FUNCTION OF SPHINGOLIPID REMODELING IN PLANT RESPONSE TO PHOSPHORUS DEFICIENCY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022148
• Grant No.: 2020-67013-30908
• Cumulative Award Amt.: $475,000.00
• Proposal No.: 2019-05555
• Project Start Date: Jun 1, 2020
• Project End Date: May 31, 2025
• Grant Year: 2020
• Program Code: [A1152]- Physiology of Agricultural Plants

Recipient Organization
UNIVERSITY OF MISSOURI SYSTEM
ONE UNIVERSITY BLVD
SAINT LOUIS,MO 63121

Performing Department
Biology

Non Technical Summary
Low phosphate (Pi) availability in soils and low phosphorus use efficiency (PEU) by plants are two major constraints for crop production.Approxiiemately 70% of global cultivated land is deficient in Pi. Pi fertilizers are widely used to increase crop production, but this practice is unsustainable because it accelerates not only production costs, but also depletion of non-renewable P reserves. The global Pi fertilizer market was estimated to be $61.6 billion in 2018. Using a fraction less of Pi fertilizers would amount to hundreds of millions of dollars in savings for agricultural production in the United States. Therefore, developing crops with tolerance to low Pi and increased PUE would bring tremendous societal benefits economically and environmentally. A better understanding of the mechanisms by which crop plants adapt to Pi deficiency will facilitate greatly the development of cultivars with decreased P dependency. However, current knowledge is rather limited regarding the mechanism by which plants optimize P use, and the role of P deprivation-induced metabolic adaptation in crop plant production. This project focuses on a new membrane lipid remodeling process involving sphingolipids in the major oilseed crop canola. Demand for canola oil in the U.S. has increased dramatically in recent years because of its health benefits. Canola oil has the least saturated fatty acids of all culinary oils and has high levels of monounsaturated and omega-3 fatty acids. Canola meals are the second largest protein meal produced in the world for animal feed. Increased demand for canola products has resulted in increasing canola acreage in the U.S. and the cultivation of canola requires nutrient-rich soil. Thus, the use of canola to investigate the metabolism and function of sphingolipid changes under Pi deficiency provides excellent opportunities to integrate basic knowledge advancement with applications to increasing crop production. The proposed study addresses the priorities of Physiology of Agricultural Plants (A1152), particularly in relation to nutrient utilization and phosphorus efficiency. The biochemical, molecular, and physiological analyses of lipid metabolism will advance understanding of the mechanisms by which plants optimize P use in response to Pi deprivation. In addition, it will explore whether lipid remodeling enables plants to be less-dependent on P for growth and production, thus decreasing use of Pi fertilizers. Such progress would facilitate the development of crop varieties with improved growth resilience to adverse environmental conditions.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1848	1020	100%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1848 - Canola;

Field Of Science
1020 - Physiology;

Keywords

brassica napus l.

canola

lipid remodeling

phospholipase

phosphorus efficiency

sphingolipids

Goals / Objectives
The goal of the project is to determine the role of a newly discovered sphingolipid hydrolysis process in canola (Brassica napus) response to P deficiency. The hypotheses are that the P deficiency-induced non-specific phospholipase C4 (NPC4) in planta hydrolyzes primarily glycosylinositol-phosphoceramide (GIPC) associated with the plasma membrane rafts under P deficiency and that the NPC4-mediated sphingolipid hydrolysis may be modulated to improve crop P efficiency under P limitation. The supporting objectives are to determine:1) NPC4's substrate selectivity between the sphingophospholipid GIPC and glycerophospholipid PC;2) NPC4's sub-membrane association and access to GIPC under P limitation;3) Effect of NPC4 on lipid remodeling under P deficiency in NPC4-altered canola; and4) Function of NPC4-mediated sphingolipid changes in canola response to P deficiency.

Project Methods
1) Use surface-dilution kinetics and competition analyses to characterize NPC4's substrate selectivity between the sphingophospholipid GIPC and glycerophospholipid PC;2) Measure the role of S-acylation of NPC4 in its association with membrane rafts and hydrolysis of GIPC to determine NPC4's sub-membrane association and access to GIPC under P limitation;3) Profile sphingolipid and glycerolipid changes in NPC4-altered canola to determine the effect of NPC4 on lipid remodeling under P deficiency; and4) Grow NPC4-altered canola lines under different levels of P to maturity to evaluate the effect of NPC4-alterations on P efficiencies and seed production.

