BASL controls asymmetric cell division in Arabidopsis.

Development in multicellular organisms requires the organized generation of differences. A universal mechanism for creating such differences is asymmetric cell division. In plants, as in animals, asymmetric divisions are correlated with the production of cellular diversity and pattern; however, structural constraints imposed by plant cell walls and the absence of homologs of known animal or fungal cell polarity regulators necessitates that plants utilize new molecules and mechanisms to create asymmetries. Here, we identify BASL, a novel regulator of asymmetric divisions in Arabidopsis. In asymmetrically dividing stomatal-lineage cells, BASL accumulates in a polarized crescent at the cell periphery before division, and then localizes differentially to the nucleus and a peripheral crescent in self-renewing cells and their sisters after division. BASL presence at the cell periphery is critical for its function, and we propose that BASL represents a plant-specific solution to the challenge of asymmetric cell division.

SEC1A is a major Arabidopsis Sec1/Munc18 gene in vesicle trafficking during pollen tube tip growth.

Pollen tubes (PTs) grow by the targeted secretion of new cell wall material to their expanding tip region. Sec1/Munc18 (SM) proteins promote membrane fusion through regulation of the SNARE complex. We have previously shown that disruption of protein glycosylation in the Arabidopsis thaliana hpat1 hpat3 double mutant leads to PT growth defects that can be suppressed by reducing secretion. Here, we identified five point mutant alleles of the SM protein SEC1A as hpat1/3 suppressors. The suppressors increased seed set, reduced PT growth defects and reduced the rate of glycoprotein secretion. In the absence of the hpat mutations, sec1a reduced pollen germination and PT elongation producing shorter and wider PTs. Consistent with a defect in membrane fusion, sec1a PTs accumulated secretory vesicles. Though sec1a had significantly reduced male transmission, homozygous sec1a plants maintained full seed set, demonstrating that SEC1A was ultimately dispensable for pollen fertility. However, when combined with a mutation in another SEC1-like SM gene, keule, pollen fertility was totally abolished. Mutation in sec1b, the final member of the Arabidopsis SEC1 clade, did not enhance the sec1a phenotype. Thus, SEC1A is the major SM protein promoting pollen germination and tube elongation, but in its absence KEULE can partially supply this activity. When we examined the expression of the SM protein family in other species for which pollen expression data were available, we found that at least one Sec1-like protein was highly expressed in pollen samples, suggesting a conserved role in pollen fertility in other species.

O-glycosylation of the extracellular domain of pollen class I formins modulates their plasma membrane mobility.

In plant cells, linkage between the cytoskeleton, plasma membrane, and cell wall is crucial for maintaining cell shape. In highly polarized pollen tubes, this coordination is especially important to allow rapid tip growth and successful fertilization. Class I formins contain cytoplasmic actin-nucleating formin homology domains as well as a proline-rich extracellular domain and are candidate coordination factors. Here, using Arabidopsis, we investigated the functional significance of the extracellular domain of two pollen-expressed class I formins: AtFH3, which does not have a polar localization, and AtFH5, which is limited to the growing tip region. We show that the extracellular domain of both is necessary for their function, and identify distinct O-glycans attached to these sequences, AtFH5 being hydroxyproline-arabinosylated and AtFH3 carrying arabinogalactan chains. Loss of hydroxyproline arabinosylation altered the plasma membrane localization of AtFH5 and disrupted actin cytoskeleton organization. Moreover, we show that O-glycans differentially affect lateral mobility in the plasma membrane. Together, our results support a model of protein sub-functionalization in which AtFH5 and AtFH3, restricted to specific plasma membrane domains by their extracellular domains and the glycans attached to them, organize distinct subarrays of actin during pollen tube elongation.

Exocyst mutants suppress pollen tube growth and cell wall structural defects of hydroxyproline O-arabinosyltransferase mutants.

