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The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed. Dissecting the genetic architecture of natural variation for maize (Zea mays L.) leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we performed an integrated genome- and transcriptome-wide association studies (GWAS and TWAS) to identify candidate genes putatively regulating variation in leaf gc. Of the 22 plausible candidate genes identified, 4 were predicted to be involved in cuticle precursor biosynthesis and export, 2 in cell wall modification, 9 in intracellular membrane trafficking, and 7 in the regulation of cuticle development. A gene encoding an INCREASED SALT TOLERANCE1-LIKE1 (ISTL1) protein putatively involved in intracellular protein and membrane trafficking was identified in GWAS and TWAS as the strongest candidate causal gene. A set of maize nested near-isogenic lines that harbor the ISTL1 genomic region from eight donor parents were evaluated for gc, confirming the association between gc and ISTL1 in a haplotype-based association analysis. The findings of this study provide insights into the role of regulatory variation in the development of the maize leaf cuticle and will ultimately assist breeders to develop drought-tolerant maize for target environments.

Significance Plant cuticles provide barriers to water loss and arose as aquatic plants adapted to the dry terrestrial environment. The cuticle components, waxes and the fatty acid-based polymer cutin, are synthesized in the plant epidermis, exported across the cell wall, and deposited on the plant surface. This study suggests a role for PHYTOCHROME light receptors during cuticle development in leaves of maize and moss, diverse species that are separated by more than 400 million y of land plant evolution. We hypothesize that phytochrome-mediated light signaling contributed to the evolution of cuticles in land plants., Plant cuticles are composed of wax and cutin and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. A weighted gene coexpression network (WGCN) analysis suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during the regulation of cuticular wax deposition. Genetic analyses reveal that phyB1 phyB2 double mutants of maize exhibit abnormal cuticle composition, supporting the predictions of our coexpression analysis. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting an ancestral role for PHYTOCHROME-mediated, light-stimulated regulation of cuticle development during plant evolution.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed at night and under water-limited conditions. Elucidating the genetic architecture of natural variation for leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we conducted a genome-wide association study ofgcof adult leaves in a maize inbred association panel that was evaluated in four environments (Maricopa, AZ, and San Diego, CA in 2016 and 2017). Five genomic regions significantly associated withgcwere resolved to seven plausible candidate genes (ISTL1, two SEC14 homologs, cyclase-associated protein, a CER7 homolog, GDSL lipase, and β-D-XYLOSIDASE 4). These candidates are potentially involved in cuticle biosynthesis, trafficking and deposition of cuticle lipids, cutin polymerization, and cell wall modification. Laser microdissection RNA sequencing revealed that all these candidate genes, with the exception of the CER7 homolog, were expressed in the zone of the expanding adult maize leaf where cuticle maturation occurs. With direct application to genetic improvement, moderately high average predictive abilities were observed for whole-genome prediction ofgcin locations (0.46 and 0.45) and across all environments (0.52). The findings of this study provide novel insights into the genetic control ofgcand have the potential to help breeders more effectively develop drought-tolerant maize for target environments.Article summaryThe cuticle serves as the major barrier to water loss when stomata are closed at night and under water-limited conditions and potentially relevant to drought tolerance in crops. We performed a genome-wide association study to elucidate the genetic architecture of natural variation for maize leaf cuticular conductance. We identified epidermally expressed candidate genes that are potentially involved in cuticle biosynthesis, trafficking and deposition, cutin polymerization, and cell wall modification. Finally, we observed moderately high predictive abilities for whole-genome prediction of leaf cuticular conductance. Collectively, these findings may help breeders more effectively develop drought-tolerant maize.

Background and AimsPrior work has examined cuticle function, composition and ultrastructure in many plant species, but much remains to be learned about how these features are related. This study aims to elucidate relationships between these features via analysis of cuticle development in adult maize (Zea mays L.) leaves, while also providing the most comprehensive investigation to date of the composition and ultrastructure of adult leaf cuticles in this important crop plant.MethodsWe examined water permeability, wax and cutin composition via gas chromatography, and ultrastructure via transmission electron microscopy, along the developmental gradient of partially expanded adult maize leaves, and analysed the relationships between these features.Key ResultsThe water barrier property of the adult maize leaf cuticle is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously over the course of development, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, and a thin cuticle proper that acquires an inner, osmiophilic layer during development.ConclusionsCuticular waxes of the adult maize leaf are dominated by alkanes and alkyl esters. Unexpectedly, these are localized mainly in the epicuticular layer. Establishment of the water barrier during development coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper. Thus, alkyl esters and the deposition of the cutin polyester are implicated as key components of the water barrier property of adult maize leaf cuticles.

