The Structure of a Phytocyanin, the Basic Blue Protein from Cucumber, Refined at 1.8 Å Resolution

Guss, J. Mitchell; Merritt, E A; Phizackerley, R. P.; Freeman, H. C.

doi:10.1006/jmbi.1996.0545

Cited by 120 publications

(147 citation statements)

References 56 publications

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“…The 3D structure of plantacyanin is that of a Greek key ␤-barrel fold, as are all of the blue copper proteins, but in the plantacyanins, the two histidine ligands are exposed to the surface, and they have two cysteines that form a disulfide bond as well as the copper-binding cysteine. Also the barrel is open, so it is more like a ␤-taco structure (30,31).…”

Section: Discussionmentioning

confidence: 99%

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

Kim

Mollet

Dong

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

207

184

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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 9,898 Da, and the molecular mass of the mature protein (calculated from the cDNA) is 9,900.2 Da. Activity of the lily plantacyanin (named chemocyanin) is enhanced in the presence of stigma͞stylar cysteine-rich adhesin, previously identified as a pollen tube adhesin in the lily style.

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Section: Discussionmentioning

confidence: 99%

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

Kim

Mollet

Dong

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

207

184

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“…Arabidopsis plantacyanin, with one Met at the fourth ligand site, as in spinach and cucumber plantacyanins, is believed to display a high redox potential (Nersissian et al, 1998). Because of the special redox chemistry of copper, plantacyanins, with their copper exposed on the surface, as predicted by three-dimensional structure analysis (Guss et al, 1996;Einsle et al, 2000), may readily participate in reactive oxygen species (ROS) production, including hydroxyl radicals (Halliwell and Gutteridge, 1984). If Arabidopsis plantacyanin binds copper and is capable of producing ROS, the three apparently disparate phenotypes we observed in the OXPs can be explained.…”

Section: Plantacyanin a Member Of An Ancient Family Of Blue Copper Pmentioning

confidence: 99%

Plantacyanin Plays a Role in Reproduction in Arabidopsis

2005

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Plantacyanins belong to the phytocyanin family of blue copper proteins. In the Arabidopsis (Arabidopsis thaliana) genome, only one gene encodes plantacyanin. The T-DNA-tagged mutant is a knockdown mutant that shows no visible phenotype. We used both promoter-b-glucuronidase transgenic plants and immunolocalization to show that Arabidopsis plantacyanin is expressed most highly in the inflorescence and, specifically, in the transmitting tract of the pistil. Protein levels show a steep gradient in expression from the stigma into the style and ovary. Overexpression plants were generated using cauliflower mosaic virus 35S, and protein levels in the pistil were examined as well as the pollination process. Seed set in these plants is highly reduced mainly due to a lack of anther dehiscence, which is caused by degeneration of the endothecium. Callose deposits occur on the pollen walls in plants that overexpress plantacyanin, and a small percentage of these pollen grains germinate in the closed anthers. When wild-type pollen was used on the overexpression stigma, seed set was still decreased compared to the control pollinations. We detected an increase in plantacyanin levels in the overexpression pistil, including the transmitting tract. Guidance of the wild-type pollen tube on the overexpression stigma is disrupted as evidenced by the growth behavior of pollen tubes after they penetrate the papillar cell. Normally, pollen tubes travel down the papilla cell and into the style. Wildtype pollen tubes on the overexpression stigma made numerous turns around the papilla cell before growing toward the style. In some rare cases, pollen tubes circled up the papilla cell away from the style and were arrested there. We propose that when plantacyanin levels in the stigma are increased, pollen tube guidance into the style is disrupted.Pollination is a crucial step in the life cycle of flowering plants. The pistil, composed of the stigma, style, and ovary, is the female receptive organ in pollination through which the pollen tube travels to deliver the sperm cells to the egg. The stigma, as the entry into the pistil's specialized transmitting tract tissue, provides a receptive surface for compatible pollen to adhere, hydrate, germinate, and grow. Crucifers, like Arabidopsis (Arabidopsis thaliana), have dry stigmas, which do not secrete much extracellular matrix (ECM) for the pollen tube to encounter on their surface. The pollen tube must penetrate into the cell wall space of the papilla cell to enter the transmitting tract ECM. From there, the pollen tube tracks the secretory ECM to the micropyle of the ovule for fertilization. By contrast, in lily (Lilium longiflorum), pollen tubes land on a stigma with copious secretions and enter an open style to grow on an epidermal, secretory ECM, never penetrating stigma or stylar tissues. In these two extreme cases, though, pollen tubes are in contact with the ECM of the pistil transmitting tract from the stigma to the ovule for fertilization (Lord and Russell, 2002). For this reason, pollen t...

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“…in the CST structure. The accommodations of CST in this region create a larger loop (residues 14-19) plus an a-helix (residues 20-28) that is absent in CBP ( Cuss et al, 1996).…”

Section: Overall Protein Foldmentioning

confidence: 99%

A missing link in cupredoxins: Crystal structure of cucumber stellacyanin at 1.6 Å resolution

et al. 1996

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Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 8, resolution. The crystallographic R-value for all 18,488 reflections ((T > 0) between 50-1.6 8, is 0.195. The overall fold is organized in two P-sheets, both with four P-strands. Two cy-helices are found in loop regions between P-strands. The P-sheets form a P-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the P-barrel is more flattened, there is an extra N-terminal a-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.

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The Structure of a Phytocyanin, the Basic Blue Protein from Cucumber, Refined at 1.8 Å Resolution

Cited by 120 publications

References 56 publications

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

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

Plantacyanin Plays a Role in Reproduction in Arabidopsis

A missing link in cupredoxins: Crystal structure of cucumber stellacyanin at 1.6 Å resolution

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