HYDROXYPROLINE O-ARABINOSYLTRANSFERASEs (HPATs) initiate a post-translational protein modification (Hyp-Ara) found abundantly on cell wall structural proteins. In Arabidopsis thaliana, HPAT1 and HPAT3 are redundantly required for full pollen fertility. In addition to the lack of Hyp-Ara in hpat1/3 pollen tubes (PTs), we also found broadly disrupted cell wall polymer distributions, particularly the conversion of the tip cell wall to a more shaft-like state. Mutant PTs were slow growing and prone to rupture and morphological irregularities. In a forward mutagenesis screen for suppressors of the hpat1/3 low seed-set phenotype, we identified a missense mutation in exo70a2, a predicted member of the vesicle-tethering exocyst complex. The suppressed pollen had increased fertility, fewer morphological defects and partially rescued cell wall organization. A transcriptional null allele of exo70a2 also suppressed the hpat1/3 fertility phenotype, as did mutants of core exocyst complex member sec15a, indicating that reduced exocyst function bypassed the PT requirement for Hyp-Ara. In a wild-type background, exo70a2 reduced male transmission efficiency, lowered pollen germination frequency and slowed PT elongation. EXO70A2 also localized to the PT tip plasma membrane, consistent with a role in exocyst-mediated secretion. To monitor the trafficking of Hyp-Ara modified proteins, we generated an HPAT-targeted fluorescent secretion reporter. Reporter secretion was partially dependent on EXO70A2 and was significantly increased in hpat1/3 PTs compared with the wild type, but was reduced in the suppressed exo70a2 hpat1/3 tubes.

Hydroxyproline O-arabinosyltransferase mutants oppositely alter tip growth in Arabidopsis thaliana and Physcomitrella patens.

Hydroxyproline O-arabinosyltransferases (HPATs) are members of a small, deeply conserved family of plant-specific glycosyltransferases that add arabinose sugars to diverse proteins including cell wall-associated extensins and small signaling peptides. Recent genetic studies in flowering plants suggest that different HPAT homologs have been co-opted to function in diverse species-specific developmental contexts. However, nothing is known about the roles of HPATs in basal plants. We show that complete loss of HPAT function in Arabidopsis thaliana and the moss Physcomitrella patens results in a shared defect in gametophytic tip cell growth. Arabidopsis hpat1/2/3 triple knockout mutants suffer from a strong male sterility defect as a consequence of pollen tubes that fail to fully elongate following pollination. Knocking out the two HPAT genes of Physcomitrella results in larger multicellular filamentous networks due to increased elongation of protonemal tip cells. Physcomitrella hpat mutants lack cell-wall associated hydroxyproline arabinosides and can be rescued with exogenous cellulose, while global expression profiling shows that cell wall-associated genes are severely misexpressed, implicating a defect in cell wall formation during tip growth. Our findings point to a major role for HPATs in influencing cell elongation during tip growth in plants.

Transcription factor control of asymmetric cell divisions that establish the stomatal lineage.

The establishment of new cell lineages during development often requires a symmetry-breaking event. An asymmetric division in the epidermis of plants initiates a lineage that ultimately produces stomatal guard cells. Stomata are pores in the epidermis that serve as the main conduits for gas exchange between plants and the atmosphere; they are critical for photosynthesis and exert a major influence on global carbon and water cycles. Recent studies implicated intercellular signalling in preventing the inappropriate production of stomatal complexes. Genes required to make stomata, however, remained elusive. Here we report the identification of a gene, SPEECHLESS (SPCH), encoding a basic helix-loop-helix (bHLH) transcription factor that is necessary and sufficient for the asymmetric divisions that establish the stomatal lineage in Arabidopsis thaliana. We demonstrate that SPCH and two paralogues are successively required for the initiation, proliferation and terminal differentiation of cells in the stomatal lineage. The stomatal bHLHs define a molecular pathway sufficient to create one of the key cell types in plants. Similar molecules and regulatory mechanisms are used during muscle and neural development, highlighting a conserved use of closely related bHLHs for cell fate specification and differentiation.

The hydroxyproline O-arabinosyltransferase FIN4 is required for tomato pollen intine development.

The pollen grain cell wall is a highly specialized structure composed of distinct layers formed through complex developmental pathways. The production of the innermost intine layer, composed of cellulose, pectin and other polymers, is particularly poorly understood. Here we demonstrate an important and specific role for the hydroxyproline O-arabinosyltransferase (HPAT) FIN4 in tomato intine development. HPATs are plant-specific enzymes which initiate glycosylation of certain cell wall structural proteins and signaling peptides. FIN4 was expressed throughout pollen development in both the developing pollen and surrounding tapetal cells. A fin4 mutant with a partial deletion of the catalytic domain displayed significantly reduced male fertility in vivo and compromised pollen hydration and germination in vitro. However, fin4 pollen that successfully germinated formed morphologically normal pollen tubes with the same growth rate as the wild-type pollen. When we examined mature fin4 pollen, we found they were cytologically normal, and formed morphologically normal exine, but produced significantly thinner intine. During intine deposition at the late stages of pollen development we found fin4 pollen had altered polymer deposition, including reduced cellulose and increased detection of pectin, specifically homogalacturonan with both low and high degrees of methylesterification. Therefore, FIN4 plays an important role in intine formation and, in turn pollen hydration and germination and the process of intine formation involves dynamic changes in the developing pollen cell wall.