The cuticle is a hydrophobic layer on the outer surface plant shoots, which serves as an important interaction interface with the environment. It consists of the lipid polymer cutin, embedded with and covered by waxes, and provides protection against stresses including desiccation, UV radiation, and pathogen attack. Bulliform cells form in longitudinal strips on the adaxial leaf surface, and have been implicated in the leaf rolling response observed in drought stressed grass leaves. In this study, we show that bulliform cells of the adult maize leaf epidermis have a specialized cuticle, and we investigate its function along with that of bulliform cells themselves. Analysis of natural variation was used to relate bulliform strip pattering to leaf rolling rate, providing evidence of a role for bulliform cells in leaf rolling. Bulliform cells displayed increased shrinkage compared to other epidermal cell types during dehydration of the leaf, providing a potential mechanism to facilitate leaf rolling. Comparisons of cuticular conductance between adaxial and abaxial leaf surfaces, and between bulliform-enriched mutants vs. wild type siblings, provided evidence that bulliform cells lose water across the cuticle more rapidly than other epidermal cell types. Bulliform cell cuticles have a distinct ultrastructure, and differences in cutin monomer content and composition, compared to other leaf epidermal cells. We hypothesize that this cell type-specific cuticle is more water permeable than the epidermal pavement cell cuticle, facilitating the function of bulliform cells in stress-induced leaf rolling observed in grasses.One sentence summaryBulliform cells in maize have a specialized cuticle, lose more water than other epidermal cell types as the leaf dehydrates, and facilitate leaf rolling upon dehydration.

Plant cuticles are composed of wax and cutin, and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. Correlations between cuticle development and cell wall biosynthesis processes were identified, as well as evidence of roles for auxin and brassinosteroids. In addition, our network analyses suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during cuticular wax deposition. Genetic analyses reveal that the phyB1 phyB2 double mutant of maize exhibits abnormal cuticle composition, supporting predictions of our coexpression analyses. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting a role for light-stimulated development of cuticular waxes during plant evolution.

Although extensive prior work has characterized cuticle composition, function, ultrastructure and development in many plant species, much remains to be learned about how these features are interrelated. Moreover, very little is known about the adult maize leaf cuticle in spite of its significance for agronomically important traits in this major crop. We analyzed cuticle composition, ultrastructure, and permeability along the developmental gradient of partially expanded adult maize leaves to probe the relationships between these features. The water barrier property is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously along the developmental gradient, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, a thin cuticle proper that acquires an inner, osmiophilic layer during development, and no cuticular layer. Cuticular waxes of the adult maize leaf are dominated by alkanes and wax esters localized mainly in the epicuticular layer. Establishment of the water barrier coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper.Higlight statementChemical, ultrastructural and functional analysis of cuticle development in partially expanded adult maize leaves revealed important roles for wax esters and an osmiophilic, likely cutin-rich, layer in protection from dehydration.

Pangloss1 (PAN1) and PAN2 are leucine-rich repeat receptor-like proteins that function cooperatively to polarize the divisions of subsidiary mother cells (SMCs) during stomatal development in maize (Zea mays). PANs colocalize in SMCs, and both PAN1 and PAN2 promote polarization of the actin cytoskeleton and nuclei in these cells. Here, we show that PAN1 and PAN2 have additional functions that are unequal or divergent. PAN1, but not PAN2, is localized to cell plates in all classes of dividing cells examined. pan1 mutants exhibited no defects in cell plate formation or in the recruitment or removal of a variety of cell plate components; thus, they did not demonstrate a function for PAN1 in cytokinesis. PAN2, in turn, plays a greater role than PAN1 in directing patterns of postmitotic cell expansion that determine the shapes of mature stomatal subsidiary cells and interstomatal cells. Localization studies indicate that PAN2 impacts subsidiary cell shape indirectly by stimulating localized cortical actin accumulation and polarized growth in interstomatal cells. Localization of PAN1, Rho of Plants2, and PIN1a suggests that PAN2-dependent cell shape changes do not involve any of these proteins, indicating that PAN2 function is linked to actin polymerization by a different mechanism in interstomatal cells compared with SMCs. Together, these results demonstrate that PAN1 and PAN2 are not dedicated to SMC polarization but instead play broader roles in plant development. We speculate that PANs may function in all contexts to regulate polarized membrane trafficking either directly or indirectly via their influence on actin polymerization.

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.

Mechanisms governing the polarization of plant cell division are poorly understood. Previously, we identified pangloss1 (PAN1) as a leucine-rich repeat-receptor-like kinase (LRR-RLK) that promotes the polarization of subsidiary mother cell (SMC) divisions toward the adjacent guard mother cell (GMC) during stomatal development in maize (Zea mays). Here, we identify pangloss2 (PAN2) as a second LRR-RLK promoting SMC polarization. Quantitative proteomic analysis identified a PAN2 candidate by its depletion from membranes of pan2 single and pan1;pan2 double mutants. Genetic mapping and sequencing of mutant alleles confirmed the identity of this protein as PAN2. Like PAN1, PAN2 has a catalytically inactive kinase domain and accumulates in SMCs at sites of GMC contact before nuclear polarization. The timing of polarized PAN1 and PAN2 localization is very similar, but PAN2 acts upstream because it is required for polarized accumulation of PAN1 but is independent of PAN1 for its own localization. We find no evidence that PAN2 recruits PAN1 to the GMC contact site via a direct or indirect physical interaction, but PAN2 interacts with itself. Together, these results place PAN2 at the top of a cascade of events promoting the polarization of SMC divisions, potentially functioning to perceive or amplify GMC-derived polarizing cues.

