Convergent glycerolipid metabolism in highland adapted maize?

After domestication from lowland teosinte in Mexican subtropic maize colonized the highlands of both Mexico and South America characterized by lower temperatures and phosphorus bioavailability. We hypothesize that low temperature and phosphorus bioavailability were major selective forces during maize adaptation to the highlands and shaped glycerolipid metabolism since low temperature and P deficiency have opposite effects on the content of glycerolipid species.

To test this hypothesis grew a maize RIL mapping population (developed using a Mexican highland landrace Palomero Toluqueño and B73) and a 120 landrace diversity panel (composed of lowland and highland Mesoamerican and South American landraces) in common garden experiments at sea level and at 2600 masl. in Mexico and measures glycerolipid species (125 different compounds) that were used as phenotypes to identify genotype-phenotype associations and plasticity between highland and lowland conditions. I will present this results and in particular on an interesting QTL in ch3 that explains phosphatidylcholine to lysophosphatidylcholine conversion and seems to be selected against in highland Mexican landraces but not in South American ones.

Transcript

1. Convergent glycerolipid metabolism in highland adapted maize? www.rrlab.org
2. Convergent phenotypic evolution occurs when the similar phenotypes are selected for during independent events of adaptation
3. A B C D E F Phenotypic convergence can be supported by genotypic convergence at the gene level A B C D E F
4. A B C D E F Phenotypic convergence can be supported by genotypic convergence at the pathway level A B C D E F
5. A B C D E F A B C D E F Phenotypic convergence can be supported by genotypic convergence at the pathway level
6. A B C D E F Phenotypic convergence can also have a divergent genotypic basis K L Ñ E M P X
7. Selection of the same (biochemical) phenotype can occur at different levels A B C D F A B C D E F single mutation big effect several mutations small effect mutations in cis regulatory regions E
8. Predictable changes in a biochemical phenotype do not always have a predictable molecular basis highland lowland Natarajan et al . 2016 PubMedID 27846568 Mutations in different Hb amino acids lead to the higher O2 affinity.
9. -If the number of possible adaptive mutations is low we might expect a relatively constrained genetic path to adaptation and convergence among populations. -If there are many possible adaptive mutations, the path is less constrained and different populations may take diverse approaches to adaptation. Convergence depends on the number of possible adaptive mutations E E
10. A B C D E F Our model system is glycerolipid metabolism in highland adapted maize
11. Maize colonized highland territory after its domestication in tropical areas of Southwest Mexico Sawers et al . 2013 PubMedID 22303349 Relationships in the genus Zea Tripsicum Zea nicaraguensis Zea luxurians Zea diploperennis Zea perennis Zea mays ssp. huehuetenangensis Zea mays ssp. mexicana Zea mays ssp. parviglumis Zea mays ssp. mays After Hufford et al., 2012
12. Relationships in the genus Zea Tripsicum Zea nicaraguensis Zea luxurians Zea diploperennis Zea perennis Zea mays ssp. huehuetenangensis Zea mays ssp. mexicana Zea mays ssp. parviglumis Zea mays ssp. mays After Hufford et al., 2012 In México, there was significant gene flow between maize and highland teosinte mexicana Sawers et al . 2013 PubMedID 22303349 Hufford et al . 2013 PubMedID 23671421 maize mexicana low from mexicana in highland adaptation 3 parviglumis mexicana Geneflow from mexicana Adaptive? van Heerwaarden et al., 2011 Sawers & Sanchez, 2011 1750m parviglumis Gene flow from mexicana in highland adaptation 3 parviglumis mexicana Geneflow from mexicana Adaptive? van Heerwaarden et al., 2011 Sawers & Sanchez, 2011 1750m parviglumis
13. 3 parviglumis Geneflow from mexicana Adaptive? van Heerwaarden et al., 2011 Sawers & Sanchez, 2011 1750m parviglumis A second, independent event of highland adaptation occurred in In South America
14. 3 parviglumis Geneflow from mexicana Adaptive? van Heerwaarden et al., 2011 Sawers & Sanchez, 2011 1750m parviglumis A second, independent event of highland adaptation occurred in In South America Gene flow from mexicana in highland a parviglumis Geneflow from mexicana Adaptive? van Heerwaarden et al., 2011 Sawers & Sanchez, 2011 parvig
15. Andosol probability % 0 50 100 Maize had to adapt to colder temperatures and soils with lower P availability
16. How plants adapt to low phosphorus and cold conditions?
17. Plants use two strategies to increase P availability Phosphate Starvation ((Pi Sensing)) Local Response Systemic Response Root System Architecture Phosphate homeostasis
18. Local responses increase top soil root surface exploration area P-ineficient P-efficient Lynch et al . 2012 PubMedID 22527403 Rellan-Álvarez et al . 2015 PubMedID 26287479 shoot/root
19. Recovery Transport Recycling Systemic responses lead to P transport to where is most needed Li et al . 2007 PubMedID 17592130 logFC -P/+P root shoot Sawers et al . Unpublished Veneeklas et al . 2012 PubMedID 22691045 P P P
20. What P containing compounds is the plant recycling? Veneeklas et al . 2012 PubMedID 22691045
21. What P containing compounds is the plant recycling? Veneeklas et al . 2012 PubMedID 22691045
22. Inorganic Pi 18 What P containing compounds is the plant recycling? Veneeklas et al . 2012 PubMedID 22691045
23. Nucleic Acids 40 Inorganic Pi 18 What P containing compounds is the plant recycling? Veneeklas et al . 2012 PubMedID 22691045
24. Ester-P 18 Nucleic Acids 40 Inorganic Pi 18 What P containing compounds is the plant recycling? Veneeklas et al . 2012 PubMedID 22691045
25. Phospholipids 24 Ester-P 18 Nucleic Acids 40 Inorganic Pi 18 What P containing compounds is the plant recycling? Veneeklas et al . 2012 PubMedID 22691045
26. Glycerolipids are essential building blocks of membranes and signaling compounds Glycerol Polar Head Precursors Fatty Acids Phospholipids Galactolipids Sulfolipids adapted from: http://big.cea.fr/drf/big/english/PCV/LiPMB Basic components of glycerolipids lyso-Phospholipids
27. High P Low P Pi Recycling Cold Adaptation Glycerolipids relative abundance change in low P and in low Temperature Lambers et al . 2012 PubMedID 22937909 Degenkolbe et al . 2012 PubMedID 23061922 Phospholipids Sulfolipids Galactolipids
28. High P Low P Pi Recycling Cold Adaptation Glycerolipids relative abundance change in low P and in low Temperature Lambers et al . 2012 PubMedID 22937909 Degenkolbe et al . 2012 PubMedID 23061922 Phospholipids Sulfolipids Galactolipids High T Low T Phospholipids Sulfolipids Galactolipids
29. Hypothesis: glycerolipid metabolism was under high selective pressure and was important to maize adaptation to highlands in Mesoamerica and South America A B C D E F
30. Experimental approaches Common Garden Experiments in contrasting environments Bi-parental and diversity panel mapping populations Biochemical phenotyping Quantitative and population genetics Reverse genetics and heterologous expression
31. We grow our mapping populations in highland and lowland common garden fields Valle de Banderas (Nayarit) 25 masl Metepec (Edo. de México) 2600 masl
38. 3500 masl ~ 11500 feet pH ~ 5.1
39. 3500 masl ~ 11500 feet pH ~ 5.1 [P] ~ 4 ppm
40. Meso/South America Highland/Lowland Paired design Diversity panel aka HiLo panel Mesoamerica highland 30 x lowland 30 x highland 30 x lowland 30 x South America Daniel Runcie Matt Hufford Sherry Flint-Garcia Ruairidh Sawers
41. B73 x Palomero Toluqueño (PT) Recombinant Inbred Lines B73 PT X F1 Backcross 100 BC1S5 families (25% Palomero Toluqueño on B73 background) 5 Self Generations
