Plant Effects on Spatial and Temporal Patterns of Nitrogen Cycling in Shortgrass Steppe

Because of the water-limited nature and discontinuous plant cover of shortgrass steppe, spatial patterns in ecosystem properties are influenced more by the presence or absence of plants than by plant type. However, plant type may influence temporal patterns of nutrient cycling between plant and soil. Plants having the carbon-3 (C₃) or carbon-4 (C₄) photosynthetic pathway differ in phenology as well as other attributes that affect nitrogen (N) cycling. We estimated net N mineralization rates and traced nitrogen-15 (¹⁵N) additions among plant and soil components during May, July, and September of 1995 in native plots of C₃ plants, C₄ plants, or mixtures of C₃ and C₄. Net N mineralization was significantly greater in C₃ plots than in C₄ plots during both July and September. C₃ plots retained significantly more ¹⁵N in May than did mixed and C₄ plots; these differences in ¹⁵N retention were due to greater ¹⁵N uptake by C₃ plants than by C₄ plants during May. There were no significant differences in total ¹⁵N retention among plant communities for July and September. Soil ¹⁵N was influenced more by presence or absence of plants than by type of plant; greater quantities of ¹⁵N remained in soil interspaces between plants than in soil directly under plants for July and September. Our results indicate that plant functional type (C₃ versus C₄) can affect both the spatial and the temporal patterns of N cycling in shortgrass steppe. Further research is necessary to determine how these intraseasonal differences translate to longer-term and coarser-scale effects of plants on N cycling, retention, and storage. Received 8 December 1997; accepted 6 May 1998.     

The response of mycorrhizal colonization to elevated CO2 and climate change in Pascopyrum smithii and Bouteloua gracilis

Large intact soil cores of nearly pure stands of Pascopyrum smithii (western wheatgrass, C₃) and Bouteloua gracilis (blue grama, C₄) were extracted from the Central Plains Experimental Range in northeastern Colorado, USA and transferred to controlled environment chambers. Cores were exposed to a variety of water, temperature and CO₂ regimes for a total of four annual growth cycles. Root subsamples were harvested after the completion of the second and fourth growth cycles at a time corresponding to late winter, and were examined microscopically for the presence of mycorrhizae. After two growth cycles in the growth chambers, 54% of the root length was colonized in P. smithii, compared to 35% in blue grama. Field control plants had significantly lower colonization. Elevation of CO₂ increased mycorrhizal colonization in B. gracilis by 46% but had no effect in P. smithii. Temperatures 4° C higher than normal decreased colonization in P. smithii by 15%. Increased annual precipitation decreased colonization in both species. Simulated climate change conditions of elevated CO₂, elevated temperature and lowered precipitation decreased colonization in P. smithii but had less effect on B. gracilis. After four growth cycles in P. smithii, trends of treatments remained similar, but overall colonization rate decreased.     

Differential Controls of Water Input on Litter Decomposition and Nitrogen Dynamics in the Patagonian Steppe

Studies of the effects of precipitation on litter decomposition and nitrogen mineralization in arid and semiarid environments have demonstrated contradictory results. We conducted a manipulative experiment with rainout shelters in the semiarid Patagonian steppe, aimed at assessing the direct effects of water availability on litter decomposition and net nitrogen mineralization while isolating the indirect effects. We created four levels of precipitation input: control and three levels (30, 55 and 80%) of precipitation interception and we examined litter decomposition and nutrient release of a dominant grass species, Stipa speciosa, inorganic soil nitrogen, and in situ net nitrogen mineralization over two consecutive years. Litter decomposition rates (k, year⁻¹) varied significantly (P < 0.001) among precipitation interception treatments and were positively correlated with incoming annual precipitation (APPT, mm/year) (k = 0.0007 × APPT + 0.137). In contrast, net N mineralization was not correlated with incoming precipitation. Soil NO₃⁻ significantly decreased with increasing precipitation input, whereas soil NH₄⁺ concentration did not differ among precipitation interception treatments. Controls of water input on litter decomposition appear to be different from controls on N mineralization in the semiarid Patagonian steppe. We suggest that although water availability affects both the mineralization of C and N, it differentially affects the movement and fate of the inorganic products. A consequence of the accumulation of inorganic N during dry episodes is that periods of maximum water and soil nutrient availability may occur at different times. This asynchrony in the availability of N and water in the soil may explain the observed lags in the response of primary production to increases in water availability.     

