Free-air carbon dioxide enrichment of soybean: influence of crop variety on residue decomposition.

Elevated atmospheric CO2 can result in larger plants returning greater amounts of residue to the soil. However, the effects of elevated CO2 on carbon (C) and nitrogen (N) cycling for different soybean varieties have not been examined. Aboveground residue of eight soybean [Glycine max (L.) Merr.] varieties was collected from a field study where crops had been grown under two different atmospheric CO2 levels [370 micromol mol(-1) (ambient) and 550 micromol mol(-1) (free-air carbon dioxide enrichment, FACE)]. Senesced residue material was used in a 60-d laboratory incubation study to evaluate potential C and N mineralization. In addition to assessing the overall effects of CO2 level and variety, a few specific variety comparisons were also made. Across varieties, overall residue N concentration was increased by FACE, but residue C concentration was only slightly increased. Overall residue C to N ratio was lower under FACE and total mineralized N was increased by FACE, suggesting that increased N2 fixation impacted residue decomposition; total mineralized C was also slightly increased by FACE. Across CO2 levels, varietal differences were also observed with the oldest variety having the lowest residue N concentration and highest residue C to N ratio; mineralized N was lowest in the oldest variety, illustrating the influence of high residue C to N ratio. It appears (based on our few specific varietal comparisons) that the breeding selection process may have resulted in some varietal differences in residue quality which can result in increased N or C mineralization under elevated CO2 conditions. This limited number of varietal comparisons indicated that more work investigating varietal influences on soil C and N cycling under elevated CO2 conditions is required.

Impacts of chilling temperatures on photosynthesis in warm-climate plants.

Photosynthesis in warm-climate plants is substantially reduced after chilling. Tropical and subtropical species offer the opportunity to study the effects of low temperature on photosynthetic processes undisguised by the myriad of protective responses observed in temperate species. In this article, we highlight the primary components of photosynthesis that are affected by a short chill, in both the dark and the light, and discuss what is known of the mechanisms involved. Recent work implicates impaired redox and circadian regulation among other processes.

An overnight chill induces a delayed inhibition of photosynthesis at midday in mango (Mangifera indica L.).

The effect of a cold night on photosynthesis in herbaceous chilling-sensitive crops, like tomato, has been extensively studied and is well characterized. This investigation examined the behaviour of the sub-tropical fruit tree, mango, to enable comparison with these well-studied systems. Unlike tomato, chilling between 5 degrees C and 7 degrees C overnight produced no significant inhibition of light-saturated CO(2) assimilation (A:) during the first hours following rewarming, measured either under controlled environment conditions or in the field. By midday, however, there was a substantial decline in A:, which could not be attributed to photoinhibition of PSII, but rather was associated with an increase in stomatal limitation of A: and lower Rubisco activity. Overnight chilling of tomato can cause severe disruption in the circadian regulation of key photosynthetic enzymes and is considered to be a major factor underlying the dysfunction of photosynthesis in chilling-sensitive herbaceous plants. Examination of the gas exchange of mango leaves maintained under constant conditions for 2 d, demonstrated that large depressions in A: during the subjective night were primarily the result of stomatal closure. Chilling did not disrupt the ability of mango leaves to produce a circadian rhythm in stomatal conductance. Rather, the midday increase in stomatal limitation of A: appeared to be the result of altered guard cell sensitivity to CO(2) following the dark chill.

Differential effects of chilling-induced photooxidation on the redox regulation of photosynthetic enzymes.