The cellular organization of plant tissues is determined by patterns of cell division and growth coupled with cellular differentiation. Cells proliferate mainly via symmetric division, whereas asymmetric divisions are associated with initiation of new developmental patterns and cell types. Division planes in both symmetrically and asymmetrically dividing cells are established through the action of a cortical preprophase band (PPB) of cytoskeletal filaments, which is disassembled upon transition to metaphase, leaving behind a cortical division site (CDS) to which the cytokinetic phragmoplast is later guided to position the cell plate. Recent progress has been made in understanding PPB formation and function as well as the nature and function of the CDS. In asymmetrically dividing cells, division plane establishment is governed by cell polarity. Recent work is beginning to shed light on polarization mechanisms in asymmetrically dividing cells, with receptor-like proteins and potential downstream effectors emerging as important players in this process.

Unique mechanisms are used to orient cell division planes in plants. A cortical ring of cytoskeletal filaments called the preprophase band (PPB) predicts the future division plane during G2 and is disassembled as the mitotic spindle forms, leaving behind a 'cortical division site' (CDS) that guides the placement of the new cell wall (cell plate) during cytokinesis. The molecular features of the CDS have remained elusive for decades. Recently, a few proteins have at last been identified that are specifically localized to or excluded from the CDS and that participate in the orientation, attachment or maturation of cell plates. Recent progress has also been made in identifying proteins needed for PPB formation and thus for division plane establishment.

Variants in the X-linked gene PCDH19 are associated with early infantile epileptic encephalopathy-9. This unusual condition spares hemizygous males except for psychiatric and behavioral abnormalities, and for this reason is also known as female limited epilepsy. Some cases are due to de novo PCDH19 variants, but may also be paternally inherited. Our patient is a 6-year-old male with epileptic encephalopathy. Exome sequencing revealed apparent heterozygosity in PCDH19 for a novel nonsense variant, c.605C>A (p.Ser202*), inconsistent with expectations for a male. Testing of other tissues revealed a mixture of mutant and normal alleles. These results are consistent with somatic mosaicism for p.Ser202*. This is the second male with somatic mosaicism for PCDH19 deficiency, providing further support for cellular interference as the pathogenic mechanism for this condition, which leads to this unusual mode of inheritance in which females are more severely affected than males. © 2016 Wiley Periodicals, Inc.

Pre-mitotic establishment of polarity is a key event in the preparation of mother cells for asymmetric cell divisions that produce daughters of distinct fates, and ensures correct cellular patterning of tissues and eventual organ function. Previous work has shown that two receptor-like kinases, PANGLOSS2 (PAN2) and PAN1, and the small GTPase RHO GTPASE OF PLANTS (ROP) promote mother cell polarity and subsequent division asymmetry in developing maize stomata. PAN proteins become polarized prior to asymmetric cell division, however, the mechanism of this polarization is unknown. Here we show that the SCAR/WAVE regulatory complex, which activates the actin-nucleating ARP2/3 complex, is the first known marker of polarity in this asymmetric division model and is required for PAN polarization. These findings implicate actin, and specifically branched actin networks, in PAN polarization and asymmetric cell division.

The Arp2/3 complex, a highly conserved nucleator of F-actin polymerization, plays a key role in the regulation of actin dynamics eukaryotic cells. In animal cells and yeasts, Wiskott-Aldrich Syndrome protein (WASP)/suppressor of cAMP receptor (Scar)/WASP family verprolin homologous (WAVE) family proteins activate the Arp2/3 complex in response to localized cues. Like other eukaryotes, plants have an Arp2/3 complex, which has recently been shown to play an important role in F-actin organization and cell morphogenesis. However, no activators of the Arp2/3 complex have been identified in plants, which lack obvious homologs of WASP/Scar/WAVE family proteins. Here, we identify a family of Scar/WAVE-related plant Arp2/3 activators. Like Scar/WAVE proteins, four proteins identified in Arabidopsis thaliana (AtSCAR1 to AtSCAR4) and one in maize (ZmSCAR1) have a C-terminal WASP homology 2 (WH2)/acidic (WA)-verprolin homology/cofilin homology/acidic (VCA)-like domain, which we show can activate the bovine Arp2/3 complex. At their N termini, AtSCAR1 to ATSCAR4, along with a fifth protein lacking a VCA/WA-like domain at its C terminus (At4g18600), are related to the N-terminal Scar homology domains of Scar/WAVE family proteins. Analysis of gene expression patterns suggests functional redundancy among members of the AtSCAR family. Full-length AtSCAR1 and ATSCAR3 proteins and their Scar homology domains bind in vitro to AtBRICK 1 (AtBRK1), the Arabidopsis homolog of HSPC300, a WAVE-binding protein recently identified as a component of a complex implicated in the regulation of Scar/WAVE activity. Thus, AtSCAR proteins are likely to function in association with AtBRK1, and perhaps other Arabidopsis homologs of WAVE complex components, to regulate activation of the Arp2,3 complex in vivo .

