Multilocus sequence typing for characterization of Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Four housekeeping genes with allelic polymorphisms were identified and used to develop an expanded multilocus sequence typing (MLST) scheme. The new seven-locus technique shows S. pseudintermedius to have greater genetic diversity than previous methods and discriminates more isolates based upon host origin.

The role of mecA and blaZ regulatory elements in mecA expression by regional clones of methicillin-resistant Staphylococcus pseudintermedius.

Two major regional clones of methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been identified in Europe and North America. They are designated multilocus sequence types (ST) 71 and 68 and contain staphylococcal chromosome cassette (SCCmec) types II-III and V(T), respectively. One notable difference between the two clones is a deletion in the mecI/mecR1 regulatory apparatus of ST 68 SCCmec V(T). This deletion in analogous methicillin-resistant Staphylococcus aureus (MRSA) results in more responsive and greater expression of the mecA encoded penicillin-binding protein 2a, and is associated with SCCmec types occurring in community-acquired MRSA lineages. The aim of this study was to characterize mec and bla regulatory apparatuses in MRSP and determine their effects on expression of mecA. Seventeen S. pseudintermedius isolates representing nine methicillin-resistant ST lineages were screened for the presence of the repressors blaI and mecI and sensors blaR1 and mecR1. The bla and mec operons for each isolate were sequenced and compared for homology between the repressor open-reading frames (ORF), sensor ORFs, and mecA promoter regions. A real-time reverse transcriptase PCR expression assay was developed, validated and applied to nine isolates determining the effect of oxacillin induction on mecA transcription. Significant differences were found in mecA expression between isolates with a full regulatory complement (mecI/mecR1 and blaI/blaR1) and those with truncated and/or absent regulatory elements. Isolates representative of European and North American MRSP ST regional clones have dissimilar mecA responses to oxacillin.

Identification of a predominant multilocus sequence type, pulsed-field gel electrophoresis cluster, and novel staphylococcal chromosomal cassette in clinical isolates of mecA-containing, methicillin-resistant Staphylococcus pseudintermedius.

Methicillin resistance encoded by the mecA gene is increasingly observed in Staphylococcus pseudintermedius. Little is known about the population genetics of veterinary staphylococci bearing methicillin resistance. The aim of this study was to determine the relatedness of resistant bacteria and to compare them with methicillin-susceptible isolates. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) fragment profiling were performed on methicillin-resistant S. pseudintermedius (MRSP) and methicillin-susceptible S. pseudintermedius (MSSP) isolates obtained from canine samples submitted to the veterinary teaching hospital bacteriology service between 2006 and 2008. Multilocus sequence typing detected 20 different sequence types, 16 of which were not previously described. Methicillin-resistant isolates were predominantly ST 68, possessed the Staphylococcus aureus-associated staphylococcal chromosomal cassette mec (SCCmec) type V(T) and fell within the largest PFGE cluster; whereas methicillin-susceptible strains were more genetically diverse. This suggests that most methicillin resistance within the population of isolates tested originated from a single source which has persisted and expanded for several years.

Frequent requirement of hedgehog signaling in non-small cell lung carcinoma.

Although it had previously been suggested that the hedgehog (HH) pathway might be activated in some lung tumors, the dependence of non-small cell lung carcinomas (NSCLC) for HH activity had not been comprehensively studied. During a screen of a panel of 60 human tumor cell lines with an HH antagonist, we observed that the proliferation of a subset of NSCLC cell lines was inhibited. These NSCLC cell lines express HH, as well as key HH target genes, consistent with them being activated through an autocrine mechanism. Interestingly, we also identified a number of NSCLC cell lines that express high levels of the downstream transcription factor GLI1 and harbor enhanced levels of HH activity, but appear insensitive to known HH antagonists. We hypothesized that the high levels of GLI1 in these cells would function downstream of the HH antagonist target, allowing them to bypass the antagonist-mediated block in proliferation. Consistent with this hypothesis, when the levels of GLI1 are knocked down in such cells, they become sensitive to these inhibitors. We go on to show that a large percentage of primary NSCLC samples express GLI1, consistent with constitutive activation of the HH pathway in these samples. Taken together, these results establish the involvement of the HH signaling pathway in a subset of NSCLCs.