Photosynthesis in plant species that are evolutionarily adapted for growth in warm climates is highly sensitive to illumination under cool conditions. Although it is well documented that illumination of these sensitive species under cool conditions results in the photosynthetic production of reactive oxygen molecules, the underlying mechanism for the inhibition of photosynthesis remains uncertain. Determinations of chloroplast fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase activity showed that the light-dependent, reductive activation of these key carbon reduction cycle enzymes was substantially inhibited in tomato (Lycopersicon esculentum) following illumination at 4 degrees C. However, other chloroplast enzymes also dependent on thioredoxin-mediated reductive activation were largely unaffected. We performed equilibrium redox titrations to investigate the thermodynamics of the thiol/disulfide exchange between thioredoxin f and the regulatory sulfhydryl groups of fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, NADP-glyceraldehyde phosphate dehydrogenase, and the chloroplast ATPsynthase. We determined that the redox midpoint potentials for the regulatory sulfhydryl groups of the various enzymes spanned a broad range ( approximately 50 mV at pH 7. 9). The electron-sharing equilibria among thioredoxin f and its target enzymes largely explained the differential effects of photooxidation induced at low temperature on thioredoxin-mediated activation of chloroplast enzymes in tomato. These results not only provide a plausible mechanism for the low-temperature-induced inhibition of photosynthesis in this important group of plants, but also provide a quantitative basis to evaluate the influence of thioredoxin/target enzyme electron-sharing equilibria on the differential activation and deactivation kinetics of thioredoxin-regulated chloroplast enzymes.

Circadian Regulation of Sucrose Phosphate Synthase Activity in Tomato by Protein Phosphatase Activity.

Sucrose phosphate synthase (SPS), a key enzyme in sucrose biosynthesis, is regulated by protein phosphorylation and shows a circadian pattern of activity in tomato. SPS is most active in its dephosphorylated state, which normally coincides with daytime. Applying okadaic acid, a potent protein phosphatase inhibitor, prevents SPS activation. More interesting is that a brief treatment with cycloheximide, a cytoplasmic translation inhibitor, also prevents the light activation of SPS without any effect on the amount of SPS protein. Cordycepin, an inhibitor of transcript synthesis and processing, has the same effect. Both of these inhibitors also prevent the activation phase of the circadian rhythm in SPS activity. Conversely, cycloheximide and cordycepin do not prevent the decline in circadian SPS activity that normally occurs at night. These observations indicate that SPS phosphatase activity but not SPS kinase activity is controlled, directly or indirectly, at the level of gene expression. Taken together, these data imply that there is a circadian rhythm controlling the transcription of a protein phosphatase that subsequently dictates the circadian rhythm in SPS activity via effects on this enzyme's phosphorylation state.

Factors Associated with Depression of Photosynthetic Quantum Efficiency in Maize at Low Growth Temperature.

The photosynthetic productivity of maize (Zea mays) in temperate regions is often limited by low temperatures. The factors responsible for the sensitivity of photosynthesis in maize to growth at suboptimal temperature were investigated by measuring (a) the quantum yields of CO2 fixation and photosystem II (PSII) photochemistry, (b) the pigments of the xanthophyll cycle, (c) the concentrations of active and inactive PSII reaction centers, and (d) the synthesis of core components of PSII reaction centers. Measurements were made on fully expanded leaves grown at 14[deg]C, both before and during the first 48 h after transfer of these plants to 25[deg]C. Our findings indicate that zeaxanthin-related quenching of absorbed excitation energy at PSII is, quantitatively, the most important factor determining the depressed photosynthetic efficiency in 14[deg]C-grown plants. Despite the photoprotection afforded by zeaxanthin-related quenching of absorbed excitation energy, a significant and more persistent depression of photosynthetic efficiency appears to result from low temperature-induced inhibition of the rate at which damaged PSII centers can be replaced.

The Effects of Chilling in the Light on Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Tomato (Lycopersicon esculentum Mill.).

Photosynthesis rate, ribulsoe-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation state, and ribulose bisphosphate concentration were reduced after exposing tomato (Lycopersicon esculentum Mill.) plants to light at 4[deg]C for 6 h. Analysis of lysed and reconsituted chloroplasts showed that activity of the thylakoid membrane was inhibited and that Rubisco, Rubisco activase, and other soluble factors were not affected. Leaf photosynthesis rates and the ability of chilled thylakoid membranes to promote Rubisco activation recovered after 24 h at 25[deg]C. Thylakoid membranes from control tomato plants were as effective as spinach thylakoids in activating spinach Rubisco in the presence of spinach Rubisco activase. This observation is in sharp contrast to the poor ability of spinach Rubisco activase to activate tomato Rubisco (Z.-Y. Wang, G.W. Snyder, B.D. Esau, A.R. Portis, and W.L. Ogren [1992] Plant Physiol 100: 1858-1862). The ability of thylakoids from chilled tomato plants to activate Rubisco in the assay system was greatly inhibited compared to control plants. These experiments indicate that chilling tomato plants at 4[deg]C interferes with photosynthetic carbon metabolism at two sites, thioredoxin/ferredoxin reduction (G.F. Sassenrath, D.R. Ort, and A.R. Portis, Jr. [1990] Arch Biochem Biophys 282: 302-308), which limits bisphosphatase activity, and Rubisco activase, which reduces Rubisco activation state.