42. B73 x Palomero Toluqueño (PT) Recombinant Inbred Lines 100 BC1S5 families (25% PT on B73 background) Lowland site Highland site B73 PT B73 PT LOWLAND Valle de Banderas (50masl) HIGHLAND Metepec (2600masl) Local adaptation drives maize diversity B73 B73 PT PT
43. Separation Charged Surface Hybrid Technology Detection qTOF MS/MS Identification Quantification Internal Stds LipidBlast Oliver Fiehn, UC Davis Samples are collected in the field and then analyzed using HPLC-MS to identify and quantify glycerolipid species Extraction Field sampling: - V4-V6 plants - Youngest fully developed leaf - 600 samples collected in 80 minutes and flash frozen in liquid N2 Kind et al, 2013 PubmedID 25340521 Karla Juarez
44. Separation Detection qTOF MS/MS Identification Quantification Internal Stds LipidBlast We can identify around 150 glycerolipid species Extraction Kind et al, 2013 PubmedID 25340521 Charged Surface Hybrid Technology
45. PCs and LPCs are the compounds that show higher differences between highland and lowland lines of the diversity panel haring metabolomic features B)
46. PCs and LPCs are the species that show higher differences between highland and lowland lines, but we can see some differences between Meso and South America Mesoamerica South America Low High Low High Low High Low High Mesoamerica South America Meso_high SA_high Meso_low SA_low
47. Since PCs can be converted into LPCs and viceversa we use ratios between them as a biochemical phenotype
48. Landraces with high PC/LPC ratios are mainly Mexican highland landraces ∑ ∑ ∑ Meso_high SA_high Meso_low SA_low
49. Landraces with high PC/LPC ratios are mainly Mexican highland landraces ∑ Meso_high SA_high Meso_low SA_low ∑ ∑ Meso_high SA_high Meso_low SA_low
50. PC/LPC ratios between Mexican higlands and lowlands is significative but not when we compare South American highlands and lowlands Meso_high SA_high Meso_low SA_low Mesoamerica South America ∑ ∑ Wilcoxon test p_val = 0.031 Low High Low High Wilcoxon test p_val = 0.626 Mesoamerica South-America
51. We use a B73 x Palomero Toluqueño (PT) Recombinant Inbred Lines mapping population to identify QTLs explaining glycerolipids variation B73 PT X F1 Backcross 100 BC1S5 families (25% Palomero Toluqueño on B73 background) 5 Self Generations
52. Palomero Toluqueño has lower levels of LPCs and higher PC/LPC ratios than B73 PT RILS B73
53. We run a QTL analysis using sum of PCs as the phenotype and identified a QTL in chr 3 chr3 8.6Mb ∑
54. Another QTL on the same region was identified when we used Lyso-PCs as phenotype chr3 8.6Mb ∑
55. Petersen et al, 2012 PubmedID 22672667 p-val = 9.094074e-09 PCs Metabolite ratios improve our ability to find associations between metabolite concentrations and genetic loci since they provide a better readout of enzymatic activity +
56. Metabolite ratios improve our ability to find associations between metabolite concentrations and genetic loci since they provide a better readout of enzymatic activity p-val = 9.094074e-09 p-val = 1.183811e-14 PCs PCs/LPCs Petersen et al, 2012 PubmedID 22672667 +
57. p-val = 9.094074e-09 p-val = 1.183811e-14 p-gain = 768203 PCs PCs/LPCs Metabolite ratios improve our ability to find associations between metabolite concentrations and genetic loci since they provide a better readout of enzymatic activity Petersen et al, 2012 PubmedID 22672667 +
