Stomatal closure in maize is mediated by subsidiary cells and the PAN2 receptor.

Stomata are epidermal pores that facilitate plant gas exchange. Grasses have fast stomatal movements, likely due to their dumbbell-shaped guard cells and lateral subsidiary cells. Subsidiary cells reciprocally exchange water and ions with guard cells. However, the relative contribution of subsidiary cells during stomatal closure is unresolved. We compared stomatal gas exchange and stomatal aperture dynamics in wild-type and pan1, pan2, and pan1;pan2 Zea mays (L.) (maize) mutants, which have varying percentages of aberrantly formed subsidiary cells. Stomata with 1 or 2 defective subsidiary cells cannot close properly, indicating that subsidiary cells are essential for stomatal function. Even though the percentage of aberrant stomata is similar in pan1 and pan2, pan2 showed a more severe defect in stomatal closure. In pan1, only stomata with abnormal subsidiary cells fail to close normally. In pan2, all stomata have stomatal closure defects, indicating that PAN2 has an additional role in stomatal closure. Maize Pan2 is orthologous to Arabidopsis GUARD CELL HYDROGEN PEROXIDE-RESISANT1 (GHR1), which is also required for stomatal closure. PAN2 acts downstream of Ca2+ in maize to promote stomatal closure. This is in contrast to GHR1, which acts upstream of Ca2+ , and suggests the pathways could be differently wired.

A polarized nuclear position specifies the correct division plane during maize stomatal development.

Asymmetric cell division generates different cell types and is a feature of development in multicellular organisms. Prior to asymmetric cell division, cell polarity is established. Maize (Zea mays) stomatal development serves as an excellent plant model system for asymmetric cell division, especially the asymmetric division of the subsidiary mother cell (SMC). In SMCs, the nucleus migrates to a polar location after the accumulation of polarly localized proteins but before the appearance of the preprophase band. We examined a mutant of an outer nuclear membrane protein that is part of the LINC (linker of nucleoskeleton and cytoskeleton) complex that localizes to the nuclear envelope in interphase cells. Previously, maize linc kash sine-like2 (mlks2) was observed to have abnormal stomata. We confirmed and identified the precise defects that lead to abnormal asymmetric divisions. Proteins that are polarly localized in SMCs prior to division polarized normally in mlks2. However, polar localization of the nucleus was sometimes impaired, even in cells that have otherwise normal polarity. This led to a misplaced preprophase band and atypical division planes. MLKS2 localized to mitotic structures; however, the structure of the preprophase band, spindle, and phragmoplast appeared normal in mlks2. Time-lapse imaging revealed that mlks2 has defects in premitotic nuclear migration toward the polarized site and unstable position at the division site after formation of the preprophase band. Overall, our results show that nuclear envelope proteins promote premitotic nuclear migration and stable nuclear position and that the position of the nucleus influences division plane establishment in asymmetrically dividing cells.

The OPAQUE1/DISCORDIA2 myosin XI is required for phragmoplast guidance during asymmetric cell division in maize.

Formative asymmetric divisions produce cells with different fates and are critical for development. We show the maize (Zea mays) myosin XI protein, OPAQUE1 (O1), is necessary for asymmetric divisions during maize stomatal development. We analyzed stomatal precursor cells before and during asymmetric division to determine why o1 mutants have abnormal division planes. Cell polarization and nuclear positioning occur normally in the o1 mutant, and the future site of division is correctly specified. The defect in o1 becomes apparent during late cytokinesis, when the phragmoplast forms the nascent cell plate. Initial phragmoplast guidance in o1 is normal; however, as phragmoplast expansion continues o1 phragmoplasts become misguided. To understand how O1 contributes to phragmoplast guidance, we identified O1-interacting proteins. Maize kinesins related to the Arabidopsis thaliana division site markers PHRAGMOPLAST ORIENTING KINESINs (POKs), which are also required for correct phragmoplast guidance, physically interact with O1. We propose that different myosins are important at multiple steps of phragmoplast expansion, and the O1 actin motor and POK-like microtubule motors work together to ensure correct late-stage phragmoplast guidance.

Polarly localized WPR proteins interact with PAN receptors and the actin cytoskeleton during maize stomatal development.