Progress 06/01/23 to 05/31/24

Outputs
Target Audience:Plant biology community, lipid researchers, biotechnologists, biotech industries, undergraduate and graduate students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One student and one research associate are trained in plant biochemistry, lipidology, genetic manipulation, and plant physiology. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest via publications in peer-reviewed journals and presentations in conferences, such as 2024 ASPB Annual Meeting, and 2024 International Symposium of Plant Lipids, and in seminars What do you plan to do during the next reporting period to accomplish the goals?Proceed as proposed and also include NPC4-manipulated rice to test its function is conserved in cereal crops to improve plant growth under low P.

Impacts
What was accomplished under these goals? Objective 1. Determine NPC4's substrate selectivity between the sphingophospholipid GIPC and glycerophospholipid PC (Year 1-4) 100% completion Progress: We have assayed the hydrolytic activity of NPC4 toward GIPC and PC and analyzed its enzymatic kinetics. The results show that NPC4 hydrolyzes both GIPC and PC with some preference to GIPC. In addition, we collaborated with others to determine the structure of NPC4 that provides insights to NPC4's binding to and hydrolysis of different lipids. The exposed cleft on NPC4 is larger than the molecular size of GIPC or PC, suggesting that the large-exposed cleft of NPC4 enables the recognition of various substrates with different molecular sizes. Outcomes: Analyses of NPC4 activity in vitro show that NPC4 hydrolyzes both GIPC and PC with higher activity toward GIPC. Analyses of lipid changes in NPC4-altered rapeseed and camelina indicate that NPC4 hydrolyzes both GIPC and phosphoglycerolipids. Impacts: The results show that NPC4 in plants can use both GIPC and PC as substrate and the relative activity between these lipids varies between plant species. Objective 2. Determine NPC4's sub-membrane association under P limitation (Year 1-4) 90% completion Progress: We conducted subcellular fractionation and fluorescent imaging of GFP-tagged NPC4. In addition, S-acylation and subcellular association of wildtype NPC4 were detected and mutated NPC4 with the S-acylating cysteine was changed to alanine. Outcomes: NPC4 is associated with the plasma membrane, and S-acylation is required for the plasma membrane association. The acylation and plasma membrane association are needed for NPC4 hydrolysis of GIPC under P deficiency. Impacts: The data indicate that NPC4 resides in the membrane where GIPC is. The need of S-acylation for NPC4 function in GIPC hydrolysis under P deficiency opens another avenue for manipulating NPC4 functions. Objective 3. Determine effect of NPC4 on lipid remodeling under P deficiency in NPC4-altered canola (Year 1-4) 95% completion Progress: We have grown WT and NPC4-altered canola and camelina under P-sufficient and deficient conditions and assayed the effect of NPC4 alterations on lipid remodeling and P contents in different tissues. In addition, we have assayed the effect of NPC4-alterations on P levels at different ages of leaves. Furthermore, we have monitored the expression levels of genes involved in P transport and utilization under P-replete and deplete conditions and in old and young leaves as affected by NPC4 alterations. Outcomes: Our data show that NPC4 promotes P recycling from old, senescing leaves to young leaves. NPC4 increases the expression of genes involved in P transport and utilization, particularly under low P and senescent leaves. Impacts: The results reveal a new mechanism by which NPC4 enhances plant response to low P, which is that NPC4 promotes P remobilization from old, senescent, source tissues to young, growing, sink tissues, in addition to lipid remodeling. Objective 4. Determine Function of NPC4-mediated lipid changes in crop response to P deficiency (Year 1-4) 85% completion Progress: We grew WT and NPC4-altered canola and camelina to maturity under P-sufficient and deficient conditions and analyzed plant growth, development, reproductive structures, and seed production. The results show that NPC4 promotes plant growth and seed oil and seed production, particularly under low P in both oilseed crops. Outcomes: Compared with WT, NPC4-KO plants grew smaller whereas NPC4-overexpressing rapeseed and camelina flower earlier and produced more seeds, and the magnitude of difference between WT and NPC4-altered plants is greater under P-deficient that P-sufficient conditions. Impacts: The results from two oil crops indicate that NPC4 is a positive regulator for plant growth and production under low P, identifying a new way to improve crop production with less input of P fertilizer. A patent disclosure has been filed on improving plant P remobilization and use efficiency.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Yang B, Li J, Yan J, Zhang K, Ouyang Z, Lu Y, Wei H, Li Q, Yao X, Lu S, Hong Y, Wang X*, Guo L*. NON-SPECIFIC PHOSPHOLIPASE C4 hydrolyzes phosphosphingolipids and phosphoglycerolipids and promotes rapeseed growth and yield. J. Integr. Plant Biol. 2023, 65, 2421-2436. PMID: 37642157 (*=co-corresponding author)
• Type: Journal Articles Status: Published Year Published: 2024 Citation: Li J, Yao S, Jonas M, Kim SC, Wang X. Nonspecific phospholipase c4 improves phosphorus remobilization from old to young leaves in Camelina. Plant, Cell & Environ. 2024, doi: 10.1111/pce.15122. PMID: 39253961.
• Type: Journal Articles Status: Published Year Published: 2024 Citation: Yao S, Kim SC, Li J Tang S, Wang X. Phosphatidic acid signaling and functions in nuclei. Prog Lipid Res. 2024, 93, 101267. doi: 10.1016/j.plipres.101267; PMID: 38154743.

