Final Report Summary - PLANT CELL WALL (Heat Shock Protein 70 in the regulation of plant cell wall function)
Abiotic stress conditions account for significant yield losses in various crop plants, mainly due to stress-induced growth arrest. The plant cell wall is a composite structure, made up of polysaccharides, proteins and phenolic compounds. In plant, the cell wall plays a central role in dictating the extent and direction of growth driven by the turgor-pressure. We therefore investigated the mechanism employed in the modification of cell wall deposition along the course of plant development as well as in response to abiotic stress conditions. Previous studies suggest that the FEI-SOS pathway is required for cell wall deposition in various developmental contexts, like dark-grown seedlings, seed mucilage and elongating roots. Moreover, The FEI-SOS pathway is essential for root growth maintenance under abiotic stress conditions of either high-sucrose or high-salt containing media. FEI1 and FEI2, encode for two receptor like kinases, while SALT-OVERLY SENSITIVE 5 (SOS5) encodes for an extra cellular arabinogalactan protein. Genetic studies suggest that they function as part of a linear pathway and affect cell wall deposition through a yet to be identified mechanism. In order to further elucidate the way the FEI-SOS pathway regulate cell wall deposition, a genetic screen was conducted aimed to identify suppressors of the fei1fei2 short and swollen root phenotype, observed under restrictive conditions of either high-slat or high-sucrose containing media. Three independent alleles mutated in HEAT SHOCK PROTIEN 70-1 (HSP70-1) were identified suggesting HSP70-1 as a new player involved in cell wall deposition. HSP70-1 is an ATP-dependent chaperone involved in protein homeostasis and highly induced under biotic and abiotic stress conditions. In plants HSP70s are encoded by large gene families and functional analysis is restricted by high levels of functional redundancy.
In the current study we investigated the role of the identified hsp70-1 alleles, named shou5-1 to shou5-3, in the regulation of cell wall deposition and plant growth. Number of findings suggest that the hsp70-1shou5 alleles are of gain of function character and hence able to uncover new roles for the HSP70-1 protein, these include: i) the observation that the hsp70-1 null allele, SALK_135531, does not suppress the fei1fei2 phenotypes; ii) the observation that in transgenic fei1fei2 plants expressing either the HSP70-1shou5-1 or the HSP70-1WT form only the shou5 version suppressed the fei1fei2 phenotype; iii) that the HSP70-inhibitor 2-phenylethynesulfonamide, identified as an inhibitor of mammalian HSP70, suppressed both the fei1fei2 and the sos5 root elongation phenotype under restrictive conditions. Altogether, these results pinpoint to the unique character of the hsp70-1shou5 alleles suggesting it might serve as a tool to uncover new roles for HSP70s in the regulation of plant growth and development. Close examination of the recovery in the hsp70-1shou5-induced suppression of fei1fei2 root elongation under restrictive conditions demonstrate: i) inhibition of root meristem shortening under stress conditions; ii) inhibition of ROS accumulation at the root tip; and, iii) inhibition of the ectopic lignin accumulation characteristic to fei1fei2. Interestingly, while hsp70-1shou5 suppressed root elongation in fei1fei2 and sos5 background, it did not suppress the root growth defect of either procuste, mutated in CELLULOSE SYNTHASE A 6, or cobra, mutated in extra cellular protein required for cellulose synthesis. These results, suggest that the hsp70-1shou5 can recover root growth under abiotic stress conditions, of high-salt or high-sucrose containing media, only if the core-machinery employed in cellulose deposition is functional. Similarly it can partially suppress cell wall deposition in elongating hypocotyls of dark grown seedlings as well as in seed mucilage. These results have been recently summarized in manuscript entitled ‘Dominant Negative Mutations in HEAT SHOCK PROTEIN 70-1 Restore Root Growth in FEI-SOS Mutants’, in-preparation.
The role of the FEI-SOS pathway and hsp70-1shou5 on cell wall deposition in other developmental contexts, like: stems, leaves and stomatal guard cells – No phenotype was observed. However, the important role of stomata function in plant adaptation to changing environment prompted us to utilize the set of tools developed in the current study to investigate cell wall composition in stomatal guard cells of arabidopsis and other plant species representing different points in the evolution of land plants. These studies led to the observation that in different plants species different cell wall components provide the mechanical support required for stomatal function (Shtein et al., Annals of Botany 2017; Plant Signaling and Behavior, 2017). Major research effort by our group and colleagues focused on elucidating the role of stomatal cell wall composition in order to set the stage for improved adaptation of plants to changing environment.