Sequence and function of basic helix-loop-helix proteins required for stomatal development in Arabidopsis are deeply conserved in land plants.

Stomata are a broadly conserved feature of land plants with a crucial role regulating transpiration and gas exchange between the plant and atmosphere. Stereotyped cell divisions within a specialized cell lineage of the epidermis generate stomata and define the pattern of their distribution. The behavior of the stomatal lineage varies in its detail among different plant groups, but general features include asymmetric cell divisions and an immediate precursor (the guard mother cell [GMC]) that divides symmetrically to form the pair of cells that will differentiate into the guard cells. In Arabidopsis, the closely related basic helix-loop-helix (bHLH) subgroup Ia transcription factors SPEECHLESS, MUTE, and FAMA promote asymmetric divisions, the acquisition of GMC identity and guard cell differentiation, respectively. Genome sequence data indicate that these key positive regulators of stomatal development are broadly conserved among land plants. While orthologies can be established among individual family members within the angiosperms, more distantly related groups contain subgroup Ia bHLHs of unclear affinity. We demonstrate group Ia members from the moss Physcomitrella patens can partially complement MUTE and FAMA and recapitulate gain of function phenotypes of group Ia genes in multiple steps in the stomatal lineage in Arabidopsis. Our data are consistent with a mechanism whereby a multifunctional transcription factor underwent duplication followed by specialization to provide the three (now nonoverlapping) functions of the angiosperm stomatal bHLHs.

Sequential Deposition and Remodeling of Cell Wall Polymers During Tomato Pollen Development.

The cell wall of a mature pollen grain is a highly specialized, multilayered structure. The outer, sporopollenin-based exine provides protection and support to the pollen grain, while the inner intine, composed primarily of cellulose, is important for pollen germination. The formation of the mature pollen grain wall takes place within the anther with contributions of cell wall material from both the developing pollen grain as well as the surrounding cells of the tapetum. The process of wall development is complex; multiple cell wall polymers are deposited, some transiently, in a controlled sequence of events. Tomato (Solanum lycopersicum) is an important agricultural crop, which requires successful fertilization for fruit production as do many other members of the Solanaceae family. Despite the importance of pollen development for tomato, little is known about the detailed pollen gain wall developmental process. Here, we describe the structure of the tomato pollen wall and establish a developmental timeline of its formation. Mature tomato pollen is released from the anther in a dehydrated state and is tricolpate, with three long apertures without overlaying exine from which the pollen tube may emerge. Using histology and immunostaining, we determined the order in which key cell wall polymers were deposited with respect to overall pollen and anther development. Pollen development began in young flower buds when the premeiotic microspore mother cells (MMCs) began losing their cellulose primary cell wall. Following meiosis, the still conjoined microspores progressed to the tetrad stage characterized by a temporary, thick callose wall. Breakdown of the callose wall released the individual early microspores. Exine deposition began with the secretion of the sporopollenin foot layer. At the late microspore stage, exine deposition was completed and the tapetum degenerated. The pollen underwent mitosis to produce bicellular pollen; at which point, intine formation began, continuing through to pollen maturation. The entire cell wall development process was also punctuated by dynamic changes in pectin composition, particularly changes in methyl-esterified and de-methyl-esterified homogalacturonan.

Arabinogalactan glycoprotein dynamics during the progamic phase in the tomato pistil.

KEY MESSAGE: Pistil AGPs display dynamic localization patterns in response to fertilization in tomato. SlyFLA9 (Solyc07g065540.1) is a chimeric Fasciclin-like AGP with enriched expression in the ovary, suggesting a potential function during pollen-pistil interaction. During fertilization, the male gametes are delivered by pollen tubes to receptive ovules, deeply embedded in the sporophytic tissues of the pistil. Arabinogalactan glycoproteins (AGPs) are a diverse family of highly glycosylated, secreted proteins which have been widely implicated in plant reproduction, particularly within the pistil. Though tomato (Solanum lycopersicum) is an important crop requiring successful fertilization for production, the molecular basis of this event remains understudied. Here we explore the spatiotemporal localization of AGPs in the mature tomato pistil before and after fertilization. Using histological techniques to detect AGP sugar moieties, we found that accumulation of AGPs correlated with the maturation of the stigma and we identified an AGP subpopulation restricted to the micropyle that was no longer visible upon fertilization. To identify candidate pistil AGP genes, we used an RNA-sequencing approach to catalog gene expression in functionally distinct subsections of the mature tomato pistil (the stigma, apical and basal style and ovary) as well as pollen and pollen tubes. Of 161 predicted AGP and AGP-like proteins encoded in the tomato genome, we identified four genes with specifically enriched expression in reproductive tissues. We further validated expression of two of these, a Fasciclin-like AGP (SlyFLA9, Solyc07g065540.1) and a novel hybrid AGP (SlyHAE, Solyc09g075580.1). Using in situ hybridization, we also found SlyFLA9 was expressed in the integuments of the ovule and the pericarp. Additionally, differential expression analyses of the pistil transcriptome revealed previously unreported genes with enriched expression in each subsection of the mature pistil, setting the foundation for future functional studies.