We have taken a genetic approach to investigating cytoskeleton-dependent mechanisms governing cell morphogenesis in the maize leaf epidermis. Previously, we showed that the Brick1 (Brk1) gene is required for the formation of epidermal cell lobes as well as for properly polarized divisions of stomatal subsidiary mother cells, and encodes an 8 kDa protein highly conserved in plants and animals. Here, we show that two additional Brick genes, Brk2 and Brk3, are involved in the same aspects of epidermal cell morphogenesis and division. As shown previously for Brk1, analysis of the cytoskeleton shows that Brk2 andBrk3 are required for the formation of local F-actin enrichments associated with lobe outgrowth in wild-type cells. Analysis of brk1;brk2,brk1;brk3 and brk2;brk3 double mutants shows that their phenotypes are the same as those of brk single mutants. Mosaic analysis shows that Brk1 acts non cell-autonomously over a short distance. By contrast, Brk2 and Brk3 act cell-autonomously to promote pavement cell lobe formation, but Brk3 acts non cell-autonomously, and Brk2 partially non cell-autonomously, to promote polarized subsidiary mother cell divisions. Together, these observations indicate that all three Brk genes act in a common pathway in which each Brk gene has a distinct function. Recent work demonstrating a function for the mammalian homolog of BRK1 (HSPC300) in activation of Arp2/3-dependent actin polymerization implicates theBrk pathway in local regulation of actin polymerization in plant cells.

Multiple disease resistance has important implications for plant fitness, given the selection pressure that many pathogens exert directly on natural plant populations and indirectly via crop improvement programs. Evidence of a locus conditioning resistance to multiple pathogens was found in bin 1.06 of the maize genome with the allele from inbred line “Tx303” conditioning quantitative resistance to northern leaf blight (NLB) and qualitative resistance to Stewart’s wilt. To dissect the genetic basis of resistance in this region and to refine candidate gene hypotheses, we mapped resistance to the two diseases. Both resistance phenotypes were localized to overlapping regions, with the Stewart’s wilt interval refined to a 95.9-kb segment containing three genes and the NLB interval to a 3.60-Mb segment containing 117 genes. Regions of the introgression showed little to no recombination, suggesting structural differences between the inbred lines Tx303 and “B73,” the parents of the fine-mapping population. We examined copy number variation across the region using next-generation sequencing data, and found large variation in read depth in Tx303 across the region relative to the reference genome of B73. In the fine-mapping region, association mapping for NLB implicated candidate genes, including a putative zinc finger and pan1. We tested mutant alleles and found that pan1 is a susceptibility gene for NLB and Stewart’s wilt. Our data strongly suggest that structural variation plays an important role in resistance conditioned by this region, and pan1, a gene conditioning susceptibility for NLB, may underlie the QTL.

Spatial control of cytokinesis in plant cells depends on guidance of the cytokinetic apparatus, the phragmoplast, to a cortical “division site” established before mitosis. Previously, we showed that the Tangled1 (Tan1) gene of maize is required for this process during maize leaf development (Cleary, A.L., and L.G. Smith. 1998. Plant Cell. 10:1875–1888.). Here, we show that the Tan1 gene is expressed in dividing cells and encodes a highly basic protein that can directly bind to microtubules (MTs). Moreover, proteins recognized by anti-TAN1 antibodies are preferentially associated with the MT-containing cytoskeletal structures that are misoriented in dividing cells of tan1 mutants. These results suggest that TAN1 protein participates in the orientation of cytoskeletal structures in dividing cells through an association with MTs.

Asymmetric cell divisions occur repeatedly during plant development, but the mechanisms by which daughter cells are directed to adopt different fates are not well understood [1,2]. Previous studies have demonstrated roles for positional information in specification of daughter cell fates following asymmetric divisions in the embryo [3] and root [4]. Unequally inherited cytoplasmic determinants have also been proposed to specify daughter cell fates after some asymmetric cell divisions in plants [1,2,5], but direct evidence is lacking. Here we investigate the requirements for specification of stomatal subsidiary cell fate in the maize leaf by analyzing four mutants disrupting the asymmetric divisions of subsidiary mother cells (SMCs). We show that subsidiary cell fate does not depend on proper localization of the new cell wall during the SMC division, and is not specified by positional information acting on daughter cells after completion of the division. Instead, our data suggest that specification of subsidiary cell fate depends on polarization of SMCs and on inheritance of the appropriate daughter nucleus. We thus provide evidence of a role for unequal inheritance of an intracellular determinant in specification of cell fate after an asymmetric plant cell division.