In situ evidence that chilling in the light does not cause uncoupling of photophosphorylation or detachment of coupling factor in chilling-sensitive plants.

The potential involvement of impaired photophosphorylation in the chilling sensitivity of photosynthesis in warm climate plant species has been a topic of investigation for more than two decades. With recent advances in the analysis of photosynthetic energy transduction in intact leaves, experiments are now possible that either address or avoid important uncertainties in the significance and interpretation of earlier in vitro work. Nevertheless, different laboratories using different techniques to analyze the effects of chilling in the light on photophosphorylation in intact cucumber (Cucumis sativus) leaves have come to very different conclusions regarding the role of impaired ATP formation capacity in the inhibition of net photosynthesis. In order to evaluate these discrepancies and bring this issue to a final resolution, in this investigation, we have made a detailed analysis of the decay of the flash-induced electrochromic shift and changes in chlorophyll fluorescence yield in cucumber leaves before, during and after a 5 h light-chill at chill temperatures of between 4 and 10°C. We feel that our findings address the major discrepancies in both data and interpretation as well as provide convincing evidence that photophosphorylation is not disrupted in cucumber leaves during or after light and chilling exposure. It follows that impaired photophosphorylation is not a contributing element to the inhibition of net photosynthesis that is widely observed in warm climate plants as a result of chilling in the light.

Mutants of Chloroplast Coupling Factor Reduction in Arabidopsis.

We have devised a two-step screening strategy for the selection of chloroplast coupling factor reduction mutants from an M2 population of Arabidopsis thaliana. The selection strategy relies on a lowered energetic threshold for catalytic activation of the enzyme that has been shown to accompany thioredoxin-mediated reduction of a cysteine bridge on the [gamma] subunit of coupling factor. We selected first for plants that grew poorly under low irradiance but performed satisfactorily at high irradiance when the transmembrane electrochemical potential of hydrogen ions is large and competent to maintain a high level of coupling factor activation without [gamma] subunit reduction. In the second step of the screen we monitored the flash-induced electrochromic change to select putative coupling factor reduction mutants from other sorts of mutations that shared the phenotype of poor growth and vigor when transferred from high to low irradiance. Among the mutants selected, one appears incapable of reducing coupling factor, whereas another behaves as though coupling factor is at least partially reduced even in dark-adapted plants.

In vivo pulse labeling of proteins in attached leaves with radioactive amino acids.

The pulse labeling of proteins in attached leaves by the incorporation of radioactive amino acids can be a valuable approach to study a wide range of topics pertaining to plant gene expression. Transient changes in the rate of net protein synthesis can be measured since pulse labeling permits discrimination between proteins actively synthesized during the labeling interval from those accumulated at earlier times. Specific procedures for a rapid and convenient method of in vivo pulse labeling are outlined which we believe could be broadly useful in plant biological research. A particularly important technical feature of the protocol is electroblotting of radioactive proteins from the sodium dodecyl sulfate acrylamide resolving gel matrix to polyvinylidene difluoride transfer membrane. This step permits a single polyacrylamide slab gel to be used for autoradiography, immunoblot analysis, and protein staining, thereby greatly facilitating comparison among these complementary techniques and resulting in a significant savings in time and radioactive sample. Central considerations concerning the quantitation and interpretation of in vivo pulse labeling data are discussed.

The non-photochemical reduction of plastoquinone in leaves.

