Evidence of 4-Cl-IAA and IAA Bound to Proteins in Pea Fruit and Seeds

The auxins 4-chloroindole-3-acetic acid (4-Cl-IAA) and indole-3-acetic acid (IAA) occur naturally in pea vegetative and fruit tissues (Pisum sativum L.). Previous work has shown that 4-Cl-IAA can substitute for the seeds in the stimulation of pea pericarp growth, whereas IAA is ineffective. Both auxins are found as free acids and as low-molecular-weight conjugates from organic solvent-soluble extracts from pea fruit. Here we present evidence for an additional conjugated auxin species that was not soluble in organic solvent and yielded 4-Cl-IAA and IAA after strong alkaline hydrolysis, suggestive of auxin attachment to pea seed and pericarp proteins. The solvent-insoluble conjugated 4-Cl-IAA in young pericarp was on average 15-fold greater than solvent-soluble 4-Cl-IAA. The solvent-insoluble conjugated IAA was approximately half the levels reported for the solvent-soluble IAA fraction. To identify putative 4-Cl-IAA-bound proteins, polyclonal antibodies were raised to 4-Cl-IAA linked to bovine serum albumin protein (BSA). Immunoblots probed with anti-4-Cl-IAA-BSA antiserum detected three to four unique bands (32-40 kDa) in primarily maternal tissues, and a different set of protein bands were detected in mainly embryonic tissues (ca. 65-74 kDa in mature seed). 4-Cl-IAA and IAA were also identified from protein fractions separated by polyacrylamide gel electrophoresis using GC-MS. These data show that the majority of 4-Cl-IAA, the growth-active auxin in young pea pericarp, and significant levels of IAA are linked to protein fractions. Auxin-proteins may function in regulation of free bioactive 4-Cl-IAA and IAA levels, and/or 4-Cl-IAA or IAA may be targeted to specific proteins post-translationally to modify protein function or stability.