IMMUNOCYTOCHEMICAL CHARACTERIZATION OF THE INTRAPYRENOID THYLAKOIDS OF CRYPTOMONADS1

Thylakoid lamellae extend into the pyrenoids of only two genera of cryptomonad algae, Chroomonas and Hemiselmis, We used immunoelectron microscopy to assess the photosynthetic competency of cryptomonad intrapyrenoid thylakoids. Intrapyrenoid thylakoids possess phycobiliproteins and the chlorophyll a/c₂ light-harvesting complex, both of which are associated with photosystem (PS) II in a light-harvesting capacity. In addition, thylakoids that extend into the pyrenoid of Hemiselmis brunnescens were immunolabelled by anti-PSI. These results indicate that cryptomonad intrapyrenoid thylakoids likely function in a manner analogous to thylakoids of the chloroplast stroma. Moreover, our observation that the Calvin cycle enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is pyrenoid-localized in these two cryptophytes indicates that the processes of photosynthetic O₂-evolution and ribulose 1,5-bisphosphate (RuBP) carboxylation/oxygenation are not spatially separated in these algae.     

GENETIC CORRELATIONS AMONG MORPHOMETRIC TRAITS AND RATES OF GROWTH AND DIFFERENTIATION IN THE GREEN TREE FROG,HYLA CINEREA

It is often proposed that the morphometric shape of animals often evolves as a correlated response to selection on life-history traits such as whole-body growth and differentiation rates. However, there exists little empirical information on whether selection on rates of growth or differentiation in animals could generate correlated response in morphometric shape beyond that owing to the correlation between these rates and body size. In this study genetic correlations were estimated among growth rate, differentiation rate, and body-size-adjusted head width in the green tree frog, Hyla cinerea. Head width was adjusted for size by using the residuals from log-log regressions of head width on snout-vent length. Size-adjusted head width at metamorphosis was positively genetically correlated with larval period length. Thus, size-independent shape might evolve as a correlated response to selection on a larval life-history trait. Larval growth rate was not significantly genetically correlated with size-adjusted head width. An additional morphometric trait, size-adjusted tibiofibula length, had a nonnormal distribution of breeding values, and so was not included in the analysis of genetic correlations (offspring from one sire had unusually short legs). This result is interesting because, although using genetic covariance matrices to predict long-term multivariate response to selection depends on the assumption that all loci follow a multivariate Gaussian distribution of allelic effects, few data are available on the distribution of breeding values for traits in wild populations. Size at metamorphosis was positively genetically correlated with larval period and larval growth rate. Quickly growing larvae that delay metamorphosis therefore emerge at a large size. The genetic correlation between larval growth rate and juvenile (postmetamorphic) growth rate was near zero. Growth rate may therefore be an example of a fitness-related trait that is free to evolve in one stage of a complex life cycle without pleiotropic constraints on the same trait expressed in the other stage.     

BOTANICAL AFFINITIES OF SOME TRIPROJECTACITES FOSSIL POLLEN

During Late Cretaceous to Oligocene times, fossil pollen of the Triprojectacites group (also known as Aquilapolles or triprojectates), comprised a temporally and environmentally distinctive element of palynofloras in eastern Asia and western North America. Several species of this group serve as biostratigraphic index fossils for this interval. Using electron microscope and numerical analyses, primarily of North American triprojectate pollen, it is possible to recognize the presence of three distinct subgroups. One group, corresponding to the fossil genus Mancicorpus, has no morphologically close modern representative. Characters resembling those in Santalaceae (Santalales, Rosidae) occur in the second group, which is represented by a previously underscribed fossil triprojectate genus. The third group contains retipilate, isopolar pollen and strioreticulate, isopolar pollen. Forms exhibiting the latter morphology are commonly assigned to the genus Integricorpus, while the retipilate morphology characterizes another underscribed genus. This third group may have some phylogenetic connection to Apiaceae (Apiales, Asteridae). Pollen of some other extant families exhibits triprojectate features, although no close fossil representative can be presently identified. The triprojectate morphology is thus interpreted in modern and fossil forms as resulting from convergence rather than close phylogenetic relationships at the group level.     