Increases in Desert Shrub Productivity under Elevated Carbon Dioxide Vary with Water Availability

Productivity of aridland plants is predicted to increase substantially with rising atmospheric carbon dioxide (CO₂) concentrations due to enhancement in plant water-use efficiency (WUE). However, to date, there are few detailed analyses of how intact desert vegetation responds to elevated CO₂. From 1998 to 2001, we examined aboveground production, photosynthesis, and water relations within three species exposed to ambient (around 38 Pa) or elevated (55 Pa) CO₂ concentrations at the Nevada Desert Free-Air CO₂ Enrichment (FACE) Facility in southern Nevada, USA. The functional types sampled—evergreen (Larrea tridentata), drought-deciduous (Ambrosia dumosa), and winter-deciduous shrubs (Krameria erecta)—represent potentially different responses to elevated CO₂ in this ecosystem. We found elevated CO₂ significantly increased aboveground production in all three species during an anomalously wet year (1998), with relative production ratios (elevated:ambient CO₂) ranging from 1.59 (Krameria) to 2.31 (Larrea). In three below-average rainfall years (1999–2001), growth was much reduced in all species, with only Ambrosia in 2001 having significantly higher production under elevated CO₂. Integrated photosynthesis (mol CO₂ m⁻² y⁻¹) in the three species was 1.26–2.03-fold higher under elevated CO₂ in the wet year (1998) and 1.32–1.43-fold higher after the third year of reduced rainfall (2001). Instantaneous WUE was also higher in shrubs grown under elevated CO₂. The timing of peak canopy development did not change under elevated CO₂; for example, there was no observed extension of leaf longevity into the dry season in the deciduous species. Similarly, seasonal patterns in CO₂ assimilation did not change, except for Larrea. Therefore, phenological and physiological patterns that characterize Mojave Desert perennials—early-season lags in canopy development behind peak photosynthetic capacity, coupled with reductions in late-season photosynthetic capacity prior to reductions in leaf area—were not significantly affected by elevated CO₂. Together, these findings suggest that elevated CO₂ can enhance the productivity of Mojave Desert shrubs, but this effect is most pronounced during years with abundant rainfall when soil resources are most available.     

The quantum yield for CO2 uptake in C3 and C4 grasses

The quantum yield for CO₂ uptake was measured in C₃ and C₄ monocot species from several different grassland habitats. When the quantum yield was measured in the presence of 21% O₂ and 340 cm³ m^-3 CO₂, values were very similar in C₃ monocots, C₃ dicots, and C₄ monocots (0.045–0.056 mole CO₂ · mole^-1 quanta absorbed). In the presence of 2% O₂ and 800 cm³ m^-3 CO₂, enhancements of the quantum yield values occurred for the C₃ plants (both monocots and dicots), but not for C₄ monocots. A dependence of the quantum yield on leaf temperature was observed in the C₃ grass, Agropyron smithii, but not in the C₄ grass, Bouteloua gracilis, in 21% O₂ and 340 cm³ m^-3 CO₂. At leaf temperatures between 22–25°C the quantum yield values were approximately equal in the two species.     

Partitioning evapotranspiration fluxes from a Colorado grassland using stable isotopes: Seasonal variations and ecosystem implications of elevated atmospheric CO2

The stable isotopic composition of soil water is controlled by precipitation inputs, antecedent conditions, and evaporative losses. Because transpiration does not fractionate soil water isotopes, the relative proportions of evaporation and transpiration can be estimated using a simple isotopic mass balance approach. At our site in the shortgrass steppe in semi-arid northeastern Colorado, ¹⁸O values of soil water were almost always more enriched than those of precipitation inputs, owing to evaporative losses. The proportion of water lost by evaporation (E/ET) during the growing season ranged from nil to about 40% (to >90% in the dormant season), and was related to the timing of precipitation inputs. The sum of transpiration plus evaporation losses estimated by isotopic mass balance were similar to actual evapotranspiration measured from a nearby Bowen ratio system. We also investigated the evapotranspiration response of this mixed C₃/C₄ grassland to doubled atmospheric [CO₂] using Open-Top Chambers (OTC). Elevated atmospheric [CO₂] led to increased soil-water conservation via reduced stomatal conductance, despite greater biomass growth. We used a non-invasive method to measure the ¹⁸O of soil CO₂ as a proxy for soil water, after establishing a strong relationship between ¹⁸O of soil CO₂ from non-chambered control (NC) plots and ¹⁸O of soil–water from an adjacent area of native grassland. Soil–CO₂ ¹⁸O values showed significant treatment effects, particularly during a dry summer: values in ambient chambers (AC) were more enriched than in NC and elevated chamber (EC) plots. During the dry growing season of 2000, transpiration from the EC treatment was higher than from AC and lower than from NC treatments, but during 2001, transpiration was similar on all three treatments. Slightly higher evaporation rates from AC than either EC or NC treatments in 2000 may have resulted from increased convection across the soil surface from the OTC blowers, combined with lower biomass and litter cover on the AC treatment. Transpiration-use efficiency, or the amount of above-ground biomass produced per mm water transpired, was always greatest on EC and lowest on NC treatments.     