Phylogenetic analysis of tribe Salsoleae (Chenopodiaceae) based on ribosomal ITS sequences: implications for the evolution of photosynthesis types.

Diversity in photosynthetic pathways in the angiosperm family Chenopodiaceae is expressed in both biochemical and anatomical characters. To understand the evolution of photosynthetic diversity, we reconstructed the phylogeny of representative species of tribe Salsoleae of subfamily Salsoloideae, a group that exhibits in microcosm the patterns of photosynthetic variation present in the family as a whole, and examined the distribution of photosynthetic characters on the resulting phylogenetic tree. Phylogenetic relationships were inferred from parsimony analysis of nucleotide sequences of the internal transcribed spacer regions (ITS) of the 18S-26S nuclear ribosomal DNA of 34 species of Salsola and related genera (Halothamnus, Climacoptera, Girgensohnia, Halocharis, and Haloxylon) and representative outgroups from tribes Camphorosmeae (Camphorosma lessingii, Kochia prostrata, and K. scoparia) and Atripliceae (Atriplex spongiosa). A highly resolved strict consensus tree largely agrees with photosynthetic type and anatomy of leaves and cotyledons. The sequence data provide strong support for the origin and evolution of two main lineages of plants in tribe Salsoleae, with NAD-ME and NADP-ME C(4) photosynthesis, respectively. These groups have different C(4) photosynthetic types in leaves and different structural and photosynthetic characteristics in cotyledons. Phylogenetic relationships inferred from ITS sequences generally agree with classifications based on morphological data, but deviations from the existing taxonomy were also observed. The NAD-ME C(4) lineage contains species classified in sections Caroxylon, Malpigipila, Cardiandra, Belanthera, and Coccosalsola, and the NADP-ME lineage comprises species from sections Coccosalsola and Salsola. Reconstruction of photosynthetic characters on the ITS phylogeny indicates separate NAD-ME and NADP-ME lineages and suggests two reversions to C(3) photosynthesis. Reconstruction of geographic distributions suggests Salsoleae originated and diversified in central Asia and subsequently dispersed to Africa, Europe, and Mongolia. Inferred patterns and processes of photosynthetic evolution in Salsoleae should further our understanding of biochemical and anatomical evolution in Chenopodiaceae as a whole.

C4 plants in the vegetation of Mongolia: their natural occurrence and geographical distribution in relation to climate.

The natural geographical occurrence, carbon assimilation, and structural and biochemical diversity of species with C4 photosynthesis in the vegetation of Mongolia was studied. The Mongolian flora was screened for C4 plants by using 13C/12C isotope fractionation, determining the early products of 14CO2 fixation, microscopy of leaf mesophyll cell anatomy, and from reported literature data. Eighty C4 species were found among eight families: Amaranthaceae, Chenopodiaceae, Euphorbiaceae, Molluginaceae, Poaceae, Polygonaceae, Portulacaceae and Zygophyllaceae. Most of the C4 species were in three families: Chenopodiceae (41 species), Poaceae (25 species) and Polygonaceae, genus Calligonum (6 species). Some new C4 species in Chenopodiaceae, Poaceae and Polygonaceae were detected. C4 Chenopodiaceae species make up 45% of the total chenopods and are very important ecologically in saline areas and in cold arid deserts. C4 grasses make up about 10% of the total Poaceae species and these species naturally concentrate in steppe zones. Naturalized grasses with Kranz anatomy,of genera such as Setaria, Echinochloa, Eragrostis, Panicum and Chloris, were found in almost all the botanical-geographical regions of Mongolia, where they commonly occur in annually disturbed areas and desert oases. We analyzed the relationships between the occurrence of C4 plants in 16 natural botanical-geographical regions of Mongolia and their major climatic influences. The proportion of C4 species increases with decreasing geographical latitude and along the north-to-south temperature gradient; however grasses and chenopods differ in their responses to climate. The abundance of Chenopodiaceae species was closely correlated with aridity, but the distribution of the C4 grasses was more dependent on temperature. Also, we found a unique distribution of different C4 Chenopodiaceae structural and biochemical subtypes along the aridity gradient. NADP-malic enzyme (NADP-ME) tree-like species with a salsoloid type of Kranz anatomy, such as Haloxylon ammodendron and Iljinia regelii, plus shrubby Salsola and Anabasis species, were the plants most resistant to ecological stress and conditions in highly arid Gobian deserts with less than 100 mm of annual precipitation. Most of the annual C4 chenopod species were halophytes, succulent, and occurred in saline and arid environments in steppe and desert regions. The relative abundance of C3 succulent chenopod species also increased along the aridity gradient. Native C4 grasses were mainly annual and perennial species from the Cynodonteae tribe with NAD-ME and PEP-carboxykinase (PEP-CK) photosynthetic types. They occurred across much of Mongolia, but were most common in steppe zones where they are often dominant in grazing ecosystems.

