For objective 1, a SE-specific callose synthase, CALS7, was localized using a green fluorescent protein (GFP) fusion. Its mutant cals7 was earlier shown to lack callose in SEs and sieve pores. GFP-CALS7 was used for genetic complementation and to address intracellular dynamics using confocal laser scanning microscopy (CLSM). CALS7 is specific to sieve pores from an intermediate stage on. Initially, CALS7 is secreted ubiquitously but quickly polarizes to the sieve plate and becomes exclusive to pores. This dynamic depends on endocytosis and recycling, which was manipulated using recycling inhibitor brefeldin A (BFA). BFA aggregates recycled plasma membrane (PM) proteins to intracellular BFA bodies. CALS7 is sensitive to BFA treatment in early SEs but resistant in older, where CALS7 is already at sieve pores. This means that, once at sieve pores, CALS7 forms a stable PM domain, similar to previously described scaffolding proteins in the PM of other cell types.
In close collaboration with host lab member Sofia Otero, objective 2 addressed the role of sphingolipid metabolism in PD formation. A suppressor of the previously describe cher1 mutant with constricted PD, a long-chain base kinase mutant, was described. Homologs of this mutant exist, yet higher order mutants established that the cher1 suppressor is unique in its family. Physiological and CLSM-based assays using free GFP suggest that this cher1 suppressor increases phloem unloading, putatively through widening of PD. We investigated whether CALS7 mislocalisation was responsible for the cher1 phenotype or its suppressor’s, which we excluded upon subcellular studies. These findings are relevant to PD in general, since CHER1 and its suppressor are broadly expressed. Sphingolipids are enriched at PD, which may explain their role in PD permeability.
Little was known on the molecular genetic basis of sieve pore formation. Objective 3 aimed at identifying novel genes. Such genes should be phloem-specific and encode PM- or secreted CW-proteins. Candidate genes were identified through mining high resolution transcriptomic data, available in the host lab. Focusing on CW-modifying enzymes and screening available mutants identified a novel phloem-specific pectate lyase.This mutant showed growth and development defects relatively late in development. After around 8 days, root growth stopped, and overall development was affected. We confirmed a SE-specific role through genetic complementation using another known late SE-promoter. We used GFP-CALS7 to investigate sieve pore defects and observed smaller and more heterogeneously shaped sieve pores. A functional GFP fusion of the lyase confirmed that while mostly present in peripheral regions of SEs, it transiently localizes to sieve plates late during SE differentiation. Pectin metabolisms had never been evoked in the context of SE formation. We therefore addressed the biochemical function. Enzymatic domains of pectate lyases are conserved and we identified the reactive center through homology. An inactive lyase did not complement the mutant. Finally, we performed physiological assays using micrografting to follow phloem translocation of a phloem mobile GFP from shoot to root. This showed that the lyase mutant was affected specifically in long-distance phloem transport while short-distance transport through PD was unaffected.
All parts of the project are very advanced and were presented on scientific meetings in summer 2021. Manuscripts are in preparation and submission as open access articles are projected within the next 6-9 months.