Polarization of cells prior to asymmetric cell division is crucial for correct cell divisions, cell fate, and tissue patterning. In maize (Zea mays) stomatal development, the polarization of subsidiary mother cells (SMCs) prior to asymmetric division is controlled by the BRICK (BRK)-PANGLOSS (PAN)-RHO FAMILY GTPASE (ROP) pathway. Two catalytically inactive receptor-like kinases, PAN2 and PAN1, are required for correct division plane positioning. Proteins in the BRK-PAN-ROP pathway are polarized in SMCs, with the polarization of each protein dependent on the previous one. As most of the known proteins in this pathway do not physically interact, possible interactors that might participate in the pathway are yet to be described. We identified WEAK CHLOROPLAST MOVEMENT UNDER BLUE LIGHT 1 (WEB1)/PLASTID MOVEMENT IMPAIRED 2 (PMI2)-RELATED (WPR) proteins as players during SMC polarization in maize. WPRs physically interact with PAN receptors and polarly accumulate in SMCs. The polarized localization of WPR proteins depends on PAN2 but not PAN1. CRISPR-Cas9-induced mutations result in division plane defects in SMCs, and ectopic expression of WPR-RFP results in stomatal defects and alterations to the actin cytoskeleton. We show that certain WPR proteins directly interact with F-actin through their N-terminus. Our data implicate WPR proteins as potentially regulating actin filaments, providing insight into their molecular function. These results demonstrate that WPR proteins are important for cell polarization.

Plant Biology: BASL Gives the Plant Nucleus a Sense of Direction.

Arabidopsis stomatal development requires asymmetric cell division, where the nucleus moves to the division site based on cellular polarity cues. A new study reveals the role of distinct cytoskeletal networks, both guided by the polarity factor BASL, for nuclear movement before and after division.

Flanking Support: How Subsidiary Cells Contribute to Stomatal Form and Function.

Few evolutionary adaptations in plants were so critical as the stomatal complex. This structure allows transpiration and efficient gas exchange with the atmosphere. Plants have evolved numerous distinct stomatal architectures to facilitate gas exchange, while balancing water loss and protection from pathogens that can egress via the stomatal pore. Some plants have simple stomata composed of two kidney-shaped guard cells; however, the stomatal apparatus of many plants includes subsidiary cells. Guard cells and subsidiary cells may originate from a single cell lineage, or subsidiary cells may be recruited from cells adjacent to the guard mother cell. The number and morphology of subsidiary cells varies dramatically, and subsidiary cell function is also varied. Subsidiary cells may support guard cell function by offering a mechanical advantage that facilitates guard cell movements, and/or by acting as a reservoir for water and ions. In other cases, subsidiary cells introduce or enhance certain morphologies (such as sunken stomata) that affect gas exchange. Here we review the diversity of stomatal morphology with an emphasis on multi-cellular stomata that include subsidiary cells. We will discuss how subsidiary cells arise and the divisions that produce them; and provide examples of anatomical, mechanical and biochemical consequences of subsidiary cells on stomatal function.

The SCAR/WAVE complex polarizes PAN receptors and promotes division asymmetry in maize.

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.

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.

Analysis of the enzymatic properties of a broad family of alanine aminotransferases.

Alanine aminotransferase (AlaAT) has been studied in a variety of organisms due to the involvement of this enzyme in mammalian processes such as non-alcoholic hepatocellular damage, and in plant processes such as C4 photosynthesis, post-hypoxic stress response and nitrogen use efficiency. To date, very few studies have made direct comparisons of AlaAT enzymes and fewer still have made direct comparisons of this enzyme across a broad spectrum of organisms. In this study we present a direct kinetic comparison of glutamate:pyruvate aminotransferase (GPAT) activity for seven AlaATs and two glutamate:glyoxylate aminotransferases (GGAT), measuring the K(M) values for the enzymes analyzed. We also demonstrate that recombinant expression of AlaAT enzymes in Eschericia coli results in differences in bacterial growth inhibition, supporting previous reports of AlaAT possessing bactericidal properties, attributed to lipopolysaccharide endotoxin recognition and binding. A probable lipopolysaccharide binding region within the AlaAT enzymes, homologous to a region of a lipopolysaccharide binding protein (LBP) in humans, was also identified in this study. The AlaAT enzyme differences identified here indicate that AlaAT homologues have differentiated significantly and the roles these homologues play in vivo may also have diverged significantly. Specifically, the differing kinetics of AlaAT enzymes and how this may alter the nitrogen use efficiency in plants is discussed.

Identification of PAN2 by quantitative proteomics as a leucine-rich repeat-receptor-like kinase acting upstream of PAN1 to polarize cell division in maize.

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.

Division polarity in developing stomata.

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.

Plasma membrane-associated SCAR complex subunits promote cortical F-actin accumulation and normal growth characteristics in Arabidopsis roots.

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.

Toward a systems approach to understanding plant cell walls.

One of the defining features of plants is a body plan based on the physical properties of cell walls. Structural analyses of the polysaccharide components, combined with high-resolution imaging, have provided the basis for much of the current understanding of cell walls. The application of genetic methods has begun to provide new insights into how walls are made, how they are controlled, and how they function. However, progress in integrating biophysical, developmental, and genetic information into a useful model will require a system-based approach.