Final Report Summary - IMPACT (Improved Millets for Phosphate ACquisition and Transport)
The objectives of the project as set out in Annexe 1 are listed below with a commentary on achievements for each of them.
1. To investigate the patterns of gene expression and its regulation for all members of the phosphate transporter (PHT1) family from foxtail millet (Setaria italica)
Foundation work documented the growth and yield of foxtail millet plants on different levels of supplied inorganic phosphate (Pi). From this work 300 µM Pi was chosen as representative of phosphate replete conditions and 10 µM Pi was chosen as representative of phosphate deficiency. A hydroponic system was set up to allow precise control over nutrient levels and access to roots. Growth parameters, total and inorganic phosphate levels and chlorophyll were measured in plants grown under Pi replete and depleted conditions. Primers were designed and RT-PCR experiments carried out to assess expression levels for all 12 members of the SiPHT1 family in root, leaf and shoot samples from Pi replete and Pi deficient samples. In an extension to the objective we also monitored expression of the PHT1 family in response to colonisation by the arbuscular mycorrhizal fungus Glomus mosseae. Results of initial RT-PCR investigations suggested that SiPHT1;2 is ubiquitously expressed, SiPHT1;3 is predominantly expressed in leaf and SiPHT1;4 in root in 15 day old samples of hydroponically grown plants. SiPHT1;8 and SiPHT1;9 are induced by G. mosseae colonisation. Subsequent QPCR analysis focused on these 5 genes. Two reference genes were tested to identify a suitable control gene that showed little variation in expression between different tissues and growth conditions. Elongation factor 1 alpha was determined to be the most suitable. QPCR confirmed the induction of SiPHT1;8 and SiPHT1;9 by G. mosseae. SiPHT1;2 was induced by Pi deprivation in leaf, SiPHT1;3 was not induced by these conditions but was predominantly expressed in leaf and SiPHT1;4 was induced by Pi deprivation in roots of the 15 day old hydroponic samples.
To characterise the patterns of expression of the five genes (SiPHT1;2, SiPHT1;3, SiPHT1;4, SiPHT1;8 and SiPHT1;9) at the protein level, antibodies were raised against synthetic peptides corresponding to isoform-specific sequences in the central cytoplasmic loops of each protein.
The promoters for all these genes were identified and bioinformatic analysis to identify potential phosphate regulatory elements, including P1BS and CTTC motifs, was carried out. The promoters were also compared to those of homologous genes from rice, maize, sorghum and purple false brome. The promoters of SiPHT1;2 and SiPHT1;3 were fused to GUS and shown to be functional in roots by transient expression using particle bombardment. In addition transformation of shoot apices was demonstrated using a 35S-GUS construct, so millet transformation has been successfully established in the Leeds laboratory.
A manuscript has been submitted to the open access journal PlosOne;which is currently under revision.. Part of this work was also presented in poster form at the Society for Experimental Biology meeting in Valencia, Spain during 2-4 July 2013. This objective is thus essentially complete.
2. To functionally characterise the most significant members of the transporter family via their expression in yeast and Xenopus oocytes;
We decided to focus on SiPHT1;2, SiPHT1;3 and SiPHT1;4 as examples of family members with distinct expression patterns. Initially, constructs were produced in which GFP was fused to the N-terminus of these 3 proteins for Xenopus expression. These constructs were sent to our collaborators in Canada for electrophysiological characterisation, but the expected currents associated with phosphate transport were not detected in oocytes injected with RNA transcripts. Alternative constructs for these 3 proteins lacking a GFP tag were therefore made and used for functional studies in Canada. For the latter the Fellow visited Canada to learn the functional characterization of millet phosphate transporters in Xenopus oocytes during 17th April 2014 to 1st May 2014, funded by a World Universities Network Researcher Mobility grant. Unfortunately, oocytes injected with RNA transcripts encoding the un-tagged millet transporters did not produce significantly higher phosphate-induced currents than oocytes injected with water alone.
In a complementary approach towards functional characterisation of the transporters expression in yeast was next attempted. To this end 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. These constructs were employed in an attempt to complement the phosphate uptake deficiency present in a yeast host strain lacking the high affinity transporter PHO84. Optimisation of the complementation experiments remains to be completed before definitive conclusions can be made regarding the functional activities of the millet transporters.
