissue-specific phosphorylation. For example, comparison of unmodified and phosphorylated forms of PINFORMED1 (PIN1) suggests a tissue-specific difference in phosphorylation, which correlates with changes in PIN1 polarization in epidermal cells during development. Together, our data provide insights into regulatory processes underlying maize leaf development and provide a community resource cataloging the abundance and phosphorylation status of thousands of maize proteins at four leaf developmental stages.

From Tip to Base: Parallel Proteomic and Phosphoproteomic Analyses of Successive Stages of Maize Leaf Development

Maize ( Zea mays ) leaves mature from tip to base. Thus, the entire sequence of development can be found along the length of a young, unexpanded maize leaf at the same point in time. Proliferative, symmetric cell divisions occur at the leaf base (zone 1). Distal to zone 1, symmetric divisions give

Genome‐wide transcript analysis of early maize leaf development reveals gene cohorts associated with the differentiation of C4 Kranz anatomy

Photosynthesis underpins the viability of most ecosystems, with C4 plants that exhibit 'Kranz' anatomy being the most efficient primary producers. Kranz anatomy is characterized by closely spaced veins that are encircled by two morphologically distinct photosynthetic cell types. Although Kranz anatomy evolved multiple times, the underlying genetic mechanisms remain largely elusive, with only the maize scarecrow gene so far implicated in Kranz patterning. To provide a broader insight into the regulation of Kranz differentiation, we performed a genome-wide comparative analysis of developmental trajectories in Kranz (foliar leaf blade) and non-Kranz (husk leaf sheath) leaves of the C4 plant maize. Using profile classification of gene expression in early leaf primordia, we identified cohorts of genes associated with procambium initiation and vascular patterning. In addition, we used supervised classification criteria inferred from anatomical and developmental analyses of five developmental stages to identify candidate regulators of cell-type specification. Our analysis supports the suggestion that Kranz anatomy is patterned, at least in part, by a SCARECROW/SHORTROOT regulatory network, and suggests likely components of that network. Furthermore, the data imply a role for additional pathways in the development of Kranz leaves.

Modeling Temperature Responses of Leaf Growth, Development, and Biomass in Maize with MAIZSIM

Mechanistic crop models capable of representing realistic temperature responses of key physiological processes are necessary for enhancing our ability to forecast crop yields and develop adaptive cropping solutions for achieving food security in a changing climate. Leaf growth and phenology are critical components of crop growth and yield that are sensitive to climate impacts. We developed a novel modeling approach that incorporates a set of nonlinear functions to augment traditional thermal time methods (e.g., growing degree days) for simulating temperature responses of leaf expansion and phenology in maize or corn (Zea mays L.). The resulting leaf expansion and phenology models have been implemented into a new crop model, MAIZSIM, that simulates crop growth based on key physiological and physical processes including C4 photosynthesis, canopy radiative transfer, C partitioning, water relations, and N dynamics for a maize plant. Coupled with a two‐dimensional soil process model, 2DSOIL, MAIZSIM was applied to simulate leaf growth, phenology, biomass partitioning, and overall growth of maize plants planted at two field sites on the Eastern Shore of Maryland and in Delaware for 3 yr of data. The model parameters were estimated using data from outdoor sunlit growth chambers and the literature. No calibration was performed using the field data. The MAIZSIM model simulated leaf area, leaf addition rate, leaf numbers, biomass partitioning and accumulation with reasonable accuracy. Our study provides a feasible method for integrating nonlinear temperature relationships into crop models that use traditional thermal time approaches without sacrificing their current structure for predicting the climate change impacts on crops.

Mutations in an AP2 Transcription Factor-Like Gene Affect Internode Length and Leaf Shape in Maize

BackgroundPlant height is an important agronomic trait that affects yield and tolerance to certain abiotic stresses. Understanding the genetic control of plant height is important for elucidating the regulation of maize development and has practical implications for trait improvement in plant breeding.Methodology/Principal FindingsIn this study, two independent, semi-dwarf maize EMS mutants, referred to as dwarf & irregular leaf (dil1), were isolated and confirmed to be allelic. In comparison to wild type plants, the mutant plants have shorter internodes, shorter, wider and wrinkled leaves, as well as smaller leaf angles. Cytological analysis indicated that the leaf epidermal cells and internode parenchyma cells are irregular in shape and are arranged in a more random fashion, and the mutants have disrupted leaf epidermal patterning. In addition, parenchyma cells in the dil1 mutants are significantly smaller than those in wild-type plants. The dil1 mutation was mapped on the long arm of chromosome 6 and a candidate gene, annotated as an AP2 transcription factor-like, was identified through positional cloning. Point mutations near exon-intron junctions were identified in both dil1 alleles, resulting in mis-spliced variants.ConclusionAn AP2 transcription factor-like gene involved in stalk and leaf development in maize has been identified. Mutations near exon-intron junctions of the AP2 gene give mis-spliced transcript variants, which result in shorter internodes and wrinkled leaves.