Polarization of cell division is essential for eukaryotic development, but little is known about how this is accomplished in plants. The formation of stomatal complexes in maize involves the polarization of asymmetric subsidiary mother cell (SMC) divisions toward the adjacent guard mother cell (GMC), apparently under the influence of a GMC-derived signal. We found that the maize pan1 gene promotes the premitotic polarization of SMCs and encodes a leucine-rich repeat receptor-like protein that becomes localized in SMCs at sites of GMC contact. PAN1 has an inactive kinase domain but is required for the accumulation of a membrane-associated phosphoprotein, suggesting a function for PAN1 in signal transduction. Our findings implicate PAN1 in the transmission of an extrinsic signal that polarizes asymmetric SMC divisions toward GMCs.

In plants, specialized epidermal cells are arranged in semiordered patterns. In grasses such as maize, stomata and other specialized cell types differentiate in linear patterns within the leaf epidermis. A variety of mechanisms have been proposed to direct patterns of epidermal cell differentiation. One class of models proposes that patterns of cellular differentiation depend on the lineage relationships among epidermal cells. Another class of models proposes that epidermal patterning depends on positional information rather than lineage relationships. In the dicot epidermis, cell lineage is an important factor in the patterning of stomata, but not trichomes. In this study, the role of cell lineage in the linear patterning of stomata and bulliform cells in the maize leaf epidermis is investigated. Clones of epidermal cells in juvenile leaves were marked by excision of dSpm from gl15-m and in adult leaves by excision of Ds2 from bz2-m. These clones were analyzed in relation to patterns of stomata and bulliform cells, testing specific predictions of clonal origin hypotheses for the patterning of these cell types. We found that the great majority of clones analyzed failed to satisfy these predictions. Our results clearly show that lineage does not account for the linear patterning of stomata and bulliform cells, implying that positional information must direct the differentiation patterns of these cell types in maize.

Plant cells divide in two by constructing a new cell wall (cell plate) between daughter nuclei after mitosis. Golgi-derived vesicles are transported to the equator of a cytoskeletal structure called a phragmoplast, where they fuse together to form the cell plate. Orientation of new cell walls involves actin-dependent guidance of phragmoplasts and associated cell plates to cortical sites established prior to mitosis. Recent work has provided new insights into how actin filaments and other proteins in the phragmoplast and cell plate contribute to cytokinesis. Newly discovered mutations have identified a variety of genes required for cytokinesis or its spatial regulation.

A variety of approaches has recently been employed to investigate how sister cells adopt distinct fates following asymmetric divisions during plant development. Surgical and drug studies have been used to analyze asymmetric divisions during both early embryogenesis in brown algae and pollen development in tobacco. Genetic screens have been used to identify genes in Arabidopsis thaliana that are required for specific asymmetric cell divisions during pollen and root development. These studies indicate that cell polarity and division orientation are closely tied to the process of cell fate specification, and suggest that differential inheritance of determinants and positional information may both be involved in the specification of cell fates following asymmetric cell division.

The product of the maize homeobox gene, knotted1 (kn1), localizes to the nuclei of cells in shoot meristems, but is absent from portions of the meristem where leaf primordia or floral organs initiate. Recessive mutant alleles of kn1 were obtained by screening for loss of the dominant leaf phenotype in maize. Mutant kn1 alleles carrying nonsense, splicing and frame shift mutations cause severe inflorescence and floral defects. Mutant tassels produce fewer branches and spikelets. Ears are often absent, and when present, are small with few spikelets. In addition, extra carpels form in female florets and ovule tissue proliferates abnormally. Less frequently, extra leaves form in the axils of vegetative leaves. These mutations reveal a role for kn1 in meristem maintenance, particularly as it affects branching and lateral organ formation.

A comprehensive knowledge of proteomic states is essential for understanding biological systems. Using mass spectrometry, we mapped an atlas of developing maize seed proteotypes comprising 14,165 proteins and 18,405 phosphopeptides (from 4,511 proteins), quantified across eight tissues. We found that many of the most abundant proteins are not associated with detectable levels of their mRNAs, and we provide evidence for three potential explanations: transport of proteins between tissues; diurnal, out-of-phase accumulation of mRNAs and cognate proteins; and differential lifetimes of mRNAs compared with proteins. Likewise, many of the most abundant mRNAs were not associated with detectable levels of their proteins. Across the entire dataset, protein abundance was poorly correlated with mRNA levels and was largely independent of phosphorylation status. Comparisons between proteotypes revealed the quantitative contribution of specific proteins and phosphorylation events to the spatially and temporally regulated starch and oil biosynthetic pathways. Reconstruction of signaling networks established associations of proteins and phosphoproteins with distinct biological processes acting during seed development. Additionally, a protein kinase substrate network was reconstructed, enabling the identification of 762 potential substrates of specific protein kinases. Finally, examination of 694 transcription factors revealed remarkable constraints on patterns of expression and phosphorylation within transcription factor families. These results provide a resource for understanding seed development in a crop that is the foundation of modern agriculture.