3. To further probe the roles of the major low Pi-inducible root and shoot transporters in vivo by investigating the effects of their down regulation in whole plants using RNAi;
We decided to focus on SiPHT1;2, SiPHT1;3 and SiPHT1;4 as examples of family members with distinct expression patterns. Bioinformatic analysis of the genes was carried out to establish suitable regions that were gene specific for RNAi. Based on the analysis, the 3'UTRs have been utilized for SiPHT1;2 and SiPHT1;4 and a 192 bp internal coding region has been used for SiPHT1;3 RNAi constructs. The constructs were successfully made for all these 3 genes using pFGC1008 as a parental vector. However, transformation and regeneration of foxtail millet plants is time consuming and it became apparent that these experiments could not be completed in Leeds before the end of incoming phase. They were therefore postponed to the third year (return phase work) in India. To this end the plasmids have been sent to Loyola College, India: suitable facilities are available in Loyola to conduct the millet transformation as part of the return phase work. The genes will be introduced by Agrobacterium-mediated transformation and the Fellow will begin this work shortly.
4. To construct and analyse homology models of all members of the SiPHT1 family;
The Fellow was instructed on how construct the homology models using bioinformatics tools, using the structures of the Piriformospora indica phosphate transporter and the Escherichia coli XylE xylose transporter proteins as templates. The necessary alignment of the sequences has already been done. This work will be completed during the return phase.
5. To carry out site-directed mutagenesis, guided by the homology models, to identify the roles of important residues via kinetic comparisons of selected mutants and the wild type proteins;
This objective could not be completed because to date it has not been possible to detect transport activity induced by the millet transporters when expressed in yeast or Xenopus oocytes, although as mentioned above optimisation of yeast expression/complementation experiments is underway. Poor expression of functional protein may reflect differences in codon usage between millet and the expression hosts so far employed.
Objective 1 has been completed as per the schedule. Objective 2 has been partially completed and work towards this objective is also still in progress. Objective 3 has almost been completed: while studies using Xenopus oocytes as an expression host did not yield any evidence of transporter function, constructs have been made for yeast expression studies and the associated complementation experiments will be done soon. Objective 4 is self contained and will be addressed during the return phase work. Apart from a few delays, all the objectives have thus been completed.
1. To investigate the patterns of gene expression and its regulation for all members of the phosphate transporter (PHT1) family from foxtail millet (Setaria italica)
Foundation work documented the growth and yield of foxtail millet plants on different levels of supplied inorganic phosphate (Pi). From this work 300 µM Pi was chosen as representative of phosphate replete conditions and 10 µM Pi was chosen as representative of phosphate deficiency. A hydroponic system was set up to allow precise control over nutrient levels and access to roots. Growth parameters, total and inorganic phosphate levels and chlorophyll were measured in plants grown under Pi replete and depleted conditions. Primers were designed and RT-PCR experiments carried out to assess expression levels for all 12 members of the SiPHT1 family in root, leaf and shoot samples from Pi replete and Pi deficient samples. In an extension to the objective we also monitored expression of the PHT1 family in response to colonisation by the arbuscular mycorrhizal fungus Glomus mosseae. Results of initial RT-PCR investigations suggested that SiPHT1;2 is ubiquitously expressed, SiPHT1;3 is predominantly expressed in leaf and SiPHT1;4 in root in 15 day old samples of hydroponically grown plants. SiPHT1;8 and SiPHT1;9 are induced by G. mosseae colonisation. Subsequent QPCR analysis focused on these 5 genes. Two reference genes were tested to identify a suitable control gene that showed little variation in expression between different tissues and growth conditions. Elongation factor 1 alpha was determined to be the most suitable. QPCR confirmed the induction of SiPHT1;8 and SiPHT1;9 by G. mosseae. SiPHT1;2 was induced by Pi deprivation in leaf, SiPHT1;3 was not induced by these conditions but was predominantly expressed in leaf and SiPHT1;4 was induced by Pi deprivation in roots of the 15 day old hydroponic samples.
To characterise the patterns of expression of the five genes (SiPHT1;2, SiPHT1;3, SiPHT1;4, SiPHT1;8 and SiPHT1;9) at the protein level, antibodies were raised against synthetic peptides corresponding to isoform-specific sequences in the central cytoplasmic loops of each protein.
