Brown algal cell walls and development.

Brown algae are complex multicellular eukaryotes whose cells possess a cell wall, which is an important structure that regulates cell size and shape. Alginate and fucose-containing sulfated polysaccharides (FCSPs) are two carbohydrate types that have major roles in influencing the mechanical properties of the cell wall (i.e. increasing or decreasing wall stiffness), which in turn regulate cell expansion, division, adhesion, and other processes; however, how brown algal cell wall structure regulates its mechanical properties, and how this relationship influences cellular growth and organismal development, is not well-understood. This chapter is focused on reviewing what we currently know about how the roles of alginates and FCSPs in brown algal developmental processes, as well as how they influence the structural and mechanical properties of cell walls. Additionally, we discuss how brown algal mutants may be leveraged to learn more about the underlying mechanisms that regulate cell wall structure, mechanics, and developmental processes, and finally we propose questions to guide future research with the use of emerging technologies.

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.

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.

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.

Notch activation is required for downregulation of HoxA3-dependent endothelial cell phenotype during blood formation.

Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to cis-inhibition of the Notch pathway, rendering the HE nonresponsive to Notch signals. While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT.