Abstract
The membrane phosphoproteome in plant seed changes dynamically during embryo development. We examined the patterns of Phaseolus vulgaris (common bean) seed membrane protein phosphorylation from the mid-maturation stage until two days after germination. Serine and threonine phosphorylation declined during seed maturation while tyrosine phosphorylation remained relatively constant. We discovered that the aquaporin PvTIP3;1 is the primary seed membrane phosphoprotein, and PvTIP3;2 shows a very low level of expression. The level of phosphorylated Ser7 in PvTIP3;1 increased four-fold after seed maturation. Since phosphorylation increases water channel activity, we infer that water transport by PvTIP3;1 is highest in dry and germinating seeds, which would be optimal for seed imbibition. By the use of isoform-specific, polyclonal peptide antibodies, we found that PvTIP3;2 is expressed in a developmental pattern similar to PvTIP3;1. Unexpectedly, PvTIP3;2 is tyrosine phosphorylated following seed maturation, which may suggest a mechanism for the regulation of PvTIP3;2 following seed germination. Analysis of protein secondary structure by circular dichroism spectroscopy indicated that the amino-terminal domain of PvTIP3;1 is generally unstructured, and phosphorylation increases polyproline II (PPII) helical structure. The carboxy-terminal domain also gains PPII character, but in a pH-dependent manner. These structural changes are a first step to understand TIP3 aquaporin regulation.