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Biomedical subjects

T D Sharkey

Publications and source records attributed to T D Sharkey.

At least 19 recordsLinked to original sources
PubMed2004

Engineering plants for elevated CO(2): a relationship between starch degradation and sugar sensing.

In the future, plants will have additional CO(2) for photosynthesis. However, plants do not take maximal advantage of this additional CO(2) and it has been hypothesized that end product synthesis limitations and sugar sensing mechanisms are important in regulating plant responses to increasing CO(2). Attempts to increase end product synthesis capacity by engineering increased sucrose-phosphate synthase activity have been generally, but not universally, successful. It was found that plants benefited from a two- to three-fold increase in SPS activity but a 10-fold increase did not increase yield. Despite the success in increasing yield, increasing SPS did not increase photosynthesis. However, carbon export from chloroplasts was increased during the day and reduced at night (when starch provides carbon for sucrose synthesis. We develop here a hypothesis that starch degradation is closely sensed by hexokinase because a newly discovered pathway required for starch to sucrose conversion that involves maltose is one of few metabolic pathways that requires hexokinase activity.

Biological Transport↗
PubMed2004

Diffusive and metabolic limitations to photosynthesis under drought and salinity in C(3) plants.

Drought and salinity are two widespread environmental conditions leading to low water availability for plants. Low water availability is considered the main environmental factor limiting photosynthesis and, consequently, plant growth and yield worldwide. There has been a long-standing controversy as to whether drought and salt stresses mainly limit photosynthesis through diffusive resistances or by metabolic impairment. Reviewing in vitro and in vivo measurements, it is concluded that salt and drought stress predominantly affect diffusion of CO(2) in the leaves through a decrease of stomatal and mesophyll conductances, but not the biochemical capacity to assimilate CO(2), at mild to rather severe stress levels. The general failure of metabolism observed at more severe stress suggests the occurrence of secondary oxidative stresses, particularly under high-light conditions. Estimates of photosynthetic limitations based on the photosynthetic response to intercellular CO(2) may lead to artefactual conclusions, even if patchy stomatal closure and the relative increase of cuticular conductance are taken into account, as decreasing mesophyll conductance can cause the CO(2) concentration in chloroplasts of stressed leaves to be considerably lower than the intercellular CO(2) concentration. Measurements based on the photosynthetic response to chloroplast CO(2) often confirm that the photosynthetic capacity is preserved but photosynthesis is limited by diffusive resistances in drought and salt-stressed leaves.

Carbon Dioxide↗
PubMed2001

Promoter strength and tissue specificity effects on growth of tomato plants transformed with maize sucrose-phosphate synthase.

When sucrose-phosphate synthase (SPS; EC 2.4.1.14) is expressed in tomato (Lycopersicon esculentum Mill.) from a ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit (rbcS) promoter, yields are often unchanged but when SPS is expressed from a Cauliflower Mosaic Virus 35S promoter, yield is enhanced up to 80%. Two explanations for this phenomenon are (i) that expression of SPS in tissues other than leaves accounts for the increased yield or (ii) that the lower level of expression directed by the 35S promoter is more beneficial than the high level of expression directed by the rbcS promoter. To test the first hypothesis, we conducted a reciprocal graft experiment, which showed that root SPS activity did not substantially affect growth. To test the second hypothesis, we conducted a field trial using a backcrossed, segregating, population of SPS-transformed plants derived from 35S and rbcS lines. The optimal dose of SPS activity for growth was approximately twice that of the wild type regardless of which promoter was used. The effect of SPS on growth was the result of a shift in partitioning of carbon among starch, sucrose, and ionic compounds (primarily amino acids), rather than of an increase in net photosynthesis. Excessive SPS activity resulted in a decreased rate of amino acid synthesis, which could explain the non-linear response of plant growth to the level of SPS expression.

Amino Acids↗
PubMed2001

Increased heat sensitivity of photosynthesis in tobacco plants with reduced Rubisco activase.