An idea underlying a great deal of research and discussion in plant cell and developmental biology is that the spatial regulation of cell division plays a key role in plant development. In this article, the role of cell division in two aspects of leaf development is analysed: morphogenesis (leaf initiation, growth, and the generation of leaf shape) and histogenesis (the differentiation of leaf cells to form the various cell types that make up a functional leaf). The point of view that emerges from this analysis is that the rate and pattern of cell division is important for leaf development, but does not dictate leaf size, shape, or cell fate.

It is often assumed that in plants, where the relative positions of cells are fixed by cell walls, division orientations are critical for the generation of organ shapes. However, an alternative perspective is that the generation of shape may be controlled at a regional level independently from the initial orientations of new cell walls. In support of this latter view, we describe here a recessive mutation of maize, tangled-1 (tan-1), that causes cells to divide in abnormal orientations throughout leaf development without altering overall leaf shape. In normal plants, leaf cells divide either transversely or longitudinally relative to the mother cell axis; transverse divisions are associated with leaf elongation and longitudinal divisions with leaf widening. In tan-1 mutant leaves, cells in all tissue layers at a wide range of developmental stages divide transversely at normal frequencies, but longitudinal divisions are largely substituted by a variety of aberrantly oriented divisions in which the new cell wall is crooked or curved. Mutant leaves grow more slowly than normal, but their overall shapes are normal at all stages of their growth. These observations demonstrate that the generation of maize leaf shape does not depend on the precise spatial control of cell division, and support the general view that mechanisms independent from the control of cell division orientations are involved in the generation of shape during plant development.

WAVE/SCAR has long been known to activate the actin-nucleating Arp2/3 complex in a Rac-dependent manner. Recent biochemical and genetic studies have revealed important roles for four WAVE-associated proteins in regulating WAVE function.

GT-2 is a DNA-binding protein with high target-sequence specificity toward functionally defined, positively acting cis elements in the rice phytochrome A gene promoter. Using immunocytochemical procedures, it is shown here that GT-2 is localized to the nucleus, consistent with a function in transcriptional regulation. Immunoblot and immunocytochemical analyses show that rice shoots contain higher levels of GT-2 protein than roots, and that no photo-induced changes in GT-2 abundance or spatial distribution are detectable in these tissues, a result consistent with the proposed constitutive activity of GT-2. In both shoots and roots, GT-2 protein is undetectable in meristematic tissue but becomes expressed at later stages of cellular development, consistent with a role in contributing to the pattern of phytochrome A gene expression. By transfecting protoplasts with a series of constructs containing deletion derivatives of GT-2 fused to beta-glucuronidase (GUS), followed by in situ localization of GUS activity, two independent, functionally active nuclear localization sequences (NLSs) have been identified in GT-2. One NLS resides within each of a pair of previously identified, spatially separate, trihelix motifs in the protein. Sequence inversion and alanine-scanning mutagenesis has identified residues within these NLSs necessary for nuclear localization. Each NLS contains two basic domains separated by 10 amino acids, conforming to the bipartite class of NLS involved in the targeting of numerous other nuclear localized proteins.

The formation of shoot and root meristems that ultimately give rise to all tissues of the plant body occurs for the first time during embryogenesis. Meristem formation has traditionally been defined in terms of the appearance of histological features of meristems; this approach has led to varying interpretations of the timing of meristem formation relative to other events in embryogenesis. Markers that would provide more objective criteria for the analysis of meristem formation have not been widely available. The maize homeobox gene, knotted1 (kn1), is expressed in shoot meristems throughout postembryonic stages of shoot development. In order to determine whether this gene is expressed in the shoot meristem from its earliest inception, we examined the expression of kn1 in embryos at a series of stages by in situ hybridization to kn1 mRNA and immunolocalization of KN1 protein. Our results show that the onset of kn1 expression is temporally and spatially coincident with the earliest histologically recognizable signs of shoot meristem formation in the embryo, and thus provides a valuable marker for this process. © 1995 Wiley-Liss, Inc.

Stomata are generated via asymmetric cell division in both dicots and monocots. Intrinsic or extrinsic polarity cues are perceived and acted upon to generate mother cell polarity and determine asymmetric division planes. Arabidopsis employs both intrinsic and extrinsic cues to orient a variable series of asymmetric stomatal divisions, using novel proteins such as BASL and POLAR to generate polarity. In contrast, maize appears to employ only extrinsic cues to orient the polarities of divisions occurring in an invariant sequence to generate stomatal complexes. Although both plants use receptor-like kinases to generate or orient division polarity in developing stomata, there are few similarities in the proteins and pathway identified to date as regulators of these processes.