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:Plant biology community, lipid researchers, agronomists and farmers, undergraduate and graduate students Changes/Problems:We have included the oil crop camelina in theresearch because camelina plants areeasily transformed and have a short life cycle, whichwill facilitate the progress of the proposedresearch. Theaddition will also test the applicability of the proposed research to other crops. What opportunities for training and professional development has the project provided?Two students and one research associate are trained in plant biochemistry, lipidology, genetic manipulation, and plant physiology. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest via publications in peer-reviewed journals and presentations in conferences, such as 2023 Gordon Research Conference on lipid functions What do you plan to do during the next reporting period to accomplish the goals?proceed as proposed and also include NPC4-maniuplated camelina to help test the function.

Impacts
What was accomplished under these goals? Objective 1.Determine NPC4's substrate selectivity between the sphingophospholipid GIPC and glycerophospholipid PC (Year 1-4) 85% completion Progress:We have assayed the hydrolytic activity of NPC4 toward GIPC and PC and analyzed its enzymatic kinetics.In addition, we collaborated with others to determine the structure of NPC4 that provide new insights to NPC4's binding to and hydrolysis of different lipids. The exposed cleft on NPC4 is larger than the molecular size of GIPC or PC, suggesting that the large-exposed cleft of NPC4 enables the recognition of various substrates with different molecular sizes. Outcomes:Analyses of NPC4 activity and altered plants indicate that NPC4 hydrolyzes both GIPC and PC with higher activity toward GIPC. Impacts:The results provide a possible mechanism for the promiscuous activity of NPC4 hydrolysis and identify a major metabolic step in sphingolipid remodeling in plant response to P deficiency. Objective 2.Determine NPC4's sub-membrane association and access to GIPC under P limitation (Year 1-4) 85% completion Progress:We conducted subcellular fractionation and fluorescent imaging of GFP-tagged NPC4. In addition, S-acylation and subcellular association of wildtype NPC4 were detected and mutated NPC4 with the S-acylating cysteine was changed to alanine. Outcomes:NPC4 is associated with the plasma membrane, and S-acylation is required for the plasma membrane association. The acylation and plasma membrane association are needed for NPC4 hydrolysis of GIPC under P deficiency. Impacts:The data indicate that NPC4 resides in the membrane where GIPC is, so when NPC4 is induced under P deficiency, it can access to and hydrolyze GIPC. Those provide a mechanistic understanding for NPC4's subcellular distribution. The finding that S-acylation is needed for NPC4 function in GIPC hydrolysis under P deficiency opens another avenue for manipulating NPC4 functions. Objective 3.Determine effect ofNPC4on lipid remodeling under P deficiency inNPC4-altered canola (Year 1-4) 65% completion Progress:We have grown WT and NPC4-altered canola under P-sufficient and deficient conditions and assayed the effect of NPC4 alterations on lipid remodeling and P contents in different tissues. In addition, we have generated NPC4-overexpressing camelina, another oilseed crop, to assess the effect of NPC4 on lipid remodeling and growth. Outcomes:Our data indicate that NPC4 promotes P release from phospholipids to increase Pi level in canola.Under field conditions, lipidomic profiling of camelina indicates that the extent of decrease in the common PC species reflect well the degree of P deficiency and seed production. P deficiency increased seed oil content but decreased total seed production. P deficiency altered seed fatty acids composition, but the effect varied with the severity of P deficiency. Impacts:The results provide evidence for a role of NPC4 in mobilizing P in plant leaves under P deficiency. In addition, the study provides information on the effect of P deficiency on seed oil production and fatty acid composition, which determines the value of oilseed crops, in field and greenhouse conditions. Objective 4.Determine Function of NPC4-mediated sphingolipid changes in canola response to P deficiency (Year 1-4) 55% completion Progress:We grew WT and NPC4-altered canola to maturity under P-sufficient and deficient conditions and analyzed plant growth, development, reproductive structures, and seed production. We have completed two separate experiments. In addition, we have tested the effect of NPC4-overexpression on camelina growth and seed production. Outcomes:Preliminary data indicate that NPC4-KO plants grew smaller whereas NPC4-overexpressing ones grew better than WT, and seed yield per plants followed thesame trend. Impacts:Further study of the effect NPC4-altered lines under different growth conditions will define the function of NPC4 in plant utilization P and adaptation to P availability with potential to producing plants with enhanced production under P limitation.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: 1. Li J, Su Y, Shapiro CA, Schachtman DP, Wang X. Phosphate Deficiency Modifies Lipid Composition and Seed Oil Production in Camelina. Plant Sci. 2023 Feb 13;:111636. doi: 10.1016/j.plantsci.2023.111636. [Epub ahead of print] PubMed PMID: 36791961.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: 2. Fan R, Zhao F, Gong Z, Chen Y, Yang B, Zhou C, Zhang J, Du Z, Wang X, Yin P, Guo L, Liu Z. Insights into the mechanism of phospholipid hydrolysis by plant non-specific phospholipase C. Nature Commun. 2023 Jan 12;14(1):194. doi: 10.1038/s41467-023-35915-4. PubMed PMID: 36635324; PubMed Central PMCID: PMC9837106.