High temperature inhibits photosynthesis by several mechanisms including deactivation of Rubisco. The inhibition of photosynthesis by high temperature and its relationship to Rubisco deactivation was studied using tobacco (Nicotiana tabaccum L. cv W38) transformed with a Rubisco activase gene inserted in the antisense orientation and untransformed controls. High temperature (42 degrees C) reduced photosynthesis in both lines of plants. However, photosynthesis recovered nearly completely in wild-type plants and very little in plants lacking Rubisco activase. The F(0)' level of chlorophyll fluorescence decreased and q(N) increased in the control plants during heating. In the antisense plants, q(N) was always high and F(0)' increased slightly during heat stress. NADP-malate dehydrogenase activation was unaffected by heat stress in control plants but was increased in the transgenic plants, consistent with a high redox status in the chloroplast. In wild-type plants, the inhibition of photosynthesis could be explained by a reversible decarbamylation of Rubisco and an acceptor-side limitation imposed on photosynthetic electron transport. However, in the anti-activase plants, carbamylation was low and constant and could not explain how photosynthesis was reduced at high temperature. Because ribulose bisphosphate was saturating at high temperature, the reduction in photosynthesis must have been caused by some impairment of Rubisco function not reflected in measurements of activation state or carbamylation status. This in vivo Rubisco impairment was not relieved upon return to lower temperature. We speculate that the reversible decarbamylation of Rubisco at moderately high temperature may be a protective mechanism by which the plant avoids more serious effects on Rubisco and the rest of the photosynthetic apparatus.

Journal Article↗
PubMed2001

Isoprene increases thermotolerance of fosmidomycin-fed leaves.

Isoprene is synthesized and emitted in large amounts by a number of plant species, especially oak (Quercus sp.) and aspen (Populus sp.) trees. It has been suggested that isoprene improves thermotolerance by helping photosynthesis cope with high temperature. However, the evidence for the thermotolerance hypothesis is indirect and one of three methods used to support this hypothesis has recently been called into question. More direct evidence required new methods of controlling endogenous isoprene. An inhibitor of the deoxyxylulose 5-phosphate pathway, the alternative pathway to the mevalonic acid pathway and the pathway by which isoprene is made, is now available. Fosmidomycin eliminates isoprene emission without affecting photosynthesis for several hours after feeding to detached leaves. Photosynthesis of fosmidomycin-fed leaves recovered less following a 2-min high-temperature treatment at 46 degrees C than did photosynthesis of leaves fed water or fosmidomycin-fed leaves in air supplemented with isoprene. Photosynthesis of Phaseolus vulgaris leaves, which do not make isoprene, exhibited increased thermotolerance when isoprene was supplied in the airstream flowing over the leaf. Other short-chain alkenes also improved thermotolerance, whereas alkanes reduced thermotolerance. It is concluded that thermotolerance of photosynthesis is a substantial benefit to plants that make isoprene and that this benefit explains why plants make isoprene. The effect may be a general hydrocarbon effect and related to the double bonds in the isoprene molecule.

Acclimatization↗
PubMed1999

Evolutionary significance of isopreneemission from mosses.

Isoprene emission has been documented and characterized from species in all major groups of vascular plants. We report in our survey that isoprene emission is much more common in mosses and ferns than later divergent land plants but is absent in liverworts and hornworts. The light and temperature responses of isoprene emission from Sphagnum capillifolium (Ehrh.) Hedw. are similar to those of other land plants. Isoprene increases thermotolerance of S. capillifolium to the same extent seen in higher plants as measured by chlorophyll fluorescence. Sphagnum species in a northern Wisconsin bog experienced large temperature fluctuations similar to those reported in tree canopies. Since isoprene has been shown to help plants cope with large, rapid temperature fluctuations, we hypothesize the thermal and correlated dessication stress experienced by early land plants provided the selective pressure for the evolution of light-dependent isoprene emission in the ancestors of modern mosses. As plants radiated into different habitats, this capacity was lost multiple times in favor of other thermal protective mechanisms.

Journal Article↗
PubMed1998

The small, methionine-rich chloroplast heat-shock protein protects photosystem II electron transport during heat stress.

Evidence suggests that the small chloroplast heat-shock protein (Hsp) is involved in plant thermotolerance but its site of action is unknown. Functional disruption of this Hsp using anti-Hsp antibodies or addition of purified Hsp to chloroplasts indicated that (a) this Hsp protects thermolabile photosystem II and, consequently, whole-chain electron transport during heat stress; and (b) this Hsp completely accounted for heat acclimation of electron transport in pre-heat-stressed plants. Therefore, this Hsp is a major adaptation to acute heat stress in plants.