Occurrence of C(3) and C(4) photosynthesis in cotyledons and leaves of Salsola species (Chenopodiaceae).

Most species of the genus Salsola (Chenopodiaceae) that have been examined exhibit C(4) photosynthesis in leaves. Four Salsola species from Central Asia were investigated in this study to determine the structural and functional relationships in photosynthesis of cotyledons compared to leaves, using anatomical (Kranz versus non-Kranz anatomy, chloroplast ultrastructure) and biochemical (activities of photosynthetic enzymes of the C(3) and C(4) pathways, (14)C labeling of primary photosynthesis products and (13)C/(12)C carbon isotope fractionation) criteria. The species included S. paulsenii from section Salsola, S. richteri from section Coccosalsola, S. laricina from section Caroxylon, and S. gemmascens from section Malpigipila. The results show that all four species have a C(4) type of photosynthesis in leaves with a Salsoloid type Kranz anatomy, whereas both C(3) and C(4) types of photosynthesis were found in cotyledons. S. paulsenii and S. richteri have NADP- (NADP-ME) C(4) type biochemistry with Salsoloid Kranz anatomy in both leaves and cotyledons. In S. laricina, both cotyledons and leaves have NAD-malic enzyme (NAD-ME) C(4) type photosynthesis; however, while the leaves have Salsoloid type Kranz anatomy, cotyledons have Atriplicoid type Kranz anatomy. In S. gemmascens, cotyledons exhibit C(3) type photosynthesis, while leaves perform NAD-ME type photosynthesis. Since the four species studied belong to different Salsola sections, this suggests that differences in photosynthetic types of leaves and cotyledons may be used as a basis or studies of the origin and evolution of C(4) photosynthesis in the family Chenopodiaceae.

Long- and short-term flooding effects on survival and sink-source relationships of swamp-adapted tree species.

About 95% of swamp tupelo (Nyssa sylvatica var. biflora (Walt.) Sarg.) and sweetgum (Liquidambar styraciflua L.) seedlings survived continuous root flooding for more than two years, whereas none of the swamp chestnut oak (Quercus michauxii Nutt.) and cherrybark oak (Q. falcata var. pagodifolia Ell.) seedlings survived one year of flooding. Death of oak seedlings occurred in phases associated with periods of major vegetative growth, e.g., after bud burst in spring, after summer stem elongation, and during the winter deciduous stage, suggesting that stored reserves and sources were inadequate to maintain the seedlings when vegetative sinks were forming. Additional evidence that flooding induced a source deficiency in oak was that leaves of flooded oak were 65 to 75% smaller than leaves of nonflooded oak. Flooded swamp tupelo seedlings had a normal leaf size and patchy stomatal opening compared with nonflooded seedlings. Flooding caused increases in alcohol dehydrogenase (ADH) specific activity in taproot cambial tissues and increases in starch concentrations of swamp tupelo seedlings that were reversed when seedlings were removed from flooding. Flooding had little effect on soluble sugar concentrations in swamp tupelo or sweetgum. In the long-term flood-dry-flood treatment, in which all species had survivors, upper canopy leaf photosynthetic rates were higher in all species during the dry period than in nonflooded controls, whereas their starch and soluble sugars concentrations were similar to those of nonflooded controls. Based on seedling survival and the sink-source relationships, the order of flood tolerance was: swamp tupelo > sweetgum > swamp chestnut oak > cherrybark oak.

