Periodic Reporting for period 1 - SiPoMorph (Genetic control and molecular mechanisms of cell wall modifications during sieve pore morphogenesis in the phloem of the plant vascular system)
Reporting period: 2019-07-01 to 2021-06-30
The phloem transports sugars from leaves to sink tissues (roots, buds, fruits). The conductive cells are sieve elements (SE) and connect to each other through the terminal cell wall (CW), the sieve plate. These plates contain sieve pores, which are large connections between adjacent SEs, forming a continuum for long-distance transport.
Sieve pores derive from plasmodesmata (PD), which connect most cells in plants, yet are much larger, requiring remodeling of the CW. Sieve pores are the major hydraulic bottleneck in phloem transport. Their size is modulated through callose deposition in response to environmental cues. Despite their importance for sugar transport and plant productivity, we still know little about mechanisms underlying their formation.
This MSC Action addressed 3 objectives: 1) Through which intracellular process is callose deposited at sieve pores? 2) How does the lipid class of sphingolipids influence cell-to-cell transport? 3) What are novel, unknown factors in sieve pore formation?
Key findings of this project are:
- Sub-cellular dynamics and localization of the SE-specific callose synthase CALS7.
- In collaboration with host lab members, description of a new mutant in sphingolipid metabolism, increasing PD permeability.
- Identifying a pectate lyase mutant, which sheds light on a previously unexpected CW remodeling process in sieve pore formation.
This project’s objectives and results are of fundamental interest to plant cell biology and physiology. They add to our understanding of phloem function but also plant CW remodeling during cellular differentiation. In a broader context, understanding formation and adaptability of sieve pores has implications to how efficiently crops allocate sugars to sink tissues. This is important considering increasing demands to crop breeding and cultivation in a warming environment, in which agricultural output needs to increase.
Sieve pores derive from plasmodesmata (PD), which connect most cells in plants, yet are much larger, requiring remodeling of the CW. Sieve pores are the major hydraulic bottleneck in phloem transport. Their size is modulated through callose deposition in response to environmental cues. Despite their importance for sugar transport and plant productivity, we still know little about mechanisms underlying their formation.
This MSC Action addressed 3 objectives: 1) Through which intracellular process is callose deposited at sieve pores? 2) How does the lipid class of sphingolipids influence cell-to-cell transport? 3) What are novel, unknown factors in sieve pore formation?
Key findings of this project are:
- Sub-cellular dynamics and localization of the SE-specific callose synthase CALS7.
- In collaboration with host lab members, description of a new mutant in sphingolipid metabolism, increasing PD permeability.
- Identifying a pectate lyase mutant, which sheds light on a previously unexpected CW remodeling process in sieve pore formation.
This project’s objectives and results are of fundamental interest to plant cell biology and physiology. They add to our understanding of phloem function but also plant CW remodeling during cellular differentiation. In a broader context, understanding formation and adaptability of sieve pores has implications to how efficiently crops allocate sugars to sink tissues. This is important considering increasing demands to crop breeding and cultivation in a warming environment, in which agricultural output needs to increase.
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.
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.
This MSCA extended our knowledge of sieve pore formation and molecular genetic factors. Notably, this project achieved:
- GFP- CALS7 marker as the first live marker for sieve pores. This attracted interest from the community and material has been shared to allow live- and time-lapse imaging, which was previously impossible.
- A cell-biological characterization of a callose synthase in its endogenous context. CALS proteins are difficult to tag, but it can be expected that this project’s work is referenced for further studies of CALS proteins.
- Advancing the genetic understanding of sphingolipid metabolism in PD function. This might be a promising tool for fine-tuning sugar transport to sink tissues.
- Pectin metabolism as a novel and unexpected factor during SE differentiation and sieve pore formation.
- A developmental and physiological role for a pectate lyase advances the field of pectin research and, potentially, of CW enzyme research in general. CW enzymes belong to big families and functional genetics are challenging due to redundancies. Identification of a SE-specific pectin mutant may provide a platform for functional genetics on pectin metabolism; material has already been shared with collaborators.
- A genetic separation of long-distance from short-distance PD transport. Most earlier described transport mutants are generic PD mutants limiting their use for phloem research.
This MSC Action produced results with potential interest to agronomy:
- A role for sphingolipid metabolism in controlling sugar allocation to sink tissues.
- Means to visualize sieve pores in living plants may enable live-imaging of phloem responses to pathogens to better understand impacts on fruit production.
- Pectin metabolism in SE function may provide novel insights into how the CW affects resource allocation.
- GFP- CALS7 marker as the first live marker for sieve pores. This attracted interest from the community and material has been shared to allow live- and time-lapse imaging, which was previously impossible.
- A cell-biological characterization of a callose synthase in its endogenous context. CALS proteins are difficult to tag, but it can be expected that this project’s work is referenced for further studies of CALS proteins.
- Advancing the genetic understanding of sphingolipid metabolism in PD function. This might be a promising tool for fine-tuning sugar transport to sink tissues.
- Pectin metabolism as a novel and unexpected factor during SE differentiation and sieve pore formation.
- A developmental and physiological role for a pectate lyase advances the field of pectin research and, potentially, of CW enzyme research in general. CW enzymes belong to big families and functional genetics are challenging due to redundancies. Identification of a SE-specific pectin mutant may provide a platform for functional genetics on pectin metabolism; material has already been shared with collaborators.
- A genetic separation of long-distance from short-distance PD transport. Most earlier described transport mutants are generic PD mutants limiting their use for phloem research.
This MSC Action produced results with potential interest to agronomy:
- A role for sphingolipid metabolism in controlling sugar allocation to sink tissues.
- Means to visualize sieve pores in living plants may enable live-imaging of phloem responses to pathogens to better understand impacts on fruit production.
- Pectin metabolism in SE function may provide novel insights into how the CW affects resource allocation.