58. PT and B73 alleles at the QTL peak have opposite effects on PCs and LPCs B73 B73 PT PT B73 B73 PT PT
59. Heterozygous phenotype shows that the B73 allele is dominant B73 B73 PT PT B73 PT
60. Is there any candidate gene in that region that makes sense with our biochemical phenotype?
61. A phospholipase A1 with predicted PC to lyso-PC activity is right at the QTL peak GRMZM2G353444 + PLD PLC PLA1 PLA2
62. There are around 75 different genes with predicted phospholipase activity in the maize genome and almost 1/2 of them are PLA1 type phospholipases Stepflug et al, (2016) www.maizegdb.org/gene_center/gene/#rnaseq www.plantcyc.org 1 2 3 4 5 6 7 8 9 10
63. There are around 75 different genes with predicted phospholipase activity in the maize genome and almost 1/2 of them are PLA1 type phospholipases Stepflug et al, (2016) www.maizegdb.org/gene_center/gene/#rnaseq Tissue FPKM www.plantcyc.org 1 2 3 4 5 6 7 8 9 10
64. Highest Expression Levels of PLA1 s are found on vegetative leaves Tissue FPKM Stepflug et al, (2016) www.maizegdb.org/gene_center/gene/#rnaseq
65. The candidate gene is one of the most highly expressed PLA1 s GRMZM2G353444 Tissue FPKM Stepflug et al, (2016) www.maizegdb.org/gene_center/gene/#rnaseq
66. The gene is highly expressed in leaves (V3-V9) and is up-regulated in cold conditions in temperate inbred lines 2523 FPKM Roots Vegetative leaves Leaves AP/Node Reproductive tissue Seeds Stepflug et al, (2016) www.maizegdb.org/gene_center/gene/#rnaseq data from Waters et al, 2016 10.1111/tpj.13414 +
67. Population differentiation (XP-CLR) values in the candidate gene region are higher in Mexican populations than in the South American ones CDS PROM Population XP-CLR nSNP Mexico 9.71 27 South America <95% 0 Li-Wang (Hufford lab) Mexico South America Whole Genome 30X
68. Maize adapted twice to highland conditions, are there any signs of convergent evolution? Lowland Meso America Lowland South America Highland South America Highland Meso America Takuno et al . 2015 PubMedID 26078279
69. Very few genes show signs of convergent highland adaptation between Meso America and South America. Only Meso America (0.89 %) Only South America (0.65 %) Mesoamerica + South America Takuno et al . 2015 PubMedID 26078279
70. 32 pathways show evidence of convergent selection to highlands Only Meso America (0.89 %) Only South America (0.65 %) Mesoamerica + South America Takuno et al . 2015 PubMedID 26078279 A B C D E F A B C D E F Mesoamerica South America
71. 1/5 of them are glycerolipid related pathways GRMZM2G014981 GRMZM2G111632 GRMZM2G481755 CDP-diacylglycerol biosynthesis I CDP-diacylglycerol biosynthesis II Phosphatidylglycerol biosynthesis I Phosphatidylglycerol biosynthesis II Phospholipid biosynthesis II Triacylglycerol biosynthesis A B C D E F A B C D E F Mesoamerica South America
72. Two of these genes add acyl groups to LPC to form PC GRMZM2G014981 GRMZM2G481755 PC Mesoamerica South America PC +
73. Some PCs and LPCs show a QTL in chr5 nearby the “South American” gene chr5@204Mb GRMZM2G481755 @ 195 Mb B73 B73 PT PT
74. This gene is expressed in leaves (V3-V9) and is down-regulated in cold conditions in temperate inbred lines Roots Vegetative leaves Leaves AP/Node Reproductive tissue Seeds Stepflug et al, (2016) www.maizegdb.org/gene_center/gene/#rnaseq data from Waters et al, 2016 10.1111/tpj.13414 526 FPKM +
75. The gene is highly expressed in leaves (V3-V9) and is up-regulated in cold conditions in temperate inbred lines 2523 FPKM Roots Vegetative leaves Leaves AP/Node Reproductive tissue Seeds Stepflug et al, (2016) www.maizegdb.org/gene_center/gene/#rnaseq data from Waters et al, 2016 10.1111/tpj.13414 +
76. In summary: High PC/LPC ratios seem to have been selected for in Mesoamerican highland maize and to a lesser extent in South American ones Genes coding for enzymes catalyzing PC -LPC conversions are differentiated between highlands and lowlands This high accumulation of PCs could be involved in highland adaptation /cold tolerance + +
77. Currently working on: -Identifying possible mutations in the candidate gene that could explain the biochemical phenotypes that we observe in B73/PT
78. Currently working on: -Identifying possible mutations in the candidate gene that could explain biochemical phenotypes that we observe in B73/PT -Developing markers for candidate gene(s) to explore phenotype-genotypes associations in diversity panels
79. Currently working on: -Developing NILs and mutants of candidate genes to test the possible effect of PT alleles on highland adaptation and under cold/low P conditions. -Identifying possible mutations in the candidate gene that could explain biochemical phenotypes that we observe in B73/PT -Developing markers for candidate gene(s) to explore phenotype-genotypes associations in diversity panels
80. Currently working on: -Developing NILs and mutants of candidate genes to test the possible effect of PT alleles on highland adaptation and under cold/low P conditions. -Identifying possible mutations in the candidate gene that could explain biochemical phenotypes that we observe in B73/PT -Developing markers for candidate gene(s) to explore phenotype-genotypes associations in diversity panels -Identifying divergence of glycerolipids between highland and lowland maize using Qst-Fst.