An integration of photosynthetic traits and mechanisms that can increase crop photosynthesis and grain production.

The hypothesis we propose is that during photosynthesis the balance between potentially detrimental and beneficial photochemically induced events can be tipped beneficially toward increased photosynthesis and toward increased crop yield. To test this hypothesis a procedure has been devised with the rice plant, Oryza sativa, that has resulted in increasing both canopy photosynthesis and rice grain yield. Two elite rice varieties selected independently in the contrasting environments of either South China or Texas, each with distinct photosynthetic traits, were crossed to produce a hybrid with an increased canopy photosynthesis and grain yield that is regularly 20 to 22% higher than the mid-yields of the parents. The photosynthetic and mechanisms which may contribute to these beneficial results in the hybrid rice are: a reduction of the midday depression of photosynthesis; a rapid development of the canopy for photosynthetic light interception and an increased canopy photosynthesis; increased amounts of carotenoids for the xanthophyll cycle; an increased protection against free radicals induced by paraquat treatment; a 6 to 12 day shorter plant reproductive life cycle; and a 8 to 10 day increase in the longevity of the flag leaf over the parents. While the hybrid rice has successfully integrated these and likely other unknown characteristics to increase both crop photosynthesis and grain yield, we propose that understanding the underlying beneficial photosynthetic mechanisms supporting these crop plant traits is worthy of thorough investigation and application in crop production.

Leaf cavity CO2 concentrations and CO2 exchange in onion, Allium cepa L.

Onion (Allium cepa L.) plants were examined to determine the photosynthetic role of CO2 that accumulates within their leaf cavities. Leaf cavity CO2 concentrations ranged from 2250 µL L(-1) near the leaf base to below atmospheric (<350 µL L(-1)) near the leaf tip at midday. There was a daily fluctuation in the leaf cavity CO2 concentrations with minimum values near midday and maximum values at night. Conductance to CO2 from the leaf cavity ranged from 24 to 202 µmol m(-2) s(-1) and was even lower for membranes of bulb scales. The capacity for onion leaves to recycle leaf cavity CO2 was poor, only 0.2 to 2.2% of leaf photosynthesis based either on measured CO2 concentrations and conductance values or as measured directly by (14)CO2 labeling experiments. The photosynthetic responses to CO2 and O2 were measured to determine whether onion leaves exhibited a typical C3-type response. A linear increase in CO2 uptake was observed in intact leaves up to 315 µL L(-1) of external CO2 and, at this external CO2 concentration, uptake was inhibited 35.4±0.9% by 210 mL L(-1) O2 compared to 20 mL L(-1) O2. Scanning electron micrographs of the leaf cavity wall revealed degenerated tissue covered by a membrane. Onion leaf cavity membranes apparently are highly impermeable to CO2 and greatly restrict the refixation of leaf cavity CO2 by photosynthetic tissue.

Vascular cambial sucrose metabolism and growth in loblolly pine (Pinus taeda L.) in relation to transplanting stress.