Surprisingly, the single hsp70-1shou5 mutant display severe growth retardance, which includes: small rosette-leaves, late flowering and reduced biomass production, as compared to either wild-type or hsp70-1135531. In-depth study of the expression of HSP70 encoding genes, using nCounter NanoString technology, revealed that while in hsp70-1135531, lack of HSP70-1 was compensated by ectopic expression of HSP70-3 and HSP70-4, in hsp70-1shou5-background, in the presence of mal-functioning HSP70-1, no ectopic activation of other HSP70 gene expression is observed. These results suggests a mutual suppression between the hsp70-1shou5 alleles and the fei1fei2 mutants demonstrating the utility of the shou5 alleles as a tool to overcome functional redundancy and uncover new role for the HSP70-mediated chaperone machinery in plant growth and development as well as under abiotic stress conditions. In order to further elucidate the mechanism of HSP70 function, transgenic plants expressing a FLAG-tagged form of either HSP70-1shou5-1 or the HSP70-1WT were generated. Immuno-precipitation followed by either clear native or blue native gel electrophoresis identified HSP70-1 as part of a 660KDa protein complex. Mass-spectrometry will be used to identify the target proteins of HSP70-1 involved in the regulation of cell wall deposition and root elongation under ambient and stress conditions. Two additional approaches were used to further investigate the machinery employed in cell wall deposition in root elongation and other developmental contexts like seed mucilage and pollen tube elongation. To that end we used: i) a next generation genetic screen named PHANTOM and designed to overcome the high levels of functional redundancy in plants; and, ii) a bioinformatic approach using tissue-specific co-expression analysis based on former data suggesting that various components of the machinery required for cell wall deposition are tightly co-expressed. The first approach is still underway and will be completed using recent funding obtained from the Israel Science Foundation (ISF) in 2017 to continue this project. The second approach led to the identification of new role for COBRA-LIKE 2 in cell wall deposition in the context of seed mucilage. Moreover it led to the identification of additional proteins required for root elongation under restrictive conditions. The in-depth study of the role of COBRA-LIKE 2 in cell wall deposition was published in Ben-Tov et al., Plant Physiology 2015; Plant Journal 2018. The plasticity of root development represents a key feature enabling plants to adapt, or alternatively - avoid, abiotic stress conditions like, salinity, draught, nutrient deficiency and others and on the other hand, efficiently forage for resources like nutrients and water. Root growth plasticity determines the rate and extent of plant biomass production and plays a central role in plant adaptation to constantly changing environment. Elucidating the wide arsenal of pathways regulating root morphogenesis under stress conditions will set the stage for the next revolution aimed to increase crop yield and biomass production in marginal soils by breading targeted to the ‘hidden half’ – the root system.
In the current study we investigated the role of the identified hsp70-1 alleles, named shou5-1 to shou5-3, in the regulation of cell wall deposition and plant growth. Number of findings suggest that the hsp70-1shou5 alleles are of gain of function character and hence able to uncover new roles for the HSP70-1 protein, these include: i) the observation that the hsp70-1 null allele, SALK_135531, does not suppress the fei1fei2 phenotypes; ii) the observation that in transgenic fei1fei2 plants expressing either the HSP70-1shou5-1 or the HSP70-1WT form only the shou5 version suppressed the fei1fei2 phenotype; iii) that the HSP70-inhibitor 2-phenylethynesulfonamide, identified as an inhibitor of mammalian HSP70, suppressed both the fei1fei2 and the sos5 root elongation phenotype under restrictive conditions. Altogether, these results pinpoint to the unique character of the hsp70-1shou5 alleles suggesting it might serve as a tool to uncover new roles for HSP70s in the regulation of plant growth and development. Close examination of the recovery in the hsp70-1shou5-induced suppression of fei1fei2 root elongation under restrictive conditions demonstrate: i) inhibition of root meristem shortening under stress conditions; ii) inhibition of ROS accumulation at the root tip; and, iii) inhibition of the ectopic lignin accumulation characteristic to fei1fei2. Interestingly, while hsp70-1shou5 suppressed root elongation in fei1fei2 and sos5 background, it did not suppress the root growth defect of either procuste, mutated in CELLULOSE SYNTHASE A 6, or cobra, mutated in extra cellular protein required for cellulose synthesis. These results, suggest that the hsp70-1shou5 can recover root growth under abiotic stress conditions, of high-salt or high-sucrose containing media, only if the core-machinery employed in cellulose deposition is functional. Similarly it can partially suppress cell wall deposition in elongating hypocotyls of dark grown seedlings as well as in seed mucilage. These results have been recently summarized in manuscript entitled ‘Dominant Negative Mutations in HEAT SHOCK PROTEIN 70-1 Restore Root Growth in FEI-SOS Mutants’, in-preparation.