THE PHYSICAL AND CHEMICAL ENVIRONMENT OF THE DEVELOPING EMBRYO OF PINUS RESINOSA

The relationship between changes in soluble protein, hexose sugar, total lipid concentration, and osmotic potential occurring in gametophytic supernatant of Pinus resinosa Ait. during in vivo embryogenesis was measured. The effects of varying sucrose levels of culture medium on in vitro embryo and gametophyte development were examined. Increases in embryo volume, and fresh and dry weight of the female gametophyte during in vivo embryogenesis coincide with increasing levels of soluble protein, hexose sugar, and total lipid in the gametophytic supernatant. In contrast, osmotic potential of the supernatant increased only slightly between the zygote and proembryo stages of embryo development, and remained constant thereafter. Gametophytes plus embryos grown in vitro achieved dry weights approaching those of in ovulo gametophytes on media containing levels of sucrose up to 21%. Gametophytes on media with sucrose concentrations up to 21% also resembled normal in ovulo gametophytes in appearance. However, embryo development appeared to be suspended on treatment media containing from 9% to 21% sucrose, while embryos degenerated on media with constant sucrose levels of 3% and 6%. A treatment medium containing approximately 12% sucrose would provide an osmotic environment that duplicates that found in ovulo. While greater sucrose levels promoted more normal gametophyte development in Pinus resinosa, we failed to achieve complete development of the embryo in vitro. Conclusions and implications drawn from these results are discussed.     

2-KETO-SUGAR ACIDS IN GREEN FLAGELLATES: A CHEMICAL MARKER FOR PRASINOPHYCEAN SCALES1,2

The chemical composition of cell walls (thecae) of three taxa of scaly green flagellates (Prasinophyceae) was investigated. The theca of Tetraselmis striata, Tetraselmis tetrathele, and Scherffelia dubia consists mainly of carbohydrate (80% of dry weight), with proteins (5%), calcium (4%), and sulfate (6%) as minor components. The principal sugars (60% of dry weight) are the 2-keto-sugar acids 3-deoxy-manno-2-octulosonic acid (KDO), 3-deoxy-manno-5-O-methyl-2-octulosonic acid (5OMeKDO), and 3-deoxy-lyxo-2-heptulosaric acid (DHA). Arabinose, gulose, galactose, galacturonic acid, and in S. dubia, xylose and rhamnose were also found. Examination of scale preparations from Mantoniella squamata, Mesostigma viride, Pyramimonas amylifera, and Nephroselmis olivacea revealed that the 2-keto-sugar acids were always associated with the presence of typical prasinophycean scales on the cell surface. In contrast, 2-keto-sugar acids were not detected in the cell wall of Chlamydomonas reinhardtii nor in polymer preparations from the culture medium of Chlamydomonas reinhardtii, Dunaliella bioculata, Dunaliella primolecta, Asteromonas gracilis, Hafniomonas reticulate, Pedinomonas tuberculata, Monomastix sp., and Micromonas pusilla. We conclude that 2-keto-sugar acids are chemical markers for prasinophycean scales.     

Characterization of high affinity GTPase activity correlated to β-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes

β-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The β-adrenergic agonist isoproterenol (10 μM) increased the V_max of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its K_m value (≈ 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5′-(3-O-thio)triphosphate (GTPγS), isoproterenol increased cAMP formation to the same extent as that observed with AlF₄^?. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (≈ 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the ‘turn-off’ step for the adenylyl cyclase activation seen following β-adrenergic stimulation of rat parotid glands.     