81. Mesoamerica highland 30 x lowland 30 x highland 30 x lowland 30 x South America Identifying divergence of glycerolipids between highland and lowland maize using Qst-Fst
82. Currently working on: -Developing NILs and mutants of candidate genes to test the possible effect of PT alleles on highland adaptation and under cold/low P conditions. -Identifying possible mutations in the candidate gene that could explain biochemical phenotypes that we observe in B73/PT -Developing markers for candidate gene(s) to explore phenotype-genotypes associations in diversity panels -Identifying divergence of glycerolipids between highland and lowland maize using Qst-Fst. -GWAS on glycerolipids and genes involved in glycerolipids pathways using RNA-Seq ASE on B73 x LRs F1s
83. X Daniel Runcie highland site lowland site V4 stage Exome Capture RNA-Seq Lipids B73 GWAS on glycerolipids and genes involved in glycerolipids pathways using RNA-Seq ASE on B73 x LRs F1s landraces 120 F1s
84. Currently working on: -Developing NILs and mutants of candidate genes to test the possible effect of PT alleles on highland adaptation and under cold/low P conditions. -Identifying possible mutations in the candidate gene that could explain biochemical phenotypes that we observe in B73/PT -Building a Latitudinal Highland Magic Population (High-MAGIC) to analyze the genetic architecture of highland adaptation -Developing markers for candidate gene(s) to explore phenotype-genotypes associations in diversity panels -Identifying divergence of glycerolipids between highland and lowland maize using Qst-Fst. -GWAS on glycerolipids genes involved in glycerolipids pathways using RNA-Seq ASE on B73 x LRs F1s
85. We are building a Latitudinal Highland Magic Population (High-MAGIC) to analyze the genetic architecture of highland adaptation Azul Connor Palomero Bolita Sabanero Mishca Cpunti Pising Elevation Race Race Elevation 2413 Sabanero X Azul 2100 2164 Bolita X Mishca 2890 2030 Connor X Cpunti 3188 3488 Pissing X Palomero 2597 X F1s X X X F2s X X X 4 way crosses 2 rounds of inter-matting ~ 500 families to self
86. Currently working on: -Developing NILs and mutants of candidate genes to test the possible effect of PT alleles on highland adaptation and under cold/low P conditions. -Identifying possible mutations in the candidate gene that could explain biochemical phenotypes that we observe in B73/PT -Building a Latitudinal Highland Magic Population (High-MAGIC) to analyze the genetic architecture of highland adaptation -Developing markers for candidate gene(s) to explore phenotype-genotypes associations in diversity panels -Identifying divergence of glycerolipids between highland and lowland maize using Qst-Fst. -GWAS on glycerolipids genes involved in glycerolipids pathways using RNA-Seq ASE on B73 x LRs F1s -U-Mu reverse genetics to study the effect of candidate genes on glycerolipid metabolism
87. U-Mu reverse genetics to study the effect of candidate genes on glycerolipid metabolism Galactolipid synthesis Phospholipid synthesis Sulpholipid biosynthesis Phospholipid degradation Sofia Sánchez Juan Estevez
88. W22 PT X F1 Backcross x 3 300 RILS (BC3S6) 6 generations of selfing 300 RILS (BC3) To be used together with a W22 x PT RIL mapping population Matt Hufford Ruairidh Sawers
89. Funding www.highlandadaptation.org
90. It takes a whole village to grow maize… Karla Juarez Jeffrey Ross-Ibarra Matt Hufford Daniel Runcie Sherry Flint-García Juan Estevez Ruairidh Sawers Members of the Sawers and Rellan-Álvarez Labs www.highlandadaptation.org Oliver Fiehn Dave Jackson Edgar Demelsa Christoph Benning Kenny Wang Sofia Sánchez Denise Costich
91. Allele Specific Expression will allow us to confirm/discover allelic effects X 40x 40x 80 F1s Daniel Runcie highland site lowland site V4 stage Exome Capture RNA-Seq Lipids B73 Gracias!