Acclimatization↗
PubMed1996

Different sources of reduced carbon contribute to form three classes of terpenoid emitted by Quercus ilex L. leaves.

Quercus ilex L. leaves emit terpenes but do not have specialized structures for terpene storage. We exploited this unique feature to investigate terpene biosynthesis in intact leaves of Q. ilex. Light induction allowed us to distinguish three classes of terpenes: (i) a rapidly induced class including alpha-pinene; (ii) a more slowly induced class, including cis-beta-ocimene; and (iii) the most slowly induced class, including 3-methyl-3-buten-1-ol. Using 13C, we found that alpha-pinene and cis-beta-ocimene were labeled quickly and almost completely while there was a delay before label appeared in linalool and 3-methyl-3-buten-1-ol. The acetyl group of 3-methyl-3-buten-1-yl acetate was labeled quickly but label was limited to 20% of the moiety. It is suggested that the ocimene class of monoterpenes is made from one or more terpenes of the alpha-pinene class and that both classes are made entirely from reduced carbon pools inside the chloroplasts. Linalool and 3-methyl-3-buten-1-ol are made from a different pool of reduced carbon, possibly in nonphotosynthetic plastids. The acetyl group of the 3-methyl-3-buten-1-yl acetate is derived mostly from carbon that does not participate in photosynthetic reactions. Low humidity and prolonged exposure to light favored ocimenes emission and induced linalool emission. This may indicate conversion between terpene classes.

Journal Article↗
PubMed1996

Isoprene synthesis by plants and animals.

Isoprene is emitted from both plants and animals at significant rates. There is evidence for a specific enzyme, isoprene synthase, that produces isoprene from dimethyl allyl pyrophosphate, one of the intermediates involved in the synthesis of higher-order isoprenoids such as cholesterol, carotenoids and monoterpenes (for example, pine scent). The role of isoprene in animals is unknown, but there is recent evidence that isoprene helps protect plant membranes involved in photosynthesis from thermal damage. Isoprene emission from plants is a natural process that contributes more hydrocarbon to the atmosphere than all of the anthropogenic hydrocarbons.

Animals↗
PubMed1996

Field measurements of isoprene emission from trees in response to temperature and light.

The atmospheric hydrocarbon budget is important for predicting ozone episodes and the effects of pollution mitigation strategies. Isoprene emission from plants is an important part of the atmospheric hydrocarbon budget. We measured isoprene emission capacity at the bottom, middle, and top of the canopies of a white oak (Quercus alba L.) tree and a red oak (Quercus rubra L.) tree growing adjacent to a tower in the Duke University Forest. Leaves at the top of the white oak tree canopy had a three- to fivefold greater capacity for emitting isoprene than leaves at the bottom of the tree canopy. Isoprene emission rate increased with increasing temperature up to about 42 degrees C. We conclude that leaves at the top of the white oak tree canopy had higher isoprene emission rates because they were exposed to more sunlight, reduced water availability, and higher temperature than leaves at the bottom of the canopy. Between 35 and 40 degrees C, white oak photosynthesis and stomatal conductance declined, whereas red oak (Quercus rubra) photosynthesis and stomatal conductance increased over this range. Red oak had lower rates of isoprene emission than white oak, perhaps reflecting the higher stomatal conductance that would keep leaves cool. The concentration of isoprene inside the leaf was estimated with a simplified form of the equation used to estimate CO(2) inside leaves.

Journal Article↗
PubMed1992

Carbon Partitioning in a Flaveria linearis Mutant with Reduced Cytosolic Fructose Bisphosphatase.

