Optimized small-molecule pull-downs define MLBP1 as an acyl-lipid-binding protein.

Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand-binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC-MS to identify candidate binding ligands. We optimized this method using ABA-PYL interactions and show that ABA co-purifies with wild-type PYL5 but not a binding site mutant. The Kd of PYL5 for ABA is 1.1 µm, which suggests that the method has sufficient sensitivity for many ligand-protein interactions. Using this method, we surveyed a set of 37 START domain-related proteins, which resulted in the identification of ligands that co-purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co-purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein-metabolite interaction and better understand protein-ligand interactions in plants.

Chemocyanin, a small basic protein from the lily stigma, induces pollen tube chemotropism.

In plant reproduction, pollination is an essential process that delivers the sperm through specialized extracellular matrices (ECM) of the pistil to the ovule. Although specific mechanisms of guidance for pollen tubes through the pistil are not known, the female tissues play a critical role in this event. Many studies have documented the existence of diffusible chemotropic factors in the lily stigma that can induce pollen tube chemotropism in vitro, but no molecules have been isolated to date. In this study, we identified a chemotropic compound from the stigma by use of biochemical methods. We purified a lily stigma protein that is active in an in vitro chemotropism assay by using cation exchange, gel filtration, and HPLC. Tryptic digestion of the protein yielded peptides that identified the protein as a plantacyanin (basic blue protein), and this was confirmed by cloning the cDNA from the lily stigma. Plantacyanins are small cell wall proteins of unknown function. The measured molecular mass by electrospray ionization ion source MS is 9898 Da, and the molecular mass of the mature protein (calculated from the cDNA) is 9900.2 Da. Activity of the lily plantacyanin (named chemocyanin) is enhanced in the presence of stigma/stylar cysteine-rich adhesin, previously identified as a pollen tube adhesin in the lily style.

Isolation and characterization of the neutral leucine aminopeptidase (LapN) of tomato.

Tomatoes (Lycopersicon esculentum) express two forms of leucine aminopeptidase (LAP-A and LAP-N) and two LAP-like proteins. The relatedness of LAP-N and LAP-A was determined using affinity-purified antibodies to four LAP-A protein domains. Antibodies to epitopes in the most N-terminal region were able to discriminate between LAP-A and LAP-N, whereas antibodies recognizing central and COOH-terminal regions recognized both LAP polypeptides. Two-dimensional immunoblots showed that LAP-N and the LAP-like proteins were detected in all vegetative (leaves, stems, roots, and cotyledons) and reproductive (pistils, sepals, petals, stamens, and floral buds) organs examined, whereas LAP-A exhibited a distinct expression program. LapN was a single-copy gene encoding a rare-class transcript. A full-length LapN cDNA clone was isolated, and the deduced sequence had 77% peptide sequence identity with the wound-induced LAP-A. Comparison of LAP-N with other plant LAPs identified 28 signature residues that classified LAP proteins as LAP-N or LAP-A like. Overexpression of a His(6)-LAP-N fusion protein in Escherichia coli demonstrated distinct differences in His(6)-LAP-N and His(6)-LAP-A activities. Similar to LapA, the LapN RNA encoded a precursor protein with a molecular mass of 60 kD. The 5-kD presequence had features similar to plastid transit peptides, and processing of the LAP-N presequence could generate the mature 55-kD LAP-N. Unlike LapA, the LapN transcript contained a second in-frame ATG, and utilization of this potential initiation codon would yield a 55-kD LAP-N protein. The localization of LAP-N could be controlled by the balance of translational initiation site utilization and LAP-N preprotein processing.

Expression studies of SCA in lily and confirmation of its role in pollen tube adhesion.

During pollination the pollen tube grows into the style and toward the ovary via the transmitting tract. In lily the growth of pollen tubes involves tube cell adhesion to transmitting tract cells. We reported two molecules involved in this adhesion event. One is a pectic polysaccharide and the other, a 9 kDa basic protein named SCA for stigma/stylar cysteine-rich adhesin. SCA, which shows some identity with LTP (lipid transfer protein), was localized to the transmitting tract epidermis of the style where pollen tubes adhere. The present studies on the expression of SCA indicate that the protein has a similar expression pattern with LTP1 in Arabidopsis and that the protein is abundant in both the stigma and the style. For further proof of its role in pollen tube adhesion the activity of Escherichia coli-expressed protein has been studied in an in vitro adhesion assay system.