The role of the FEI-SOS pathway and hsp70-1shou5 on cell wall deposition in other developmental contexts, like: stems, leaves and stomatal guard cells – No phenotype was observed. However, the important role of stomata function in plant adaptation to changing environment prompted us to utilize the set of tools developed in the current study to investigate cell wall composition in stomatal guard cells of arabidopsis and other plant species representing different points in the evolution of land plants. These studies led to the observation that in different plants species different cell wall components provide the mechanical support required for stomatal function (Shtein et al., Annals of Botany 2017; Plant Signaling and Behavior, 2017). Major research effort by our group and colleagues focused on elucidating the role of stomatal cell wall composition in order to set the stage for improved adaptation of plants to changing environment.
Surprisingly, the single hsp70-1shou5 mutant display severe growth retardance, which includes: small rosette-leaves, late flowering and reduced biomass production, as compared to either wild-type or hsp70-1135531. In-depth study of the expression of HSP70 encoding genes, using nCounter NanoString technology, revealed that while in hsp70-1135531, lack of HSP70-1 was compensated by ectopic expression of HSP70-3 and HSP70-4, in hsp70-1shou5-background, in the presence of mal-functioning HSP70-1, no ectopic activation of other HSP70 gene expression is observed. These results suggests a mutual suppression between the hsp70-1shou5 alleles and the fei1fei2 mutants demonstrating the utility of the shou5 alleles as a tool to overcome functional redundancy and uncover new role for the HSP70-mediated chaperone machinery in plant growth and development as well as under abiotic stress conditions. In order to further elucidate the mechanism of HSP70 function, transgenic plants expressing a FLAG-tagged form of either HSP70-1shou5-1 or the HSP70-1WT were generated. Immuno-precipitation followed by either clear native or blue native gel electrophoresis identified HSP70-1 as part of a 660KDa protein complex. Mass-spectrometry will be used to identify the target proteins of HSP70-1 involved in the regulation of cell wall deposition and root elongation under ambient and stress conditions. Two additional approaches were used to further investigate the machinery employed in cell wall deposition in root elongation and other developmental contexts like seed mucilage and pollen tube elongation. To that end we used: i) a next generation genetic screen named PHANTOM and designed to overcome the high levels of functional redundancy in plants; and, ii) a bioinformatic approach using tissue-specific co-expression analysis based on former data suggesting that various components of the machinery required for cell wall deposition are tightly co-expressed. The first approach is still underway and will be completed using recent funding obtained from the Israel Science Foundation (ISF) in 2017 to continue this project. The second approach led to the identification of new role for COBRA-LIKE 2 in cell wall deposition in the context of seed mucilage. Moreover it led to the identification of additional proteins required for root elongation under restrictive conditions. The in-depth study of the role of COBRA-LIKE 2 in cell wall deposition was published in Ben-Tov et al., Plant Physiology 2015; Plant Journal 2018. The plasticity of root development represents a key feature enabling plants to adapt, or alternatively - avoid, abiotic stress conditions like, salinity, draught, nutrient deficiency and others and on the other hand, efficiently forage for resources like nutrients and water. Root growth plasticity determines the rate and extent of plant biomass production and plays a central role in plant adaptation to constantly changing environment. Elucidating the wide arsenal of pathways regulating root morphogenesis under stress conditions will set the stage for the next revolution aimed to increase crop yield and biomass production in marginal soils by breading targeted to the ‘hidden half’ – the root system.