Molecular characterization and expression of PsPK2, a PINOID-like gene from pea (Pisum sativum)

The plant hormone auxin acts as a signal for cell division, extension and differentiation during the course of plant growth and development. With the aim of elucidating the molecular mechanism of auxin responses in pea leaf development, we have isolated a putative pea PINOID (PID) homolog, PsPK2, and characterized it. The deduced protein sequence of PsPK2 had 445 amino acids and possessed 12 conserved catalytic subdomains that characterize protein kinases. The PsPK2 sequence was most closely related to PID, and together they defined a PINOID subfamily within the AGC-VIII protein kinases. PsPK2 mapped to the lower portion of linkage group VI between Pl and Gsp. By genome walking, 2.3 kb of the PsPK2 promoter was obtained and was predicted to possess auxin and gibberellin response elements. PsPK2 was strongly expressed in developing organs of wildtype (WT) pea, such as shoot tips, immature adult leaves, developing embryos and flower buds. Comparing the PsPK2 mRNA levels from tendrils and leaflets of WT as well as the other leaf form mutants, we found that irrespective of genotype, PsPK2 mRNA was much more abundant in tendrils than in leaflets. Furthermore, differential expression occurred in shoot tip RNAs of the different leaf form genotypes. Expression was slightly higher in the afila genotypes, and lower in acacia/tendrilless and tendrilled-acacia compared to WT. In an auxin induction experiment, PsPK2 mRNA levels increased most after methyl-IAA treatment compared to IAA, 4-Cl-IAA, IBA and NAA treatments. Time course and concentration dependent experiments showed that PsPK2 mRNA levels were the highest after 30 min of methyl-IAA treatment compared to control and other treatments, and peaked at 10 μM methyl-IAA. In addition, PsPK2 mRNA levels peaked after 4 h of GA3 treatment and were highest in response to 75 μM GA3. Therefore, auxin and gibberellin positively regulate PsPK2 expression and it may play a role in the control of auxin transport and leaf form generation in pea.