Plant Protein O-Arabinosylation.

A wide range of proteins with diverse functions in development, defense, and stress responses are O-arabinosylated at hydroxyprolines (Hyps) within distinct amino acid motifs of continuous stretches of Hyps, as found in the structural cell wall extensins, or at non-continuous Hyps as, for example, found in small peptide hormones and a variety of plasma membrane proteins involved in signaling. Plant O-glycosylation relies on hydroxylation of Prolines to Hyps in the protein backbone, mediated by prolyl-4-hydroxylase (P4H) which is followed by O-glycosylation of the Hyp C4-OH group by either galactosyltransferases (GalTs) or arabinofuranosyltranferases (ArafTs) yielding either Hyp-galactosylation or Hyp-arabinosylation. A subset of the P4H enzymes with putative preference to hydroxylation of continuous prolines and presumably all ArafT enzymes needed for synthesis of the substituted arabinose chains of one to four arabinose units, have been identified and functionally characterized. Truncated root-hair phenotype is one common denominator of mutants of Hyp formation and Hyp-arabinosylation glycogenes, which act on diverse groups of O-glycosylated proteins, e.g., the small peptide hormones and cell wall extensins. Dissection of different substrate derived effects may not be regularly feasible and thus complicate translation from genotype to phenotype. Recently, lack of proper arabinosylation on arabinosylated proteins has been shown to influence their transport/fate in the secretory pathway, hinting to an additional layer of functionality of O-arabinosylation. Here, we provide an update on the prevalence and types of O-arabinosylated proteins and the enzymatic machinery responsible for their modifications.

Isolation and Cloning of Suppressor Mutants with Improved Pollen Fertility.

Mutant screens remain among the most powerful unbiased methods for identifying key genes in a pathway or process of interest. However, mutants impacting pollen function pose special challenges due to their genetic behavior. Here we describe an approach for isolating pollen mutants based on screening for suppressors of a low pollen fertility starting genotype. By identifying suppressor mutants with improved pollen fertility, we are able to identify new genes which are functionally relevant to pathway(s) causing low seed set in the original background. With this method, the low fertility of the genetic background may be due to one or more mutations or transgenes disrupting any aspect of pollen development or function. Furthermore, screening for improved pollen fertility biases toward recovery of the desired mutants due to their enhanced male transmission. The causative mutation is cloned using next-generation sequencing. The procedure uses both genetic and bioinformatics approaches to ultimately yield a very small list of candidate causative mutations speeding the transition from mutant phenotype to underlying gene.

Partial purification and immunodetection of cell surface glycoproteins from plants.

Cell surface glycoproteins in plants were first described more than 50 years ago, and yet, the precise mechanisms by which they operate remain elusive to this day. Studying glycoproteins is often challenging due to their subcellular localization (many secreted or membrane associated) and the extent of glycosylation present on the protein backbone, which can have profound effects on protein structure and behavior. In plants, additional layers of complexity exist as cell surface glycoproteins are in close contact, and in some cases, establish direct linkages with the polysaccharide networks present in the cell wall. In this chapter, we guide the reader through a protocol aimed to address the glycosylation status of a presumed cell surface glycoprotein. First, we discuss the advantages and disadvantages of using plants as homologous expression systems for recombinant glycoprotein production. Next, we describe a protocol for microsomal enrichment, followed by partial purification by affinity chromatography and finally glycodetection by immunoblotting using monoclonal antibodies targeting cell wall glycans. We particularly focus on the hydroxyproline-rich glycoprotein (HRGP) family, the most abundant family of glycoproteins in the plant cell wall. We provide examples of two putative HRGP chimeric proteins, one akin to extensins and the second an arabinogalactan protein (AGP)-like protein. For the latter, we provide an AGP-specific protocol to ensure enrichment of members of this group, which can be used independently or in conjunction with the described protocol. Throughout the chapter, we provide recommendations for the handling of plant glycoproteins and highlight special considerations for experimental design, along with troubleshooting suggestions.

