Final Report Summary - IMPACT (Improved Millets for Phosphate ACquisition and Transport)
The objectives of the project for the period 18/06/2014 to 17/06/2015 as set out in Annexe 1 are listed below with a commentary on achievements for each of them.
Objective 6: To evaluate polymorphism of PHT1 loci in Indian varieties of foxtail millet with different phosphate use efficiencies (months 25-30: return phase)
This objective has been fully achieved. In total 54 different genotypes of foxtail millet have been collected from ICRISAT, Patancheru, Hyderabad, India. These genotypes were grown on perlite as a nutrient free inert medium and supplied with nutrient solution containing low (10 µM) and high (300 µM) Pi. Extensive phenotyping of seedlings were performed for both levels of Pi. The genotypes showed good phenotypic variation in both low and high Pi. The genotype 1851 produced the superior response of fresh and dry weights of shoot and root, length of root and shoot and root hair under low Pi based on the overall ranking; this was followed by 1305 and 1820 and 1209. The genotype Maxima which was used in molecular studies produced only below average response in low Pi conditions. The genotypes CO-7, 1302, 507, 480, 1299 and 1892 produced moderate response. Very least response was obtained with genotypes 748 and 1474 under same low Pi. Most notably the genotype 748 also produced inferior response under high Pi as well.
As an extension of this objective, the seed yield of these varieties has also been analyzed in field conditions by growing these varieties in natural soils having low and high Pi. A number of plant traits was analysed viz. plant height, number of tillers, number of productive tillers, length of leaf, number of clusters per flower, length of flower, 25 seed yield and total seed yield per flower. Based on the overall ranking, the genotype 663 produced superior response in low Pi soil; this was followed by CO-7, 1338, 869, 1736 and CO-5. The genotype 1851 which also produced superior response in greenhouse assays falls in the category of best performing genotypes in field assays under low Pi. The Maxima genotype used for molecular study produced only least response in field study too. The genotype 132 produced superior response under high Pi soil.
Twenty six simple sequence repeat (SSR) markers of foxtail millet were used to find the genetic polymorphism (genotyping) and to detect the marker associated with phosphate use efficiency among these genotypes. These markers produced good polymorphic bands. Association mapping was performed with phenotyping and genotyping data of 54 genotypes using TASSEL-V2.1 software. Several markers have been found to be associated with important traits of foxtail millet for low Pi with above 95% confidence level. The markers b59, SIGMS4692 and b165 have been associated with root hair density under low Pi. SIGMS4692 has also been associated with root hair length and SIGMS8556 has been associated with 25 seed yield under low Pi. Markers SIGMS4692, b269 and bb225, SIGMS12819 were associated with seed yield in low Pi. Some markers have also been associated with seed yield in high Pi group these include SIGMS8556 for 25 seed weight and b190 for total seed yield. Overall the marker SIGMS4692 has been found to be the most significant marker for traits associated with low Pi tolerance in foxtail millet. But none of the markers hit on any SiPHT1 genes of foxtail millet.
The Ph.D fellow Mr M. Ramakrishnan, who has worked in the Department of Biotechnology (DBT), project on finger millet to evaluate genetic diversity in different varieties and its relatedness to fungal disease resistance in which the fellow is the co-investigator, has helped in this study.
Objective 7: To transform foxtail millet with wild type PHT1 family members and selected mutants and to test the resultant performance on different phosphorus regimes (months 31-36: return phase)
This objective has been partially achieved as the mutants were not obtained during incoming phase at Leeds. The foxtail millet was transformed with RNAi clones of SiPHT1;2, SiPHT1;3 and SiPHT1;4 separately by Agrobacterium; the constructs were made at Leeds during incoming phase. The transformation and regeneration of transgenic foxtail millet had been a very challenging task. A novel tissue culture based direct regeneration method was developed using shoot apex as starting explants for the production of RANi transgenic lines in Maxima variety. The phosphate uptake assay was performed in T1 progenies of RNAi lines under low and high Pi using hydroponics system. The total phosphate and inorganic phosphate contents were low in root and shoot samples of SiPHT1;2-RNAi lines when compared to wild type under both sufficient (300 µM) and deficient (10 µM) Pi. The total phosphate and inorganic phosphate contents of the root samples was low in SiPHT1;4-RNAi lines as this transporter has been found to be induced by low Pi in root samples. The down-regulation of SiPHT1;3 transporter did not show much variation in total phosphate and inorganic phosphate contents under sufficient Pi.