Sucrose synthase (SS) was the dominant enzyme of sucrose metabolism in both stem and root vascular cambial zone tissues of nursery-grown loblolly pine (Pinus taeda L.) seedlings. Acid invertase (AI) and neutral invertase (NT) activities were generally less than 10% of the SS activity in both tissues. In both cambial tissues, seasonal patterns of enzyme activity were observed for SS but not for AI or NI. The seasonal patterns of SS activity in stem and root cambia paralleled the periodic growth of stems and roots. Stems had high SS activity and growth during summer and early fall. Roots had substantial SS activity and growth during summer and fall, but SS activity and growth were even higher in winter. When seedlings were transplanted, about eight months elapsed before stem and root cambia resumed rates of growth and sucrose metabolism similar to those in control nontransplanted seedlings. Two months after transplanting, root SS was at its lowest, whereas AI activity in transplants was 50% higher than in control nontransplanted seedlings. In stems, SS activity decreased in response to transplanting, whereas AI and NI activities did not change appreciably. In loblolly pine tissues, SS was specific for uridylates, whereas the nucleotide triphosphate-dependent phosphofructokinase (NTP-PFK) had similar activity with either UTP or ATP. Except in winter, the NTP-PFK was less active than the pyrophosphate-dependent phosphofructokinase (PPi-PFK) during all seasons. The PPi-dependent PFK activity in nontransplanted seedlings followed similar seasonal and spatial patterns to those of SS activity. In actively growing tissues, such as stem cambial tissues in summer and root cambial tissues in winter, the measured total PFK to SS ratio ranged between 1.5/1 and 3/1. In contrast, in less actively growing tissues or transplanted seedlings, a greater decrease occurred in SS than in PFK activity, hence the ratio rose to as high as 12/1. It was concluded that: (1) SS was the dominant enzyme for sucrose metabolism in root and stem cambial tissues of loblolly pine seedlings; (2) both SS and PPi-PFK in the cambial tissues can be used as biochemical indicators of growth sink strength in stems and roots; and (3) both enzymes can be used as indicators of seedling stress caused by events such as transplanting and winter freezing.

Novel characteristics of cassava, Manihot esculenta Crantz, a reputed C3-C 4 intermediate photosynthesis species.

The cassava plant, Manihot esculenta, grows exceptionally well in low fertility and drought prone environments, but the mechanisms that allow this growth are unknown. Earlier, and sometimes contradictory, work speculated about the presence of a C4-type photosynthesis in cassava leaves. In the present work we found no evidence for a C4 metabolism in mature attached cassava leaves as indicated i) by the low, 2 to 8%, incorporation of (14)CO2 into C4 organic acids in short time periods, 10 s, and the lack of (14)C transfer from C4 acids to other compounds in (12)CO2, ii) by the lack of C4 enzyme activity changes during leaf development and the inability to detect C4 acid decarboxylases, and iii) by leaf CO2 compensation values between 49 and 65 µl of CO2 1(-1) and by other infrared gas exchange photosynthetic measurements. It is concluded that the leaf biochemistry of cassava follows the C3 pathway of photosynthesis with no indication of a C3-C4 mechanism.However, cassava leaves exhibit several novel characteristics. Attached leaves have the ability to effectively partition carbon into sucrose with nearly 45% of the label in sucrose in about one min of (14)CO2 photosynthesis, contrasting with 34% in soybean (C3) and 25% in pigweed (C4). Cassava leaves displayed a strong preference for the synthesis of sucrose versus starch. Field grown cassava leaves exhibited high rates of photosynthesis and curvilinear responses to increasing sunlight irradiances with a tendency to saturate only at high irradiances, above 1500 µmol m(-2) s(-1). Morphologically, the cassava leaf has papillose epidermal cells on its lower mesophyll surface that form 'fence-like' arrangements encircling guard cells. It is proposed that the active synthesis of sugars has osmotic functions in the cassava plant and that the papillose epidermal cells function to maintain a healthy leaf water status in various environments.

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : II. Inhibition of Apparent Photosynthesis by a Phosphoenolpyruvate Carboxylase Inhibitor.

Hybrids have been made between species of Flaveria exhibiting varying levels of C(4) photosynthesis. The degree of C(4) photosynthesis expressed in four interspecific hybrids (Flaveria trinervia [C(4)] x F. linearis [C(3)-C(4)], F. brownii [C(4)-like] x F. linearis, and two three-species hybrids from F. trinervia x [F. brownii x F. linearis]) was estimated by inhibiting phosphoenolpyruvate carboxylase in vivo with 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP). The inhibitor was fed to detached leaves at a concentration of 4 mm, and apparent photosynthesis was measured at atmospheric levels of CO(2) and at 20 and 210 mL L(-1) of O(2). Photosynthesis at 210 mL L(-1) of O(2) was inhibited 32% by DCDP in F. linearis, by 60% in F. brownii, and by 87% in F. trinervia. Inhibition in the hybrids ranged from 38 to 52%. The inhibition of photosynthesis by 210 mL L(-1) of O(2) was increased when DCDP was used, except in the C(4) species, F. trinervia, in which photosynthesis was insensitive to O(2). Except for F. trinervia, control plants with less O(2) sensitivity (more C(4)-like) exhibited a progressively greater change in O(2) inhibition of photosynthesis when treated with DCDP. This increased O(2) inhibition probably resulted from decreased CO(2) concentrations in bundle sheath cells due to inhibition of phosphoenolpyruvate carboxylase. The inhibition of photosynthesis by DCDP is concluded to underestimate the degree of C(4) photosynthesis in the interspecific hybrids because increased direct assimilation of atmospheric CO(2) by ribulose bisphosphate carboxylase may compensate for inhibition of phosphoenolpyruvate carboxylase.