Molecular genetics provides a promising alternative to other experimental approaches for furthering our understanding of the mechanisms controlling leaf development. We investigated the molecular basis of dominant Knotted (Kn1) mutations in maize, which cause cells associated with the lateral veins of the leaf blade to acquire characteristics of sheath or auricle and sporadically form outgrowths called knots. The kn1 gene encodes a homeodomain, a DNA-binding domain shared by many transcription factors that regulate developmental processes in animals and fungi. In normal plants, the expression of kn1 is confined to the shoot apex, but in Kn1 mutants, the gene is also expressed ectopically in the veins of developing leaves, apparently causing cells to change their developmental fates. The kn1 gene may function in the shoot apex of normal plants to promote indeterminate growth. Consistent with this hypothesis, when kn1 is expressed constitutively at high levels in the leaves of transgenic tobacco, shoots are formed on the leaf surface. Thus, our results indicate that while the kn1 gene may normally have no function in leaf development, it can alter the development of maize and tobacco leaves when it is expressed in the leaf inappropriately. Genes that normally play a role in leaf development are more likely to be defined by recessive mutations that alter leaf morphogenesis and histogenesis. Key words: leaf development, molecular genetics, Knotted.

TANGLED (TAN) is the founding member of a family of plant-specific proteins required for correct orientation of the division plane. Arabidopsis thaliana TAN is localized before prophase until the end of cytokinesis at the cortical division site (CDS), where it appears to help guide the cytokinetic apparatus towards the cortex. We show that TAN is actively recruited to the CDS by distinct mechanisms before and after preprophase band (PPB) disassembly. Colocalization with the PPB is mediated by one region of TAN, whereas another region mediates its recruitment to the CDS during cytokinesis. This second region binds directly to POK1, a kinesin that is required for TAN localization. Although this region of TAN is recruited to the CDS during cytokinesis without first colocalizing with the PPB, pharmacological evidence indicates that the PPB is nevertheless required for both early and late localization of TAN at the CDS. Finally, we show that phosphatase activity is required for maintenance of early but not late TAN localization at the CDS. We propose a new model in which TAN is actively recruited to the CDS by several mechanisms, indicating that the CDS is dynamically modified from prophase through to the completion of cytokinesis.

Dominant mutations of the Knotted-1 (Kn1) homeobox gene of maize alter the differentiation and growth of cells associated with leaf veins. By analyzing Kn1 transcripts and KN1 protein, we show that the gene is not expressed at high levels during the development of wildtype leaves. Instead, Kn1 is expressed in apical meristems of vegetative and floral shoots, and is downregulated as leaves and floral organs are initiated. Kn1 is also expressed in relatively undifferentiated cells within developing vascular bundles, as well as ground tissue, in immature, unelongated axes of wild-type vegetative and floral shoots. In Kn1-N2 mutant plants, quantitative, but not qualitative differences are apparent in Kn1 transcripts and KN1 protein, consistent with previous observations that dominant Kn1 mutations map to noncoding regions of the gene. Kn1 is expressed ectopically in vascular bundles within developing mutant leaves in a pattern that correlates with the phenotypic alterations produced by the Kn1-N2 mutation. Thus, Kn1 apparently alters the fates of leaf cells in which it is ectopically expressed from an early stage of leaf development. Based on these observations, we hypothesize that Kn1 functions in its wild-type context as a regulator of cell determination.

Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkorper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.

The ARP2/3 complex, a highly conserved nucleator of F-actin polymerization, and its activator, the SCAR complex, have been shown to play important roles in leaf epidermal cell morphogenesis in Arabidopsis. However, the intracellular site(s) and function(s) of SCAR and ARP2/3 complex-dependent actin polymerization in plant cells remain unclear. We demonstrate that putative SCAR complex subunits BRK1 and SCAR1 are localized to the plasma membrane at sites of cell growth and wall deposition in expanding cells of leaves and roots. BRK1 localization is SCAR-dependent, providing further evidence of an association between these proteins in vivo. Consistent with plasma membrane localization of SCAR complex subunits, cortical F-actin accumulation in root tip cells is reduced in brk1 mutants. Moreover, mutations disrupting the SCAR or ARP2/3 complex reduce the growth rate of roots and their ability to penetrate semi-solid medium, suggesting reduced rigidity. Cell walls of mutant roots exhibit abnormal structure and composition at intercellular junctions where BRK1 and SCAR1 are enriched in the adjacent plasma membrane. Taken together, our results suggest that SCAR and ARP2/3 complex-dependent actin polymerization promotes processes at the plasma membrane that are important for normal growth and wall assembly.

Polarization of cell division is essential for eukaryotic development, but little is known about how this is accomplished in plants. The formation of stomatal complexes in maize involves the polarization of asymmetric subsidiary mother cell (SMC) divisions toward the adjacent guard mother cell (GMC), apparently under the influence of a GMC-derived signal. We found that the maize pan1 gene promotes the premitotic polarization of SMCs and encodes a leucine-rich repeat receptor-like protein that becomes localized in SMCs at sites of GMC contact. PAN1 has an inactive kinase domain but is required for the accumulation of a membrane-associated phosphoprotein, suggesting a function for PAN1 in signal transduction. Our findings implicate PAN1 in the transmission of an extrinsic signal that polarizes asymmetric SMC divisions toward GMCs.