Although it is generally assumed that the plastoquinone pool of thylakoid membranes in leaves of higher plants is rapidly oxidized upon darkening, this is often not the case. A multiflash kinetic fluorimeter was used to monitor the redox state of the plastoquinone pool in leaves. It was found that in many species of plants, particularly those using the NAD-malic enzyme C4 system of photosynthesis, the pool actually became more reduced following a light to dark transition. In some Amaranthus species, plastoquinone remained reduced in the dark for several hours. Far red light, which preferentially drives Photosystem I turnover, could effectively oxidize the plastoquinone pool. Plastoquinone was re-reduced in the dark within a few seconds when far red illumination was removed. The underlying mechanism of the dark reduction of the plastoquinone pool is still uncertain but may involve chlororespiratory activity.

Spatial Distribution of Photosynthesis during Drought in Field-Grown and Acclimated and Nonacclimated Growth Chamber-Grown Cotton.

Inhomogeneous photosynthetic activity has been reported to occur in drought-stressed leaves. In addition, it has been suggested that these water stress-induced nonuniformities in photosynthesis are caused by "patchy" stomatal closure and that the phenomenon may have created the illusion of a nonstomatal component to the inhibition of photosynthesis. Because these earlier studies were performed with nonacclimated growth chamber-grown plants, we sought to determine whether such "patches" existed in drought-treated, field-grown plants or in chamber-grown plants that had been acclimated to low leaf water potentials (psi(leaf)). Cotton (Gossypium hirsutum L.) was grown in the field and subjected to drought by withholding irrigation and rain from 24 d after planting. The distribution of photosynthesis, which may reflect the stomatal aperture distribution in a heterobaric species such as cotton, was assayed by autoradiography after briefly exposing attached leaves of field-grown plants to (14)CO(2). A homogeneous distribution of radioactive photosynthate was evident even at the lowest psi(leaf) of -1.34 MPa. "Patchiness" could, however, be induced by uprooting the plant and allowing the shoot to air dry for 6 to 8 min. In parallel studies, growth chamber-grown plants were acclimated to drought by withholding irrigation for three 5-d drought cycles interspersed with irrigation. This drought acclimation lowered the psi(leaf) value at which control rates of photosynthesis could be sustained by approximately 0.7 MPa and was accompanied by a similar decline in the psi(leaf) at which patchiness first appeared. Photosynthetic inhomogeneities in chamber-grown plants that were visible during moderate water stress and ambient levels of CO(2) could be largely removed with elevated CO(2) levels (3000 muL L(-1)), suggesting that they were stomatal in nature. However, advanced dehydration (less than approximately 2.0 MPa) resulted in "patches" that could not be so removed and were probably caused by nonstomatal factors. The demonstration that patches do not exist in drought-treated, field-grown cotton and that the presence of patches in chamber-grown plants can be altered by treatments that cause an acclimation of photosynthesis leads us to conclude that spatial heterogeneities in photosynthesis probably do not occur frequently under natural drought conditions.

Low temperature interrupts circadian regulation of transcriptional activity in chilling-sensitive plants.

Impaired chloroplast function is responsible for nearly two-thirds of the inhibition to net photosynthesis caused by dark chilling in tomato (Lycopersicon esculentum Mill.), yet it has not been possible to localize the dysfunction to specific chloroplast reactions. We report here on an effect that low-temperature exposure has in tomato on the expression of certain nuclear-encoded chloroplast proteins, which may be directly related to the chilling sensitivity of photosynthesis. Transcriptional activity of genes for both the chlorophyll a/b binding protein of photosystem II (Cab) as well as for ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase was found to be controlled by an endogenous rhythm. For Cab this rhythm was also visible at the level of newly synthesized protein, indicating that the circadian control of transcriptional activity normally ensures that this protein is synthesized only during daylight hours. However, low-temperature treatment suspended the timing of the rhythm in tomato so that, upon rewarming, the circadian control was reestablished but was displaced from the actual time of day by the length of the chilling exposure. In addition, we found that the normal turnover of Cab and Rubisco activase mRNA was suspended during the low-temperature treatment, but, upon rewarming, this stabilized message was not translated into protein. We believe that the low-temperature-induced mistiming of gene expression together with its effect on the translatability of existing transcripts may be an important clue in unraveling the basis for the chilling sensitivity of photosynthesis in tomato.

Photophosphorylation in Attached Leaves of Helianthus annuus at Low Water Potentials.

