Stomata are epidermal structures that modulate gas exchange between a plant and its environment. During development, stomata are specified and positioned nonrandomly by the integration of asymmetric cell divisions and intercellular signaling. The Arabidopsis mitogen-activated protein kinase kinase kinase gene, YODA, acts as part of a molecular switch controlling cell identities in the epidermis. Null mutations in YODA lead to excess stomata, whereas constitutive activation of YODA eliminated stomata. Transcriptome analysis of seedlings with altered YODA activity was used to identify potential stomatal regulatory genes. A putative transcription factor from this set was shown to regulate the developmental behavior of stomatal precursors.

In higher plant cytokinesis, plasma membrane and cell wall originate by vesicle fusion in the plane of cell division. The Arabidopsis KNOLLE gene, which is required for cytokinesis, encodes a protein related to vesicle-docking syntaxins. We have raised specific rabbit antiserum against purified recombinant KNOLLE protein to show biochemically and by immunoelectron microscopy that KNOLLE protein is membrane associated. Using immunofluorescence microscopy, KNOLLE protein was found to be specifically expressed during mitosis and, unlike the plasma membrane H+-ATPase, to localize to the plane of division during cytokinesis. Arabidopsis dynamin-like protein ADL1 accumulates at the plane of cell plate formation in knolle mutant cells as in wild-type cells, suggesting that cytokinetic vesicle traffic is not affected. Furthermore, electron microscopic analysis indicates that vesicle fusion is impaired. KNOLLE protein was detected in mitotically dividing cells of various parts of the developing plant, including seedling root, inflorescence meristem, floral meristems and ovules, and the cellularizing endosperm, but not during cytokinesis after the male second meiotic division. Thus, KNOLLE is the first syntaxin-like protein that appears to be involved specifically in cytokinetic vesicle fusion.

The Arabidopsis zygote divides asymmetrically into an embryonic apical cell and a basal cell with mostly extra-embryonic fate. This fundamental asymmetry sets the stage for further embryonic development, but the events mediating it are poorly understood. We have identified a MAPKK kinase gene, named YODA, that promotes extra-embryonic cell fates in the basal lineage. In loss-of-function mutants, the zygote does not elongate properly, and the cells of the basal lineage are eventually incorporated into the embryo instead of differentiating the extra-embryonic suspensor. Gain-of-function alleles cause exaggerated growth of the suspensor and can suppress embryonic development to a degree where no recognizable proembryo is formed. Our results imply that a MAP kinase cascade acts as a molecular switch promoting extra-embryonic fate.

Positional (or map-based) cloning techniques are widely used to identify the protein products of genes defined by mutation. In Arabidopsis the information generated by the Genome Initiative is giving this approach a decisive boost. A wealth of sequence polymorphisms and molecular markers is now available and can be exploited for fine mapping with technically simple and robust polymerase chain reaction-based methods. As a result it has become possible to complete positional cloning projects in a short time and with relatively little effort.

The embryo of the flowering plant Arabidopsis develops by a regular pattern of cell divisions and cell shape changes. Mutations in the KNOLLE (KN) gene affect the rate and plane of cell divisions as well as cell morphology, resulting in mutant seedlings with a disturbed radial organization of tissue layers. At the cellular level, mutant embryos are characterized by incomplete cross walls and enlarged cells with polyploid nuclei. The KN gene was isolated by positional cloning. The predicted KN protein has similarity to syntaxins, a protein family involved in vesicular trafficking. During embryogenesis, KN transcripts are detected in patches of single cells or small cell groups. Our results suggest a function for KN in cytokinesis.

The YODA (YDA) mitogen-activated protein kinase pathway promotes elongation of the Arabidopsis zygote and development of its basal daughter cell into the extra-embryonic suspensor. Here, we show that the interleukin-1 receptor-associated kinase (IRAK)/Pelle-like kinase gene SHORT SUSPENSOR (SSP) regulates this pathway through a previously unknown parent-of-origin effect. SSP transcripts are produced in mature pollen but do not appear to be translated. Instead, they are delivered via the sperm cells to the zygote and the endosperm, where SSP protein transiently accumulates. Ectopic expression of SSP protein in the leaf epidermis is sufficient to activate YDA-dependent signaling. We propose that SSP protein produced from paternal transcripts upon fertilization triggers zygotic YDA activity, providing an essential temporal cue for the regulation of the asymmetric first division.

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1, has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.

The cell types of the plant root are first specified early during embryogenesis and are maintained throughout plant life. Auxin plays an essential role in embryonic root initiation, in part through the action of the ARF5/MP transcription factor and its auxin-labile inhibitor IAA12/BDL. MP and BDL function in embryonic cells but promote auxin transport to adjacent extraembryonic suspensor cells, including the quiescent center precursor (hypophysis). Here we show that a cell-autonomous auxin response within this cell is required for root meristem initiation. ARF9 and redundant ARFs, and their inhibitor IAA10, act in suspensor cells to mediate hypophysis specification and, surprisingly, also to prevent transformation to embryo identity. ARF misexpression, and analysis of the short suspensor mutant, demonstrates that lineage-specific expression of these ARFs is required for normal embryo development. These results imply the existence of a prepattern for a cell-type-specific auxin response that underlies the auxin-dependent specification of embryonic cell types.