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:Plant biology community, lipid researchers, agronomists, undergraduate and graduate students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate student and one research associate are trained in plant biochemistry, lipidology, genetic manipulation, and plant physiology. How have the results been disseminated to communities of interest?Via publications in peer-reviewed journals,seminars, presentations in conferences, and conversations/forums with interested groups, such as Missouri 100 and Illinois Agricultural Leadership Class. What do you plan to do during the next reporting period to accomplish the goals?proceed as proposed

Impacts
What was accomplished under these goals? Objective 1. Determine NPC4's substrate selectivity between the sphingophospholipid GIPC and glycerophospholipid PC (Year 1-4) 70% completion Progress: We have assayed the hydrolytic activity of NPC4 toward GIPC and PC and analyzed its enzymatic kinetics. Outcomes: The data indicate that NPC4 hydrolyzes both GIPC and PC with higher activity toward GIPC. Impacts: The results identify a major metabolic step in sphingolipid remodeling in plant response to P deficiency. Objective 2. Determine NPC4's sub-membrane association and access to GIPC under P limitation (Year 1-4) 75% completion Progress: We conducted subcellular fractionation and fluorescent imaging of GFP-tagged NPC4. In addition, we detected S-acylation and subcellular association of WT NPC4 and mutated NPC4 that changed the S-acylating cysteine to alanine. The mutated NPC4 was introduced NPC4-KO plants to test the function of the acylation on plant response to P deficiency. Outcomes: NPC4 is associated with the plasma membrane, and S-acylation is required for the plasma membrane association. The acylation and plasma membrane association is required for NPC4 hydrolysis of GIPC under P deficiency. Impacts: The data indicate that NPC4 resides in the membrane where GIPC is, so when NPC4 is induced under P deficiency, it can access to and hydrolyze GIPC. This membrane association is enabled by acylation. Those provide a mechanistic understanding for NPC4's subcellular distribution. The finding that S-acylation is needed for NPC4 function in GIPC hydrolysis under P deficiency opens another avenue for manipulating NPC4 functions. Objective 3. Determine effect of NPC4 on lipid remodeling under P deficiency in NPC4-altered canola (Year 1-4) 40% completion Progress: We have grown WT and NPC4-altered canola under P-sufficient and deficient conditions and compared lipid and P contents. In addition, we have assessed lipid remodeling using camelina, another oilseed crop in field and laboratory conditions. Outcomes: Our data indicate that NPC4 promotes P release from phospholipids to increase Pi level in canola. Under field conditions, lipidomic profiling of camelina indicates that the extent of decrease in the common PC species reflect well the degree of P deficiency and seed production. P deficiency increased seed oil content but decreased total seed production. P deficiency altered seed fatty acids composition, but the effect varied with the severity of P deficiency. Impacts: The results provide evidence for a role of NPC4 in mobilizing P in plant leaves under P deficiency. In addition, the study provides information on the effect of P deficiency on seed oil production and fatty acid composition, which determines the value of oilseed crops, in field and greenhouse conditions. Objective 4. Determine Function of NPC4-mediated sphingolipid changes in canola response to P deficiency (Year 1-4) 35% completion Progress: We grew WT and NPC4-altered canola to maturity under P-sufficient and deficient conditions and analyzed plant growth, development, reproductive structures, and seed production. We have completed one set of the study and the second repeat to increase the sample size is under way. Outcomes: Preliminary data indicate that NPC4-KO plants grew smaller whereas NPC4-overexpressing ones grew better than WT, and seed yield per plants followed the same trend. Impacts: Further study of the effect NPC4-altered lines under different growth conditions will define the function of NPC4 in plant utilization P and adaptation to P availability with potential to producing plants with enhanced production under P limitation.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Yang B, Zhang K, Jin X, Yan J, Lu S, Shen Q, Guo L, Hong Y*, Wang X*, Guo L*. Acylation of non-specific phospholipase C4 determines its function in plant response to phosphate deficiency. Plant J. 2021 Jun;106(6):1647-1659
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Yao S, Peng S, Wang X. Phospholipase D? interacts with autophagy-related protein 8 and promotes autophagy in Arabidopsis response to nitrogen deficiency. Plant J. 2022 Mar;109(6):1519-1534