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : I. CO(2) Assimilation and Metabolism and Activities of Phosphoenolpyruvate Carboxylase and NADP-Malic Enzyme.

The degree of C(4) photosynthesis was assessed in four hybrids among C(4), C(4)-like, and C(3)-C(4) species in the genus Flaveria using (14)C labeling, CO(2) exchange, (13)C discrimination, and C(4) enzyme activities. The hybrids incorporated from 57 to 88% of the (14)C assimilated in a 10-s exposure into C(4) acids compared with 26% for the C(3)-C(4) species Flaveria linearis, 91% for the C(4) species Flaveria trinervia, and 87% for the C(4)-like Flaveria brownii. Those plants with high percentages of (14)C initially fixed into C(4) acids also metabolized the C(4) acids quickly, and the percentage of (14)C in 3-phosphoglyceric acid plus sugar phosphates increased for at least a 30-s exposure to (12)CO(2). This indicated a high degree of coordination between the carbon accumulation and reduction phases of the C(4) and C(3) cycles. Synthesis and metabolism of C(4) acids by the species and their hybrids were highly and linearly correlated with discrimination against (13)C. The relationship of (13)C discrimination or (14)C metabolism to O(2) inhibition of photosynthesis was curvilinear, changing more rapidly at C(4)-like values of (14)C metabolism and (13)C discrimination. Incorporation of initial (14)C into C(4) acids showed a biphasic increase with increased activities of phosphoenolpyruvate carboxylase and NADP-malic enzyme (steep at low activities), but turnover of C(4) acids was linearly related to NADP-malic enzyme activity. Several other traits were closely related to the in vitro activity of NADP-malic enzyme but not phosphoenolpyruvate carboxylase. The data indicate that the hybrids have variable degrees of C(4) photosynthesis but that the carbon accumulation and reduction portions of the C(4) and C(3) cycles are well coordinated.

Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls.

Alkaline invertase from sprouting soybean (Glycine max) hypocotyls was purified to apparent electrophoretic homogeneity by consecutive use of DEAE-cellulose, green 19 dye, and Cibacron blue 3GA dye affinity chromatography. This protocol produced about a 100-fold purification with about a 11% yield. The purified protein had a specific activity of 48 mumol of glucose produced mg-1 protein min-1 (pH 7.0) and showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (58 kDa) and in native PAGE, as indicated by both protein and activity staining. The native enzyme molecular mass was about 240 kDa, suggesting a homotetrameric structure. The purified enzyme exhibited hyperbolic saturation kinetics with a Km (sucrose) near 10 mM and the enzyme did not utilize raffinose, maltose, lactose, or cellibose as a substrate. Impure alkaline invertase preparations, which contained acid invertase activity, on contrast, showed biphasic curves versus sucrose concentration. Combining equal activities of purified alkaline invertase with acid invertase resulted in a biphasic response, but there was a transition to hyperbolic saturation kinetics when the activity ratio, alkaline: acid invertase, was increased above unity. Alkaline invertase activity was inhibited by HgCl2, pridoxal phosphate, and Tris with respective Ki values near 2 microM, 5 microM, and 4 mM. Glycoprotein staining (periodic acid-Schiff method) was negative and alkaline invertase did not bind to two immobilized lectins, concanavalin A and wheat germ agglutinin; hence, the enzyme apparently is not a glycoprotein. The purified alkaline invertase, and a purified soybean acid invertase, was used to raise rabbit polyclonal antibodies. The alkaline invertase antibody preparation was specific for alkaline invertase and cross-reacted with alkaline invertases from other plants. Neither purified soybean alkaline invertases nor the crude enzyme from several plants cross-reacted with the soybean acid invertase antibody.