Carryover of objectives from incoming phase
Some of the objectives of incoming phase could not be achieved due to lack of time and these were performed during return phase in India. The details are given below
Objective 2: To functionally characterise the most significant members of the transporter family via their expression in yeast
The yeast expression constructs were made for the transporters SiPHT1;1, SiPHT1;2, SiPHT1;3, SiPHT1;4, SiPHT1;7 and SiPHT1;8, in addition to one encoding the yeast phosphate transporter PHO84 during incoming phase at Leeds and sent to Loyola College, Chennai. The complementation assay was performed during the return phase. The yeast clones were grown in selective growth media containing low (0.1 mM) and high (1 mM) Pi. The OD of the yeast cultures was measured periodically for all the clones.
All clones grown under low Pi (0.1 mM) grow slowly than those grown under high Pi (1 mM). The transporters SiPHT1;2 and SiPHT1;7 showed higher growth that are near to those obtained with positive control (PHO84) under low Pi. The transporters SiPHT1;1 and SiPHT1;4 showed lower growth. All other transporters also showed higher growth than empty vector control. The same assay was also performed with a time course. Interestingly, SiPHT1;2 grows even better than positive control (PHO84). The GFP and SiPHT1;4 gave low growth responses as in previous assay after 20 hrs of growth. Future assays will be performed if possible to measure Pi uptake directly using 32P or similar isotopes.
Objective 4: To construct and analyse homology models of all members of the SiPHT1 family
This objective has been fully achieved during the return phase as there was no time to perform this study and the fellow was instructed by supervisors in Leeds on how to perform the homology modelling. The homology modelling was performed using a fungal (Piriformospora indica) phosphate transporter as template whose crystal structure has been solved recently. The models were made for all 12 SiPHT1s. The structure was produced with bound Pi. The active site residues have been well conserved in most of the SiPHT1s and were also similar as in template; these were Gln-174, Tyr-147, Tyr-315, Asp-311 and Asn-424. The transporters SiPHT1;2, SiPHT1;5, SiPHT1;7, SiPHT1;9 and SiPHT1;12 showed conserved residues. It is also interesting to note that transporter SiPHT1;2 showed constitutive expression in preliminary studies and also showed superior transport activities in yeast complementation assays. The transporter SiPHT1;9 has been found to be induced by AMF. The transporters SiPHT1;5 and SiPHT1;7 are highly homologous transporters. The transporter SiPHT1;11 showed greater variation in Pi binding site.
These preliminary molecular studies on foxtail millet will help to improve the phosphate use efficiency in this and other crops plants and offer a sustainable agriculture with reduced supply of synthetic phosphate fertilizers.
Objective 6: To evaluate polymorphism of PHT1 loci in Indian varieties of foxtail millet with different phosphate use efficiencies (months 25-30: return phase)
This objective has been fully achieved. In total 54 different genotypes of foxtail millet have been collected from ICRISAT, Patancheru, Hyderabad, India. These genotypes were grown on perlite as a nutrient free inert medium and supplied with nutrient solution containing low (10 µM) and high (300 µM) Pi. Extensive phenotyping of seedlings were performed for both levels of Pi. The genotypes showed good phenotypic variation in both low and high Pi. The genotype 1851 produced the superior response of fresh and dry weights of shoot and root, length of root and shoot and root hair under low Pi based on the overall ranking; this was followed by 1305 and 1820 and 1209. The genotype Maxima which was used in molecular studies produced only below average response in low Pi conditions. The genotypes CO-7, 1302, 507, 480, 1299 and 1892 produced moderate response. Very least response was obtained with genotypes 748 and 1474 under same low Pi. Most notably the genotype 748 also produced inferior response under high Pi as well.
As an extension of this objective, the seed yield of these varieties has also been analyzed in field conditions by growing these varieties in natural soils having low and high Pi. A number of plant traits was analysed viz. plant height, number of tillers, number of productive tillers, length of leaf, number of clusters per flower, length of flower, 25 seed yield and total seed yield per flower. Based on the overall ranking, the genotype 663 produced superior response in low Pi soil; this was followed by CO-7, 1338, 869, 1736 and CO-5. The genotype 1851 which also produced superior response in greenhouse assays falls in the category of best performing genotypes in field assays under low Pi. The Maxima genotype used for molecular study produced only least response in field study too. The genotype 132 produced superior response under high Pi soil.