The in situ response of photophosphorylation and coupling factor activity to low leaf water potential (psi(L)) was investigated using kinetic spectroscopy to measure the flash-induced electrochromic absorption change in attached sunflower (Helianthus annuus L. cv IS894) leaves. The electrochromic change is caused by the formation of an electric potential across the thylakoid membrane associated with proton uptake. Since depolarization of the thylakoid membrane following flash excitation is normally dominated by proton efflux through the coupling factor during ATP formation, this measurement can provide direct information about the catalytic activity of the coupling factor. Under low psi(L) conditions in which a clear nonstomatal limitation of net photosynthesis could be demonstrated, we found a strong inhibition of coupling factor activity in dark-adapted leaves which was probably caused by an increase in the energetic threshold for the activation of the enzyme at low psi(L). While this result supported earlier in vitro findings, we further discovered that the light-dependent reduction of coupling factor reversed any observable effect of low psi(L) on the energetics of activation or on photophosphorylation competence. Furthermore, coupling factor was reduced, even in severely droughted sunflower, almost immediately upon illumination. Based on these measurements, we conclude that the nonstomatal limitation of photosynthesis observed by us and others in droughted plants cannot be explained by impaired coupling factor activity.

Impaired reductive activation of stromal bisphosphatases in tomato leaves following low-temperature exposure at high light.

Photosynthesis in domestic tomato (Lycopersicon esculentum L.) is highly sensitive to low temperature, particularly when accompanied by high light. Since previous studies have established that the inhibited plants retain photosynthetic electron transfer and ATP formation competence, we sought to identify specific steps in the photosynthetic carbon reduction pathway that could account for the lost photosynthetic capacity. Measurements of steady-state photosynthetic metabolite pool sizes showed an accumulation of fructose 1,6-bisphosphate and sedohepulose 1,7-bisphosphate following chilling in the light. Measurements of in vivo turnover rates of the metabolite pools accompanied by direct determinations of enzymatic activity showed that the capacity of the stromal bisphosphatases was substantially reduced following chilling in the light and was the cause of the bisphosphate accumulation. The time course of the loss of phosphatase activity closely mimicked that of the inhibition of net photosynthesis, further indicating that impaired phosphatase function is the underlying cause of the sensitivity of photosynthesis in tomato to light and chilling. Fructose 1,6-bisphosphatase extracted from inhibited tomato plants could be fully activated in the presence of dithiothreitol, indicating that chilling in the light disrupts the normal, thioredoxin-dependent, activation pathway of the stromal bisphosphatases. This disruption could involve a change in the redox potential of the functional disulfide on the phosphatases.

The involvement of the photoinhibition of photosystem II and impaired membrane energization in the reduced quantum yield of carbon assimilation in chilled maize.

In this study we investigated the basis for the reduction in the quantum yield of carbon assimilation in maize (Zea mays L. cv. LG11) caused by chilling in high light. After chilling attached maize leaves at 5° C for 6 h at high irradiance (1000 µmol photons·m(-2)·s(-1)) chlorophyll fluorescence measurements indicated a serious effect on the efficiency of photochemical conversion by photosystem II (PSII) and measurements of [(14)C]atrazine binding showed that the plastoquinone binding site was altered in more than half of the PSII reaction centres. Although there were no direct effects of the chilling treatment on coupling-factor activity, ATP-formation capacity was affected because the photoinhibition of PSII led to a reduced capacity to energize the thylakoid membranes. In contrast to chilling at high irradiance, no photoinhibition of PSII accompanied the 20% decrease in the quantum yield of carbon assimilation when attached maize leaves were chilled in low light (50 µmol photons·m(-2)·s(-1)). Thus it is clear that photoinhibition of PSII is not the sole cause of the light-dependent, chillinduced decrease in the quantum yield of carbon assimilation. During the recovery of photosynthesis from the chilling treatment it was observed that full [(14)C]atrazinebinding capacity and membrane-energization capacity recovered significantly more slowly than the quantum yield of carbon assimilation. Thus, not only is photoinhibition of PSII not the sole cause for the decreased quantum yield of carbon assimilation, apparently an appreciable population of photoinhibited PSII centres can be tolerated without any reduction in the quantum yield of carbon assimilation.