CARBON FIXATION IN CULTURED MARINE BENTHIC DIATOMS1

The enzyme activity of ribulose 1, 5-bisphosphate carboxylase-oxygenase (RuBisCO) and phosphoenolpyruvate carboxylase (PEPC) was measured in four species of marine benthic diatoms isolated from subtidal sediments of Graveline Bayou, Mississippi. Enzyme activities were measured in cultures of Amphora micrometra Giffen, A. tenerrima Aleem and Hustedt, Nitzschia fontifuga Cholnoky, and Nitzschia vermicularis Grunow that were grown at light levels supporting μ_max and at light-limiting irradiances. All four species exhibited similar RuBisCO: PEP ratios (range = 1–1.8) at μ_max the lowest ratio (0.4) was observed in A. micrometra. Reduced light levels increased PEPC relative to that measured at μ_max in two species. Two-dimensional paper chromatography was used to determine the first products of carbon fixation in A. micrometra After a 15 s incorporation period, the first product of photosynthetic carbon fixation was 3-phosphoglycerate even though this alga had a PEPC activity that was three times higher than that of RuBisCO. After 30 s, over 50% of the recovered radioactivity was still in this compound. Stable carbon isotope analyses of a mixture of the four pennate diatoms also suggest the predominant carbon fixation pathway in these benthic diatoms was similar to C3 plants.     

THE COMPOSITION AND PHYLOGENETIC SIGNIFICANCE OF THE MOUGEOTIA (CHAROPHYCEAE) CELL WALL1

The two-layered, fibrillar cell wall of Mougeotia C. Agardh sp. consisted of 63.6% non-cellulosic carbohydrates and 13.4% cellulose. The orientation of cellulose microfibrils in the native cell wall agrees with the multinet growth hypothesis, which has been employed to explain the shift in microfibril orientation from transverse (inner wall) toward axial (outer wall). Monosaccharide analysis of isolated cell walls revealed the presence of ten sugars with glucose, xylose and galactose most abundant. Methylation analysis of the acid-modified, 1 N NaOH insoluble residue fraction showed that it was composed almost exclusively of 4-linked glucose, confirming the presence of cellulose. The major hemicellulosic carbohydrate was semi-purified by DEAE Sephacel (Cl^?) anion-exchange chromatography of the hot 1 N NaOH soluble fraction. This hemicellulose was a xylan consisting of a 4-xylosyl backbone and 2,4-xylosyl branch points. The major hot water soluble neutral polysaccharide was identified as a 3-linked galactan. Mougeotia cell wall composition is similar to that of (Charophyceae) and has homologies with vascular plant cell walls. Our observations support transtructural evidence which suggests that members of the Charophyceae represent the phylogenetic line that gave rise to vascular plants. Therefore, the primary cell walls of vascular plants many have evolved directly from structures typical of the filamentous green algal cell walls found in the Charophyceae.     

THE DISTRIBUTION OF LIFE-HISTORY VARIATION IN THE DAPHNIA PULEX COMPLEX

In the midwestern United States the Daphnia pulex complex consists of a mosaic of sexual and asexual populations, providing a useful model system for studying the evolutionary forces underlying the maintenance of sex. One asexual and two sexual populations were surveyed for genetic variation for isozymes, mitochondrial DNA, and life-history characters. While the sexual populations exhibited substantial levels of genetic variance for fitness characters, no variation was detected in the asexual population at any level. However, a parallel survey among asexual clones derived from other ponds revealed large amounts of quantitative variation among clones, even among those with the same molecular profile. As a group, the asexuals are more variable for life histories than are the sexual populations. The molecular data indicate a relatively recent origin for the extant asexual D. pulex. The polyphyletic origin of these clones, combined with their microevolutionary potential, provides an explanation for their broad geographic distribution. The distribution of sex in the complex cannot be explained with the standard models that assume an invariant asexual population in reproductive isolation from the parental species. Although the frequency of asexuality may be driven by the spread of a sex-limited meiosis suppressor through sexual populations, the complete displacement of sexuality may be prevented by ecological distinctions between the two classes of individuals. On average, the asexuals are larger but produce smaller clutches than the sexuals.