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Plant biology community, lipid researchers, agronomists, undergraduate and graduate students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student,one research associate, and one undergraduate are trained in plant biochemistry, lipidology, genetic manipulation, and plant physiology. How have the results been disseminated to communities of interest?Peer-reviewed publications and seminars to other research institutions. What do you plan to do during the next reporting period to accomplish the goals?proceed as proposed

Impacts
What was accomplished under these goals? Objective 1. Determine NPC4's substrate selectivity between the sphingophospholipid GIPC and glycerophospholipid PC (Year 1-4) 30% completion Progress: We produced and purified catalytically active NPC4 and assayed its activity toward GIPC and PC. Outcomes: NPC4 hydrolyzes in vitro sphingophospholipid GIPC and glycerophospholipid PC. Kinetic analysis indicates that NPC4 prefers the hydrolysis of GIPC to PC. Impacts: The results identify a new sphingolipid metabolic reaction and potentially a major metabolic step in sphingolipid remodeling in plant response to P deficiency. Objective 2. Determine NPC4's sub-membrane association and access to GIPC under P limitation (Year 1-4) 25% completion Progress: We analyzed NPC sequences for potential sites of S-acylation, performed site-specific mutagenesis made mutation of the cysteine to alanine to obtain acylation-defective mutants, and worked out assay conditions to monitor protein S-acylation. Outcomes: S-Acylation sites have been identified. Mutants with the potential acylating residue cysteine changed to alanine have been obtained to acylation-defective mutants. A resin-capture assay is adapted to assay protein S-acylation. Impacts: The results will place NPC4 directly in close access to the substrate GIPC, provide mechanistic understanding for NPC4's submembrane association, uncover a new mechanism of NPC4 regulation, and lead to a new avenue for manipulating NPC4 functions. Objective 3. Determine effect of NPC4 on lipid remodeling under P deficiency in NPC4-altered canola (Year 1-4) 10% completion Progress: We are obtaining transgenic plants with altered NPC4 expression in canola. In addition, to speed up the generation of transgenic lines and test the effect on another crop, we are producing camelina lines with increased NPC4 expression. Outcomes: Progress is underway to having multiple lines with altered NPC4 expression in camelina and canola. Impacts: Those transgenic lines will be used to determine the effect of NPC4 on sphingolipid and glycerolipid remodeling under P deficiency. Objective 4. Determine Function of NPC4-mediated sphingolipid changes in canola response to P deficiency (Year 1-4) 5% completion Progress: We are in the process to obtain homozygous lines of transgenic camelina and canola, and testing growth conditions that can bring plants to maturity at different levels of P. Outcomes: We have defined a greenhouse condition that can grow plants to maturity under different levels of P deficiency and collected preliminary data on growth performance. Impacts: The NPC4-altered lines and growth conditions will help greatly the study to define the function of NPC4 and to test whether there is a correlation between the level of NPC4 expression and tolerance to P deficiency.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Yang B, Li M, Phillips A, Li L, Ali U, Li Q, Lu S, Hong Y, Wang X, Guo L. Nonspecific phospholipase C4 hydrolyzes phosphosphingolipids and sustains plant root growth during phosphate deficiency. Plant Cell. 2021 Jan 13; PubMed PMID: 33793851.