Oxygen sensitivity and partitioning of carbon was measured in a mutant line of Flaveria linearis that lacks most of the cytosolic fructose-1,6-bisphosphatase found in wild-type lines. Photosynthesis of leaves of the mutant line was nearly insensitive to O(2), as found before. The mutant plants partitioned 2.5 times less carbon into sucrose than the wild type in a pulse chase experiment, with the extra carbon going mainly to starch but also to amino acids. From 10 to 50 min postlabeling, radioactivity chased out of the amino acid fraction to starch in both lines. In the middle of the light period, starch grains were larger in the mutant than in the wild type and covered 30% of the chloroplast area as seen with an electron microscope. Starch grains were found in both mesophyll and bundle sheath chloroplasts in both lines in these C(3)-C(4) intermediate plants. At the end of the dark period, the starch levels were considerably reduced from what they were in the middle of the light in both lines. The concentration of sucrose was higher in the mutant line despite the lack of cytosolic fructose-1,6-bisphosphatase. The amino acid fraction accounted for about 30% of all label following a 10-min chase period. In the mutant line, most of the label was in the glycine + serine fraction, with 10% in the alanine fraction. In wild-type leaves, 35% of the label in amino acids was in alanine. These results indicate that this mutant survives the reduced cytosolic fructose-1,6-bisphosphatase activity by partitioning more carbon to starch and less to sucrose during the day and remobilizing the excess starch at night. However, these results raise two other questions about this mutant. First, why is the sucrose concentration high in a plant that partitions less carbon to sucrose, and second, why is alanine heavily labeled in the wild-type plants but not in the mutant plants?

Journal Article↗
PubMed1992

Regulation of Photosynthesis in Triazine-Resistant and -Susceptible Brassica napus.

The response of photosynthetic carbon assimilation and chlorophyll fluorescence quenching to changes in intercellular CO(2) partial pressure (C(i)), O(2) partial pressure, and leaf temperature (15-35 degrees C) in triazine-resistant and -susceptible biotypes of Brassica napus were examined to determine the effects of the changes in the resistant biotype on the overall process of photosynthesis in intact leaves. Three categories of photosynthetic regulation were observed. The first category of photosynthetic response, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-limited photosynthesis, was observed at 15, 25, and 35 degrees C leaf temperatures with low C(i). When the carbon assimilation rate was Rubisco-limited, there was little difference between the resistant and susceptible biotypes, and Rubisco activity parameters were similar between the two biotypes. A second category, called feedback-limited photosynthesis, was evident at 15 and 25 degrees C above 300 microbars C(i). The third category, photosynthetic electron transport-limited photosynthesis, was evident at 25 and 35 degrees C at moderate to high CO(2). At low temperature, when the response curves of carbon assimilation to C(i) indicated little or no electron transport limitation, the carbon assimilation rate was similar in the resistant and susceptible biotypes. With increasing temperature, more electron transport-limited carbon assimilation was observed, and a greater difference between resistant and susceptible biotypes was observed. These observations reveal the increasing importance of photosynthetic electron transport in controlling the overall rate of photosynthesis in the resistant biotype as temperature increases. Photochemical quenching of chlorophyll fluorescence (q(P)) in the resistant biotype never exceeded 60%, and triazine resistance effects were more evident when the susceptible biotype had greater than 60% q(P), but not when it had less than 60% q(P).

Journal Article↗
PubMed1992

Theoretical Considerations when Estimating the Mesophyll Conductance to CO(2) Flux by Analysis of the Response of Photosynthesis to CO(2).

The conductance for CO(2) diffusion in the mesophyll of leaves can limit photosynthesis. We have studied two methods for determining the mesophyll conductance to CO(2) diffusion in leaves. We generated an ideal set of photosynthesis rates over a range of partial pressures of CO(2) in the stroma and studied the effect of altering the mesophyll diffusion conductance on the measured response of photosynthesis to intercellular CO(2) partial pressure. We used the ideal data set to test the sensitivity of the two methods to small errors in the parameters used to determine mesophyll conductance. The two methods were also used to determine mesophyll conductance of several leaves using measured rather than ideal data sets. It is concluded that both methods can be used to determine mesophyll conductance and each method has particular strengths. We believe both methods will prove useful in the future.

Journal Article↗
PubMed1992

Estimation of Mesophyll Conductance to CO(2) Flux by Three Different Methods.

