POLLEN-TUBE COMPETITION AND MALE FITNESS IN HIBISCUS MOSCHEUTOS

The stigmas of animal-pollinated flowers often capture more pollen than is needed to fertilize all available ovules, and mixed-donor pollen loads are probably common. When this is the case, variation in average pollen-tube growth rates can potentially affect the number of seeds sired by a given plant. Despite considerable interest in effects of postpollination processes on male fitness, little is known about the extent of variation in pollen performance among plants from natural populations. To examine this question in Hibiscus moscheutos (rose mallow), we conducted mixed-donor hand-pollination experiments with 39 pollen donors bearing distinctive isozyme markers. Pairs of competing donors were compared on sets of 11 to 15 recipient plants per pair. These donors often differed in the proportions of seeds they sired, with the maximum deviation from an expected ratio of 50:50 being 68:32. Furthermore, three intensively studied plants exhibited consistent trends in relative pollen performance when each was tested against (1) the same three competitors, and (2) groups of 14 competitors chosen at random from the study population. In a separate experiment, we investigated the effects of salinity stress and high soil nutrients on pollen performance. These environmental factors had anticipated effects on leaf production, flower production, and petal length, but style length and (most importantly) the number of seeds sired relative to a standard pollen donor were not affected. In summary, this study provides the strongest evidence to date that pollen-tube competitive ability varies among coexisting plants and may be an important component of male fitness in plants.     

1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Summary Sequence-specific backbone ¹H and ¹⁵N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D ¹H-¹⁵N-correlated spectra obtained from uniformly ¹⁵N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.     

A spectral correlation function for efficient sequential NMR assignments of uniformly 15N-labeled proteins

Summary A new computer-based approach is described for efficient sequence-specific assignment of uniformly ¹⁵N-labeled proteins. For this purpose three-dimensional ¹⁵N-correlated [¹H, ¹H]-NOESY spectra are divided up into two-dimensional ¹H-¹H strips which extend over the entire spectral width along one dimension and have a width of ca. 100 Hz, centered about the amide proton chemical shifts along the other dimension. A spectral correlation function enables sorting of these strips according to proximity of the corresponding residues in the amino acid sequence. Thereby, starting from a given strip in the spectrum, the probability of its corresponding to the C-terminal neighboring residue is calculated for all other strips from the similarity of their peak patterns with a pattern predicted for the sequentially adjoining residue, as manifested in the scalar product of the vectors representing the predicted and measured peak patterns. Tests with five different proteins containing both -helices and -sheets, and ranging in size from 58 to 165 amino acid residues show that the discrimination achieved between the sequentially neighboring residue and all other residues compares well with that obtained with an unguided interactive search of pairs of sequentially neighboring strips, with important savings in the time needed for complete analysis of 3D ¹⁵N-correlated [¹H, ¹H]-NOESY spectra. The integration of this routine into the program package XEASY ensures that remaining ambiguities can be resolved by visual inspection of the strips, combined with reference to the amino acid sequence and information on spin-system types obtained from additional NMR spectra.Abbreviations 1D, 2D, 3D, 4D one-, two-, three-, four-dimensional - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - COSY correlation spectroscopy - TOCSY total correlation spectroscopy     

Polymorphism of legumin subunits from field bean (Vicia faba L. var. minor) and its relation to the corresponding multigene family

Legumin, which amounts to approximately 55% of the seed protein in field beans (Vicia faba L. var. minor), is a representative of the 12S storage globulin family. The 12S storage globulins are hexameric holoprotein molecules composed of different types of polymorphic subunits encoded by a multigene family. Type-A legumin subunits contain methionine whereas type-B are methionine-free subunits. Sequencing of two different type A-specific cDNAs, as well as an FPLC/HPLC-based improvement of subunit fractionation and peptide mapping with subsequent partial amino-acid sequencing, permit the assignment of some of the polymorphic legumin subunits to members of the multigene family. Two different type A subunits (A1 and A2) correspond to the two different cDNA clones pVfLa129 (A2) and 165 (A1), but microheterogeneity in the amino-acid sequences indicates that polymorphic variants of both representatives of this type may exist. Two groups of published type B-specific gene sequences (LeB7, and LeB2, LeB4, LeB6, respectively) are represented by two polymorphic subunit fractions (B3I, B3II, and B4I, B4II). A seventh clone, LeB3, encodes one of the large legumin subunits that is only a minor component of the legumin seed protein complex.     

Sequential1H-NMR assignments of neurotoxin III from the sea anemoneHeteractis macrodactylus and structural comparison with related toxins

The complete sequence-specific assignment of resonances in the¹H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemoneHeteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III fromHeteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The CH resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected CH resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7–16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I fromStichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

Spatial autocorrelation invalidates the Dow-Cheverud test

Dow and Cheverud (Am. J. Phys. Anthropol. 68:367–373, 1985) have published a statistical test for comparing any three similarity matrices. Using both simulations and analytical arguments, I establish that the presence of spatial autocorrelation, a common feature of geographically based anthropological and biological data, causes this test to reject too often. Increasing the spatial autocorrelation increases the spurious rejection rate. About 20% of the papers that reference Dow and Cheverud's paper have used their test with spatially autocorrelated data. Mantel's (Cancer Res. 27:209–220, 1967) method, when used as a test of spatial autocorrelation, is unaffected by these considerations, since its null hypothesis is that the data are uncorrelated. © 1992 Wiley-Liss, Inc.     

RELATIVE SUCCESS OF SELF AND OUTCROSS POLLEN COMPARING MIXED- AND SINGLE-DONOR POLLINATIONS IN AQUILEGIA CAERULEA

Flowers frequently receive both self (S) and outcross (OC) pollen, but S pollen often sires proportionally fewer seeds. Failure of S pollen can reflect evolved mechanisms that promote outcrossing and/or inbreeding depression expressed during seed development. The relative importance of these two processes was investigated in Aquilegia caerulea, a self-compatible perennial herb. In the field I performed single-donor (S or OC) and mixed-donor (S plus OC) pollinations to compare the relative success of both pollen types at various stages from pollen germination to seed maturity. Single-donor S pollinations produced significantly fewer and lighter seeds (x decrease = 12% and 3%, respectively) than OC pollinations. Abortion rates differed by an average of 38% whereas fertilization rates differed by only 5%, indicating that most differences in seed number arose postzygotically. This suggests that inbreeding depression was responsible for most failure of S pollen. One prezygotic effect measured was that 10% fewer S than OC pollen tubes reached ovaries after 42 hr, suggesting S pollen might fertilize proportionately fewer ovules after mixed pollination. Using allozyme markers, I found mixed-donor pollinations produced significantly more and heavier outcrossed than selfed seeds. However, the proportion of selfed seed, fertilized ovules, and aborted seeds for mixed-donor fruits were each predictable from pollen performance in single-donor fruits, suggesting that differential paternity is best explained by inbreeding depression during seed development. Even given these similarities between mixed- and single-donor fruits in the relative performance of S and OC pollen, both individual seed weight and seed set were significantly higher in multiply-sired fruits.