Molecular cloning and characterization of phosphate (Pi) responsive genes in Gulf ryegrass (Lolium multiflorum L.): a Pi hyperaccumulator.

Gulf annual ryegrass has been identified as potential Pi hyperaccumulator, however the molecular mechanism remains largely unknown. A suppression subtractive hybridization (SSH) analysis was used to evaluate the phosphate (Pi) responsive genome expression pattern changes in Gulf annual ryegrass (Lolium multiflorum L.). Differential screening identified 384 putative Pi-starvation induced cDNAs. Bioinformatic analysis revealed that 116 cDNAs are nonredundant unigenes of which 108 exhibited high similarities with Genbank entries. The differential expression patterns of 13 cDNAs, representing diverse functional categories, were confirmed by RNA gel blot analysis. Further, detailed molecular analysis of three genes (LmPAP1, LmIPS1 and LmIDS1) was carried out by cloning and characterization of full-length cDNAs. LmPAP1 is 1,414 bp in length with an open reading frame (ORF) of 1,188 bp capable of encoding an N-terminal signal peptide of 26 amino acids. LmIPS1 gene is a member of TPSI1/Mt4 family that contains 3 short ORFs. The cDNA of LmIDS1 is 346 bp in size including a single ORF of 222 nucleotides that encodes 74 amino acid proteins, exhibiting homology with IDS1 with similarity to type 2 metallothionein like protein. In our preliminary screening of different genotypes of annual ryegrass for hyperaccumulation of Pi in their shoots, Gulf and Urugrary showed significant differences with values of 1.0% and 0.7%, respectively. Since it is logical to assume a plausible correlation that may exist between Pi-accumulation in the shoots and the expression of Pi-responsive genes, the expression of LmPAP1, LmIPS1 and LmIDS1 was evaluated in these two genotypes grown under different Pi regimes. Although there was a significant induction of these genes in both the genotypes grown under Pi-deprived condition, the abundance of LmPAP1 transcripts was relatively higher in the Gulf genotype as compared to that in the Urugrary genotype. A similar trend was observed in qRT-PCR data of other tested genotypes of annual ryegrasses. This suggests the potential role of LmPAP1 in accumulation of Pi in Gulf grass. In addition, Gulf grass genotype revealed higher levels of total P, (33)Pi uptake, and APase activity as compared to Urugrary. Together, these results suggest that the Gulf ryegrass has evolved mechanisms to acquire and hyperaccumulate more Pi under different Pi regimes by activating multiple Pi acquisition and mobilization mechanisms.