The resistance to diffusion of CO(2) from the intercellular airspaces within the leaf through the mesophyll to the sites of carboxylation during photosynthesis was measured using three different techniques. The three techniques include a method based on discrimination against the heavy stable isotope of carbon, (13)C, and two modeling methods. The methods rely upon different assumptions, but the estimates of mesophyll conductance were similar with all three methods. The mesophyll conductance of leaves from a number of species was about 1.4 times the stomatal conductance for CO(2) diffusion determined in unstressed plants at high light. The relatively low CO(2) partial pressure inside chloroplasts of plants with a low mesophyll conductance did not lead to enhanced O(2) sensitivity of photosynthesis because the low conductance caused a significant drop in the chloroplast CO(2) partial pressure upon switching to low O(2). We found no correlation between mesophyll conductance and the ratio of internal leaf area to leaf surface area and only a weak correlation between mesophyll conductance and the proportion of leaf volume occupied by air. Mesophyll conductance was independent of CO(2) and O(2) partial pressure during the measurement, indicating that a true physical parameter, independent of biochemical effects, was being measured. No evidence for CO(2)-accumulating mechanisms was found. Some plants, notably Citrus aurantium and Simmondsia chinensis, had very low conductances that limit the rate of photosynthesis these plants can attain at atmospheric CO(2) level.

Journal Article↗
PubMed1992

Identification and levels of 2'-carboxyarabinitol in leaves.

2'-Carboxyarabinitol 1-phosphate (CA1P) is a naturally occurring inhibitor of ribulose-1,5 bisphosphate carboxylase/oxygenase activity. A chloroplast phosphatase has previously been identified that degrades CA1P in vitro to carboxyarabinitol (CA) plus phosphate, but CA has not yet been detected in plants. Here, we detail procedures to isolate and assay CA from leaves and utilize mass spectrometry to demonstrate for the first time that CA is present in plants. CA was present in leaves of all 13 species examined, including those of C(3), C(4), and Crassulacean acid metabolism photosynthetic subgroups. CA was present both in species with high levels of CA1P (e.g. Phaseolus vulgaris, Lycopersicon esculentum, Beta vulgaris) as well as in species with low levels of CA1P (e.g. Spinacea oleracea, Triticum aestivum). CA levels in the light were sometimes greater than those in the dark. Bean leaves had the most CA of any species tested, with levels in the light approaching 1 micromole per milligram of chlorophyll. In illuminated bean leaves, about 63% of the CA is located outside the chloroplast. CA is one of only a few branched chain sugar acids to be identified from plants.

Journal Article↗
PubMed1991

Fractionation of Carbon Isotopes during Biogenesis of Atmospheric Isoprene.

The stable carbon isotope composition of isoprene emitted from leaves of red oak (Quercus rubra L.) was measured. Isoprene was depleted in (13)C relative to carbon recently fixed by photosynthesis. The difference in isotope composition between recently fixed carbon and emitted isoprene was independent of the isotopic composition of the source CO(2). beta-Carotene, an isoprenoid plant constituent, was depleted in (13)C relative to whole leaf carbon to the same degree as isoprene, but fatty acids were more depleted. Isoprene emitted from leaves fed abscisic acid was much less depleted in (13)C than was isoprene emitted from unstressed leaves. We conclude that isoprene is made from an isoprenoid precursor that is derived from acetyl-CoA made from recent photosynthate. The carbon isotope composition of isoprene in the atmosphere is likely to be slightly more negative (less (13)C) than C(3) plant material but when plants are stressed the isotopic composition could vary.

Journal Article↗
PubMed1990

Low humidity can cause uneven photosynthesis in olive (Olea europea L.) leaves.

We examined the photosynthetic responses of olive (Olea europea L.) leaves exposed to either (a) two hours of high leaf-to-air vapor pressure difference (vpd) or (b) four 30-min cycles of high vpd separated by 15-min periods of recovery at low vpd. Neither treatment affected photosynthesis when vpd was less than 3.0 kPa. Photosynthesis by mature leaves was also insensitive to higher vpd, but photosynthesis of young leaves was reduced by both treatments at a vpd higher than 3.2 kPa. This effect of vpd was much smaller under high intercellular CO(2) pressure. Autoradiograms showed that under a vpd of 3.2 kPa, mature leaves photosynthesized uniformly, but patches of reduced CO(2) fixation occurred in the distal part of young leaves. We conclude that heterogeneities in photosynthesis along the length of the leaf caused the apparent reduction of photosynthesis in our experiments. This pattern of patchy photosynthesis was different from that observed in mesophytic herbs, but the effect on gas exchange analysis was the same. In this case, apparent biochemical effects of low humidity on photosynthesis of young olive leaves are likely an artifact.

Journal Article↗