GLUCAN SYNTHASE-LIKE 8: A Key Player in Early Seedling Development in Arabidopsis

Plants’ cell walls have unique chemical composition and features which enable them to play essential roles during plant development as shaping the cells and providing intercellular communication between adjacent cells. Polysaccharides, including callose, and glycoproteins are known as the main constituents of the cell wall. Callose, a linear β-1,3-glucan polymer, is accumulated at the cell plate during cytokinesis, in plasmodesmata, where it regulates cell-to-cell communication, in dormant phloem, where it seals sieve plates after mechanical injury and pathogen attack, and in male and female gametophytes. GLUCAN SYNTHASE-LIKE (GSL) genes in Arabidopsis comprise a family of 12 members. A new allele of GSL8, essp8, was identified as having seedling-lethal phenotype through a genetic screen for Arabidopsis mutants showing ectopic expression of seed storage proteins (essp). The gene responsible for the observed mutant phenotype was detected using a combination of bulked-segregant analysis, rough-mapping, and next-generation mapping. An EMS-induced point mutation was identified at an intron splice site of GSL8, predicted to introduce a premature STOP-codon. essp8 seedlings exhibit pleiotropic phenotypic defects, including disruption of root tissue patterning, dwarfism and seedling lethality. Histochemical detection of callose and cell-to-cell diffusion assays showed reduction of callose deposition at the cell plates and plasmodesmata, cytokinesis defects and significant increase in size exclusion limit of plasmodesmata in essp8 seedlings. Further investigation showed that the increase in size exclusion limit leads to an alteration in symplastic trafficking in primary roots of essp8 seedlings. Plasmodesmata defects in essp8 induce ectopic movement of two non-cell-autonomous factors, SHORT ROOT and microRNA165/6, both required for root radial patterning during embryonic root development. Attempts to identify the components of a hypothetical callose synthase complex revealed the interaction of GSL8 with two plasmodesmata-associated proteins, PLASMODESMATA-LOCALIZED PROTEIN 5 and β-1,3-GLUCANASE, as well as SUCROSE SYNTHASE 1, suggesting that they all might be parts of a single complex. The proposed putative complex might regulate callose deposition at the plasmodesmata and thereby determines the size exclusion limit. In summary, my findings suggest that GSL8 is required for cell wall integrity, maintaining the basic ploidy level and regulation of symplastic movement during early seedling development in Arabidopsis

Analysis of a novel mutant allele of GSL8 reveals its key roles in cytokinesis and symplastic trafficking in Arabidopsis

Abstract Background Plant cell walls are mainly composed of polysaccharides such as cellulose and callose. Callose exists at a very low level in the cell wall; however, it plays critical roles at different stages of plant development as well as in defence against unfavorable conditions. Callose is accumulated at the cell plate, at plasmodesmata and in male and female gametophytes. Despite the important roles of callose in plants, the mechanisms of its synthesis and regulatory properties are not well understood. Results CALLOSE SYNTHASE (CALS) genes, also known as GLUCAN SYNTHASE-LIKE (GSL), comprise a family of 12 members in Arabidopsis thaliana. Here, we describe a new allele of GSL8 (named essp8) that exhibits pleiotropic seedling defects. Reduction of callose deposition at the cell plates and plasmodesmata in essp8 leads to ectopic endomitosis and an increase in the size exclusion limit of plasmodesmata during early seedling development. Movement of two non-cell-autonomous factors, SHORT ROOT and microRNA165/6, both required for root radial patterning during embryonic root development, are dysregulated in the primary root of essp8. This observation provides evidence for a molecular mechanism explaining the gsl8 root phenotype. We demonstrated that GSL8 interacts with PLASMODESMATA-LOCALIZED PROTEIN 5, a β-1,3-glucanase, and GSL10. We propose that they all might be part of a putative callose synthase complex, allowing a concerted regulation of callose deposition at plasmodesmata. Conclusion Analysis of a novel mutant allele of GSL8 reveals that GSL8 is a key player in early seedling development in Arabidopsis. GSL8 is required for maintaining the basic ploidy level and regulating the symplastic trafficking. Callose deposition at plasmodesmata is highly regulated and occurs through interaction of different components, likely to be incorporated into a callose biosynthesis complex. We are providing new evidence supporting an earlier hypothesis that GSL8 might have regulatory roles apart from its enzymatic function in plasmodesmata regulation