Twenty six simple sequence repeat (SSR) markers of foxtail millet were used to find the genetic polymorphism (genotyping) and to detect the marker associated with phosphate use efficiency among these genotypes. These markers produced good polymorphic bands. Association mapping was performed with phenotyping and genotyping data of 54 genotypes using TASSEL-V2.1 software. Several markers have been found to be associated with important traits of foxtail millet for low Pi with above 95% confidence level. The markers b59, SIGMS4692 and b165 have been associated with root hair density under low Pi. SIGMS4692 has also been associated with root hair length and SIGMS8556 has been associated with 25 seed yield under low Pi. Markers SIGMS4692, b269 and bb225, SIGMS12819 were associated with seed yield in low Pi. Some markers have also been associated with seed yield in high Pi group these include SIGMS8556 for 25 seed weight and b190 for total seed yield. Overall the marker SIGMS4692 has been found to be the most significant marker for traits associated with low Pi tolerance in foxtail millet. But none of the markers hit on any SiPHT1 genes of foxtail millet.
The Ph.D fellow Mr M. Ramakrishnan, who has worked in the Department of Biotechnology (DBT), project on finger millet to evaluate genetic diversity in different varieties and its relatedness to fungal disease resistance in which the fellow is the co-investigator, has helped in this study.
Objective 7: To transform foxtail millet with wild type PHT1 family members and selected mutants and to test the resultant performance on different phosphorus regimes (months 31-36: return phase)
This objective has been partially achieved as the mutants were not obtained during incoming phase at Leeds. The foxtail millet was transformed with RNAi clones of SiPHT1;2, SiPHT1;3 and SiPHT1;4 separately by Agrobacterium; the constructs were made at Leeds during incoming phase. The transformation and regeneration of transgenic foxtail millet had been a very challenging task. A novel tissue culture based direct regeneration method was developed using shoot apex as starting explants for the production of RANi transgenic lines in Maxima variety. The phosphate uptake assay was performed in T1 progenies of RNAi lines under low and high Pi using hydroponics system. The total phosphate and inorganic phosphate contents were low in root and shoot samples of SiPHT1;2-RNAi lines when compared to wild type under both sufficient (300 µM) and deficient (10 µM) Pi. The total phosphate and inorganic phosphate contents of the root samples was low in SiPHT1;4-RNAi lines as this transporter has been found to be induced by low Pi in root samples. The down-regulation of SiPHT1;3 transporter did not show much variation in total phosphate and inorganic phosphate contents under sufficient Pi.
Carryover of objectives from incoming phase
Some of the objectives of incoming phase could not be achieved due to lack of time and these were performed during return phase in India. The details are given below
Objective 2: To functionally characterise the most significant members of the transporter family via their expression in yeast
The yeast expression constructs were made for the transporters SiPHT1;1, SiPHT1;2, SiPHT1;3, SiPHT1;4, SiPHT1;7 and SiPHT1;8, in addition to one encoding the yeast phosphate transporter PHO84 during incoming phase at Leeds and sent to Loyola College, Chennai. The complementation assay was performed during the return phase. The yeast clones were grown in selective growth media containing low (0.1 mM) and high (1 mM) Pi. The OD of the yeast cultures was measured periodically for all the clones.
All clones grown under low Pi (0.1 mM) grow slowly than those grown under high Pi (1 mM). The transporters SiPHT1;2 and SiPHT1;7 showed higher growth that are near to those obtained with positive control (PHO84) under low Pi. The transporters SiPHT1;1 and SiPHT1;4 showed lower growth. All other transporters also showed higher growth than empty vector control. The same assay was also performed with a time course. Interestingly, SiPHT1;2 grows even better than positive control (PHO84). The GFP and SiPHT1;4 gave low growth responses as in previous assay after 20 hrs of growth. Future assays will be performed if possible to measure Pi uptake directly using 32P or similar isotopes.
Objective 4: To construct and analyse homology models of all members of the SiPHT1 family
This objective has been fully achieved during the return phase as there was no time to perform this study and the fellow was instructed by supervisors in Leeds on how to perform the homology modelling. The homology modelling was performed using a fungal (Piriformospora indica) phosphate transporter as template whose crystal structure has been solved recently. The models were made for all 12 SiPHT1s. The structure was produced with bound Pi. The active site residues have been well conserved in most of the SiPHT1s and were also similar as in template; these were Gln-174, Tyr-147, Tyr-315, Asp-311 and Asn-424. The transporters SiPHT1;2, SiPHT1;5, SiPHT1;7, SiPHT1;9 and SiPHT1;12 showed conserved residues. It is also interesting to note that transporter SiPHT1;2 showed constitutive expression in preliminary studies and also showed superior transport activities in yeast complementation assays. The transporter SiPHT1;9 has been found to be induced by AMF. The transporters SiPHT1;5 and SiPHT1;7 are highly homologous transporters. The transporter SiPHT1;11 showed greater variation in Pi binding site.
These preliminary molecular studies on foxtail millet will help to improve the phosphate use efficiency in this and other crops plants and offer a sustainable agriculture with reduced supply of synthetic phosphate fertilizers.