Sucrose Synthase in Wild Tomato, Lycopersicon chmielewskii, and Tomato Fruit Sink Strength.

Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers. To check for novel characteristics, the wild tomato fruit sucrose synthase was purified, by (NH(4))(2)SO(4) fraction and chromatography with DE-32, Sephadex G-200, and PBA-60, to one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following characteristics were obtained: native protein relative molecular weight 380,000; subunit relative molecular weight 89,000; K(m) values with: sucrose 53 millimolar, UDP 18.9 micromolar, UDP-glucose 88 micromolar, fructose 8.4 millimolar; pH optima between 6.2 to 7.3 for sucrose breakdown and 7 to 9 for synthesis; and temperature optima near 50 degrees C. The enzyme exhibited a high affinity and a preference for uridylates. The enzyme showed more sensitivity to divalent cations in the synthesis of sucrose than in its breakdown. Sink strength in tomato fruits also was investigated in regard to sucrose breakdown enzyme activities versus fruit weight gain. Sucrose synthase activity was consistently related to increases in fruit weight (sink strength) in both wild and commercial tomatoes. Acid and neutral invertases were not, because the published invertase activity values were too variable for quantitative analyses regarding the roles of invertases in tomato fruit development. In rapidly growing fruits of both wild and commercially developed tomato plants, the activity of sucrose synthase per growing fruit, i.e. sucrose synthase peak activity X fruit size, was linearly related to final fruit size; and the activity exceeded fruit growth and carbon import rates by at least 10-fold. In mature, nongrowing fruits, sucrose synthase activities approached nil values. Therefore, sucrose synthase can serve as an indicator of sink strength in growing tomato fruits.

Assessing the degree of c(4) photosynthesis in c(3)-c(4) species using an inhibitor of phosphoenolpyruvate carboxylase.

An analog of phosphoenolpyruvate, 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP), was used to inhibit phosphoenolpyruvate carboxylase and, therefore, assess the contribution of the C(4) pathway to photosynthesis in detached leaves of several C(3)-C(4) intermediate species. There was no effect of 4 millimolar DCDP on apparent photosynthesis or on inhibition of apparent photosynthesis by 210 milliliters per liter of O(2) for the C(3)-C(4) species Panicum milioides, Moricandia arvensis, and Neurachne minor or the C(3) species Flaveria pringlei. However, when leaves of Flaveria linearis (C(3)-C(4)), Flaveria brownii (C(4)-like), and Flaveria trinervia (C(4)) were fed 4 millimolar DCDP, photosynthesis was reduced 32, 60, and 90%, respectively. Photosynthetic inhibition by 210 milliliter per liter of O(2) was also significantly increased in 4 millimolar DCDP-fed leaves of F. linearis and F. brownii but not in F. trinervia when compared with control values. These results with DCDP clearly demarcate C(3)-C(4) species into species including Panicum, Moricandia, and Neurachne whose reduced photorespiration occurs without any C(4) photosynthetic involvement and species of Flaveria in which C(4) photosynthesis contributes to CO(2) assimilation.

Changes in beta-1,3-Glucan Synthase Activity in Developing Lima Bean Plants.

A plasma membrane-enriched fraction was isolated from various tissues of developing lima bean seedlings, Phaseolus lunatus var Cangreen, to study beta-1,3-glucan synthase activity changes. All tissues contained an active beta-glucan synthase, including the cotyledons that will be senescent in mature lima bean plants. Young primary leaves exhibited a very active beta-glucan synthase; but this activity dropped markedly, about fivefold, as the leaves gained weight and became photosynthetic. Some tissues, such as the hypocotyl and young stem, exhibited an increase in beta-glucan synthase activity as the tissues were growing and a decrease as the growth rate slowed. Roots exhibited a high activity early in development that only decreased slightly, about 30%, as root growth increased. Surprisingly the senescent cotyledons contained an activity equivalent to some other tissues that was maintained over our measurement time of 21 days. Perhaps this callose synthesis activity is related to translocation processes as the cotyledons transfer their reserves to the growing seedling. We concluded that beta-glucan synthase was not a good indicator of sink strength in these lima bean tissues. The plasma membrane fractions also were tested for other enzymes that might be present because an electron microscope study revealed a low contamination by other types of membranes. The membrane fractions had low but detectable activities of sucrose synthase, UDPglucose pyrophosphorylase, UDPase, alkaline invertase, and a general phosphatase; but these enzymes exhibited no consistent pattern(s) of activity change with plant development.