In plants, cell wall placement during cytokinesis is determined by the position of the preprophase band (PPB) and the subsequent expansion of the phragmoplast, which deposits the new cell wall, to the cortical division site delineated by the PPB. New cell walls are often incorrectly oriented during asymmetric cell divisions in the leaf epidermis of maize (Zea mays) discordia1 (dcd1) mutants, and this defect is associated with aberrant PPB formation in asymmetrically dividing cells. dcd1 was cloned and encodes a putative B'' regulatory subunit of the PP2A phosphatase complex highly similar to Arabidopsis thaliana FASS/TONNEAU2, which is required for PPB formation. We also identified alternative discordia1 (add1), a second gene in maize nearly identical to dcd1. While loss of add1 function does not produce a noticeable phenotype, knock down of both genes in add1(RNAi) dcd1(RNAi) plants prevents PPB formation and causes misorientation of symmetric and asymmetric cell divisions. Immunolocalization studies with an antibody that recognizes both DCD1 and ADD1 showed that these proteins colocalize with PPBs and remain at the cortical division site through metaphase. Our results indicate that DCD1 and ADD1 function in PPB formation, that this function is more critical in asymmetrically dividing cells than in symmetrically dividing cells, and that DCD1/ADD1 may have other roles in addition to promoting PPB formation at the cortical division site.

The maize leaf has a simple cellular architecture that is amenable to cell biological study. Combined with the spatially defined growth gradients in the leaf, maize cells are useful for investigating how cell division and expansion are controlled spatially and temporally. Here we present recent advances in our understanding of molecular controls of cell division and expansion, particularly as mediated through the dynamic functions of the plant cytoskeleton and via analysis of mutants. We contend that the maize leaf epidermis is a particularly useful platform for study due its ordered growth pattern and the linear array of cells. Current advances and emerging tools are discussed that will allow the power of maize genetics to be fully realized at a cell biological level.

This review discusses current knowledge on division plane determination in plant cells and how divisionwithin this plane is executed during cytokinesis. Plants cells are unusual among eukaryotes in that theirplanes of division are established prior to the onset of mitosis. Factors that contribute to the initialselection of the division plane include extra-cellular signals, cell geometry and polarity, and nuclearposition. During the G2 phase of the cell cycle, the formation of the preprophase band (PPB), a corticalassembly of microtubules and microfilaments, signals the future location of the division plane. Factorsimportant for PPB formation and maturation include the actin cytoskeleton, changes in microtubule dynamics,and protein dephosphorylation. Prior to its disassembly at prometaphase, the PPB functions in more thanone way to determine subsequent placement of the new cell wall. First, the PPB influences the initial orientationof the spindle, which facilitates subsequent cell wall formation within the division plane. Second, thePPB is thought to direct the formation of a cortical division site that persists after PPB breakdownand interacts during cytokinesis with the expanding phragmoplast, a cytoskeletal assembly that directsthe deposition of the partitioning cell wall. Both negative and positive cortical markers are implicatedin maintaining the memory of the former PPB site throughout mitosis and cytokinesis.

The Arp2/3 complex, a highly conserved nucleator of F-actin polymerization,is essential for a variety of eukaryotic cellular processes, including epidermal cell morphogenesis in Arabidopsis thaliana. Efficient nucleation of actin filaments by the Arp2/3 complex requires the presence of an activator such as a member of the Scar/WAVE family. In mammalian cells, a multiprotein complex consisting of WAVE, PIR121/Sra-1, Nap1, Abi-2 and HSPC300 mediates responsiveness of WAVE to upstream regulators such as Rac. Essential roles in WAVE complex assembly or function have been demonstrated for PIR121/Sra-1, Nap1 and Abi-2, but the significance of HSPC300 in this complex is unclear. Plant homologs of all mammalian WAVE complex components have been identified, including HSPC300, the mammalian homolog of maize BRICK1 (BRK1). We show that, like mutations disrupting the Arabidopsis homologs of PIR121/Sra-1, Nap1 and Scar/WAVE, mutations in the Arabidopsis BRK1gene result in trichome and pavement cell morphology defects (and associated alterations in the F-actin cytoskeleton of expanding cells) similar to those caused by mutations disrupting the ARP2/3 complex itself. Analysis of double mutants provides genetic evidence that BRK1 functions in a pathway with the ARP2/3 complex. BRK1 is required for accumulation of SCAR1 protein in vivo,potentially explaining the apparently essential role of BRK1 in ARP2/3 complex function.

The cytoskeleton plays important roles in plant cell shape determination by influencing the patterns in which cell wall materials are deposited. Cortical microtubules are thought to orient the direction of cell expansion primarily via their influence on the deposition of cellulose into the wall, although the precise nature of the microtubule-cellulose relationship remains unclear. In both tip-growing and diffusely growing cell types, F-actin promotes growth and also contributes to the spatial regulation of growth. F-actin has been proposed to play a variety of roles in the regulation of secretion in expanding cells, but its functions in cell growth control are not well understood. Recent work highlighted in this review on the morphogenesis of selected cell types has yielded substantial new insights into mechanisms governing the dynamics and organization of cytoskeletal filaments in expanding plant cells and how microtubules and F-actin interact to direct patterns of cell growth. Nevertheless, many important questions remain to be answered.