Analysis of a novel mutant allele of GSL8 reveals its key roles in cytokinesis and symplastic trafficking in Arabidopsis

Abstract Background Plant cell walls are mainly composed of polysaccharides such as cellulose and callose. Callose exists at a very low level in the cell wall; however, it plays critical roles at different stages of plant development as well as in defence against unfavorable conditions. Callose is accumulated at the cell plate, at plasmodesmata and in male and female gametophytes. Despite the important roles of callose in plants, the mechanisms of its synthesis and regulatory properties are not well understood. Results CALLOSE SYNTHASE (CALS) genes, also known as GLUCAN SYNTHASE-LIKE (GSL), comprise a family of 12 members in Arabidopsis thaliana. Here, we describe a new allele of GSL8 (named essp8) that exhibits pleiotropic seedling defects. Reduction of callose deposition at the cell plates and plasmodesmata in essp8 leads to ectopic endomitosis and an increase in the size exclusion limit of plasmodesmata during early seedling development. Movement of two non-cell-autonomous factors, SHORT ROOT and microRNA165/6, both required for root radial patterning during embryonic root development, are dysregulated in the primary root of essp8. This observation provides evidence for a molecular mechanism explaining the gsl8 root phenotype. We demonstrated that GSL8 interacts with PLASMODESMATA-LOCALIZED PROTEIN 5, a β-1,3-glucanase, and GSL10. We propose that they all might be part of a putative callose synthase complex, allowing a concerted regulation of callose deposition at plasmodesmata. Conclusion Analysis of a novel mutant allele of GSL8 reveals that GSL8 is a key player in early seedling development in Arabidopsis. GSL8 is required for maintaining the basic ploidy level and regulating the symplastic trafficking. Callose deposition at plasmodesmata is highly regulated and occurs through interaction of different components, likely to be incorporated into a callose biosynthesis complex. We are providing new evidence supporting an earlier hypothesis that GSL8 might have regulatory roles apart from its enzymatic function in plasmodesmata regulation

Localization of Arabidopsis Glucan Synthase-Like 5, 8, and 12 to plasmodesmata and the GSL8-dependent role of PDLP5 in regulating plasmodesmal permeability

Cell-to-cell communication via membranous channels called plasmodesmata (PD) plays critical roles during plant development and in response to biotic and abiotic stresses. Several enzymes and receptor-like proteins (RLPs), including Arabidopsis thaliana glucan synthase-likes (GSLs), also known as callose synthases (CALSs), and PD-located proteins (PDLPs), have been implicated in plasmodesmal permeability regulation and intercellular communication. Localization of PDLPs to punctate structures at the cell periphery and their receptor-like identity have raised the hypothesis that PDLPs are involved in the regulation of symplastic trafficking during plant development and in response to endogenous and exogenous signals. Indeed, it was shown that PDLP5 could limit plasmodesmal permeability through inducing an increase in callose accumulation at PD. However, mechanistically, how this is achieved remains to be elucidated. To address this key issue in understanding the regulation of PD, physical and functional interactions between PDLPs and GSLs (using the PDLP5–GSL8/CALS10 pair as a model) were investigated. Our results show that GSL8/CALS10 plays essential roles and is required for the function and plasmodesmal localization of PDLP5. Furthermore, it was demonstrated that the localization of PDLP5 to PD and its function in inducing callose deposition are GSL8-dependent. Importantly, our transgenic study shows that three key members of the GSL family, i.e., GSL5/CALS12, GSL8/CALS10, and GSL12/CALS3, localize to PD and co-localize with PDLP5, suggesting that GSL8/CALS10 might not be the only callose synthase with the determining role in PD regulation. These findings, together with our previous observation showing the direct interaction of GSL8/CALS10 with PDLP5, indicate the pivotal role of the GSL8/CALS10-PDLP5 interplay in regulating PD permeability. Future work is needed to investigate whether the PDLP5 functionality and localization are also disrupted in gsl5 and gsl12, or it is just gsl8-specific