Banana ripening: implications of changes in glycolytic intermediate concentrations, glycolytic and gluconeogenic carbon flux, and fructose 2,6-bisphosphate concentration.

In ripening banana (Musa sp. [AAA group, Cavendish subgroup] cv Valery) fruit, the concentration of glycolytic intermediates increased in response to the rapid conversion of starch to sugars and CO(2). Glucose 6-phosphate (G-6-P), fructose 6-phosphate (Fru 6-P), and pyruvate (Pyr) levels changed in synchrony, increasing to a maximum one day past the peak in ethylene synthesis and declining rapidly thereafter. Fructose 1,6-bisphosphate (Fru 1,6-P(2)) and phosphoenolpyruvate (PEP) levels underwent changes dissimilar to those of G 6-P, Fru 6-P, and Pyr, indicating that carbon was regulated at the PEP/Pyr and Fru 6-P/Fru 1,6-P(2) interconversion sites. During the climacteric respiratory rise, gluconeogenic carbon flux increased 50- to 100-fold while glycolytic carbon flux increased only 4- to 5-fold. After the climacteric peak in CO(2) production, gluconeogenic carbon flux dropped dramatically while glycolytic carbon flux remained elevated. The steady-state fructose 2,6-bisphosphate (Fru 2,6-P(2)) concentration decreased to (1/2) that of preclimacteric fruit during the period coinciding with the rapid increase in gluconeogenesis. Fru 2,6-P(2) concentration increased thereafter as glycolytic carbon flux increased relative to gluconeogenic carbon flux. It appears likely that the initial increase in respiration in ripening banana fruit is due to the rapid influx of carbon into the cytosol as starch is degraded. As starch reserves are depleted and the levels of intermediates decline, the continued enhancement of respiration may, in part, be maintained by an increased steady-state Fru 2,6-P(2) concentration acting to promote glycolytic carbon flux at the step responsible for the interconversion of Fru 6-P and Fru 1,6-P(2).

Characterization of Sucrolysis via the Uridine Diphosphate and Pyrophosphate-Dependent Sucrose Synthase Pathway.

The breakdown of sucrose to feed both hexoses into glycolytic carbon flow can occur by the sucrose synthase pathway. This uridine diphosphate (UDP) and pyrophosphate (PPi)-dependent pathway was biochemically characterized using soluble extracts from several plants. The sucrolysis process required the simultaneous presence of sucrose, UDP, and PPi with their respective K(m) values being about 40 millimolar, 23 micromolar, and 29 micromolar. UDP was the only active nucleotide diphosphate. Slightly alkaline pH optima were observed for sucrose breakdown either to glucose 1-phosphate or to triose phosphate. Sucrolysis incrased with increasing temperature to near 50 degrees C and then a sharp drop occurred between 55 and 60 degrees C. The breakdown of sucrose to triose-P was activated by fructose 2,6-P(2) which had a K(m) value near 0.2 micromolar. The cytoplasmic phosphofructokinase and fructokinase in plants were fairly nonselective for nucleotide triphosphates (NTP) but glucokinase definitely favored ATP. A predicted stoichiometric relationship of unity for UDP and PPi was measured when one also measured competing UDPase and pyrophosphatase activity. The cycling of uridylates, UDP to UTP to UDP, was demonstrated both with phosphofructokinase and with fructokinase. Enzyme activity measurements indicated that the sucrose synthase pathway has a major role in plant sucrose sink tissues. In the cytoplasmic sucrose synthase breakdown pathway, a role for the PPi-phosphofructokinase was to produce PPi while a role for the NTP-phosphofructokinase and for the fructokinase was to produce UDP.