Origin and function of stomata in the moss Physcomitrella patens.

Stomata are microscopic valves on plant surfaces that originated over 400 million years (Myr) ago and facilitated the greening of Earth's continents by permitting efficient shoot-atmosphere gas exchange and plant hydration1. However, the core genetic machinery regulating stomatal development in non-vascular land plants is poorly understood2-4 and their function has remained a matter of debate for a century5. Here, we show that genes encoding the two basic helix-loop-helix proteins PpSMF1 (SPEECH, MUTE and FAMA-like) and PpSCREAM1 (SCRM1) in the moss Physcomitrella patens are orthologous to transcriptional regulators of stomatal development in the flowering plant Arabidopsis thaliana and essential for stomata formation in moss. Targeted P. patens knockout mutants lacking either PpSMF1 or PpSCRM1 develop gametophytes indistinguishable from wild-type plants but mutant sporophytes lack stomata. Protein-protein interaction assays reveal heterodimerization between PpSMF1 and PpSCRM1, which, together with moss-angiosperm gene complementations6, suggests deep functional conservation of the heterodimeric SMF1 and SCRM1 unit is required to activate transcription for moss stomatal development, as in A. thaliana7. Moreover, stomata-less sporophytes of ΔPpSMF1 and ΔPpSCRM1 mutants exhibited delayed dehiscence, implying stomata might have promoted dehiscence in the first complex land-plant sporophytes.

A cascade of arabinosyltransferases controls shoot meristem size in tomato.

Shoot meristems of plants are composed of stem cells that are continuously replenished through a classical feedback circuit involving the homeobox WUSCHEL (WUS) gene and the CLAVATA (CLV) gene signaling pathway. In CLV signaling, the CLV1 receptor complex is bound by CLV3, a secreted peptide modified with sugars. However, the pathway responsible for modifying CLV3 and its relevance for CLV signaling are unknown. Here we show that tomato inflorescence branching mutants with extra flower and fruit organs due to enlarged meristems are defective in arabinosyltransferase genes. The most extreme mutant is disrupted in a hydroxyproline O-arabinosyltransferase and can be rescued with arabinosylated CLV3. Weaker mutants are defective in arabinosyltransferases that extend arabinose chains, indicating that CLV3 must be fully arabinosylated to maintain meristem size. Finally, we show that a mutation in CLV3 increased fruit size during domestication. Our findings uncover a new layer of complexity in the control of plant stem cell proliferation.

Synchronization of the flowering transition by the tomato TERMINATING FLOWER gene.

The transition to flowering is a major determinant of plant architecture, and variation in the timing of flowering can have profound effects on inflorescence architecture, flower production and yield. Here, we show that the tomato mutant terminating flower (tmf) flowers early and converts the multiflowered inflorescence into a solitary flower as a result of precocious activation of a conserved floral specification complex encoded by ANANTHA (AN) and FALSIFLORA (FA). Without TMF, the coordinated flowering process is disrupted, causing floral identity genes, such as AN and members of the SEPALLATA (SEP) family, to activate precociously, while the expression of flowering transition genes, such as FRUITFULL (FUL), is delayed. Indeed, driving AN expression precociously is sufficient to cause early flowering, and this expression transforms multiflowered inflorescences into normal solitary flowers resembling those of the Solanaceae species petunia and tobacco. Thus, by timing AN activation, TMF synchronizes flower formation with the gradual reproductive transition, which, in turn, has a key role in determining simple versus complex inflorescences.

Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS.

Stomata, epidermal structures that modulate gas exchange between plants and the atmosphere, play critical roles in primary productivity and the global climate. Positively acting transcription factors and negatively acting mitogen-activated protein kinase (MAPK) signaling control stomatal development in Arabidopsis; however, it is not known how the opposing activities of these regulators are integrated. We found that a unique domain in a basic helix-loop-helix (bHLH) stomatal initiating factor, SPEECHLESS, renders it a MAPK phosphorylation target in vitro and modulates its function in vivo. MAPK cascades modulate a diverse set of activities including development, cell proliferation, and response to external stresses. The coupling of MAPK signaling to SPEECHLESS activity provides cell type specificity for MAPK output while allowing the integration of multiple developmental and environmental signals into the production and spacing of stomata.