The promoters for all these genes were identified and bioinformatic analysis to identify potential phosphate regulatory elements, including P1BS and CTTC motifs, was carried out. The promoters were also compared to those of homologous genes from rice, maize, sorghum and purple false brome. The promoters of SiPHT1;2 and SiPHT1;3 were fused to GUS and shown to be functional in roots by transient expression using particle bombardment. In addition transformation of shoot apices was demonstrated using a 35S-GUS construct, so millet transformation has been successfully established in the Leeds laboratory.
A manuscript has been submitted to the open access journal PlosOne;which is currently under revision.. Part of this work was also presented in poster form at the Society for Experimental Biology meeting in Valencia, Spain during 2-4 July 2013. This objective is thus essentially complete.
2. To functionally characterise the most significant members of the transporter family via their expression in yeast and Xenopus oocytes;
We decided to focus on SiPHT1;2, SiPHT1;3 and SiPHT1;4 as examples of family members with distinct expression patterns. Initially, constructs were produced in which GFP was fused to the N-terminus of these 3 proteins for Xenopus expression. These constructs were sent to our collaborators in Canada for electrophysiological characterisation, but the expected currents associated with phosphate transport were not detected in oocytes injected with RNA transcripts. Alternative constructs for these 3 proteins lacking a GFP tag were therefore made and used for functional studies in Canada. For the latter the Fellow visited Canada to learn the functional characterization of millet phosphate transporters in Xenopus oocytes during 17th April 2014 to 1st May 2014, funded by a World Universities Network Researcher Mobility grant. Unfortunately, oocytes injected with RNA transcripts encoding the un-tagged millet transporters did not produce significantly higher phosphate-induced currents than oocytes injected with water alone.
In a complementary approach towards functional characterisation of the transporters expression in yeast was next attempted. To this end 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. These constructs were employed in an attempt to complement the phosphate uptake deficiency present in a yeast host strain lacking the high affinity transporter PHO84. Optimisation of the complementation experiments remains to be completed before definitive conclusions can be made regarding the functional activities of the millet transporters.
3. To further probe the roles of the major low Pi-inducible root and shoot transporters in vivo by investigating the effects of their down regulation in whole plants using RNAi;
We decided to focus on SiPHT1;2, SiPHT1;3 and SiPHT1;4 as examples of family members with distinct expression patterns. Bioinformatic analysis of the genes was carried out to establish suitable regions that were gene specific for RNAi. Based on the analysis, the 3'UTRs have been utilized for SiPHT1;2 and SiPHT1;4 and a 192 bp internal coding region has been used for SiPHT1;3 RNAi constructs. The constructs were successfully made for all these 3 genes using pFGC1008 as a parental vector. However, transformation and regeneration of foxtail millet plants is time consuming and it became apparent that these experiments could not be completed in Leeds before the end of incoming phase. They were therefore postponed to the third year (return phase work) in India. To this end the plasmids have been sent to Loyola College, India: suitable facilities are available in Loyola to conduct the millet transformation as part of the return phase work. The genes will be introduced by Agrobacterium-mediated transformation and the Fellow will begin this work shortly.
4. To construct and analyse homology models of all members of the SiPHT1 family;
The Fellow was instructed on how construct the homology models using bioinformatics tools, using the structures of the Piriformospora indica phosphate transporter and the Escherichia coli XylE xylose transporter proteins as templates. The necessary alignment of the sequences has already been done. This work will be completed during the return phase.
5. To carry out site-directed mutagenesis, guided by the homology models, to identify the roles of important residues via kinetic comparisons of selected mutants and the wild type proteins;
This objective could not be completed because to date it has not been possible to detect transport activity induced by the millet transporters when expressed in yeast or Xenopus oocytes, although as mentioned above optimisation of yeast expression/complementation experiments is underway. Poor expression of functional protein may reflect differences in codon usage between millet and the expression hosts so far employed.
Objective 1 has been completed as per the schedule. Objective 2 has been partially completed and work towards this objective is also still in progress. Objective 3 has almost been completed: while studies using Xenopus oocytes as an expression host did not yield any evidence of transporter function, constructs have been made for yeast expression studies and the associated complementation experiments will be done soon. Objective 4 is self contained and will be addressed during the return phase work. Apart from a few delays, all the objectives have thus been completed.