Nitrogen distribution and leaf area indices in relation to photosynthetic nitrogen use efficiency in savanna grasses

Leaf photosynthetic characteristics, distribution patterns of nitrogen content per unit leaf area (n_L) and leaf area production per unit n_Lwere measured in natural stands of a C₄ grass (Hyparrhenia rufa) from the seasonal savannas and of a C₄grass (Paspalum fasciculatum) and two C₃grasses (Leersia hexandra and Hymenachne amplexicaulis) from the flooded savannas in central Venezuela. Daily rates of canopy photosynthesis (P_cD) as well as the optimal leaf area production per unit n_Lat which P_cDfor a given total amount of nitrogen in the canopy (i.e., canopy-PNUE) is maximized were also calculated. The C₃and C₄species from the flooded savannas had similar light saturated rates of photosynthesis per unit n_L(i.e. leaf-PNUE) and similar canopy-PNUEs which was in strong contrast with previous studies. Especially H. rufa but also L. hexandra and H. amplexicaulis had leaf- and canopy-PNUEs which were considerably higher than the values calculated for most other species with the same photosynthetic pathway (i.e., C₃or C₄). In contrast to previous studies, differences in the light gradient in the canopy between stands only partially explained differences in N distribution. Measured leaf area indices were greater and the average n_L values were consequently smaller than the calculated optima. There was, however, a very strong linear correlation between the optimal and actual average n_Lindicating that even though the model overestimated average n_L, it did predict the differences in leaf area production per unit nitrogen – the inverse average n_L– very well. This result strongly indicates that leaf area production per unit of leaf nitrogen increases with leaf-PNUE and decreases with the extinction coefficient for light. Grass species from seasonal savannas have extremely high leaf-PNUEs and thus optimally produce large amounts of leaf area per unit n_L. This helps explain how stands of these species may have high leaf area indices and achieve high photosynthetic productivity despite the very low nutrient availability at which they grow.     

Plant nitrogen dynamics and nitrogen-use strategies under altered nitrogen seasonality and competition

BACKGROUND AND AIMS: Numerous studies have examined the effects of climatic factors on the distribution of C(3) and C(4) grasses in various regions throughout the world, but the role of seasonal fluctuations in temperature, precipitation and soil N availability in regulating growth and competition of these two functional types is still not well understood. This report is about the effects of seasonality of soil N availability and competition on plant N dynamics and N-use strategies of one C(3) (Leymus chinensis) and one C(4) (Chloris virgata) grass species. METHODS: Leymus chinensis and C. virgata, two grass species native to the temperate steppe in northern China, were planted in a monoculture and a mixture under three different N seasonal availabilities: an average model (AM) with N evenly distributed over the growing season; a one-peak model (OM) with more N in summer than in spring and autumn; and a two-peak model (TM) with more N in spring and autumn than in summer. KEY RESULTS: The results showed that the altered N seasonality changed plant N concentration, with the highest value of L. chinensis under the OM treatment and C. virgata under the TM treatment, respectively. N seasonality also affected plant N content, N productivity and N-resorption efficiency and proficiency in both the C(3) and C(4) species. Interspecific competition influenced N-use and resorption efficiency in both the C(3) and C(4) species, with higher N-use and resorption efficiency in the mixture than in monoculture. The C(4) grass had higher N-use efficiency than the C(3) grass due to its higher N productivity, irrespective of the N treatment or competition. CONCLUSIONS: The observations suggest that N-use strategies in the C(3) and C(4) species used in the study were closely related to seasonal dynamics of N supply and competition. N seasonality might be involved in the growth and temporal niche separation between C(3) and C(4) species observed in the natural ecosystems.     

长期不同施氮水平对草原植物群落结构的影响

为探讨施氮对贝加尔针茅草原植物群落结构的影响,该研究于内蒙古贝加尔针茅草原设置不同施氮水平0(N0)、15(N15)、30(N30)、50(N50)、100(N100)、150(N150)、200(N200)和300(N300)kg·hm-2·a-1,研究连续施氮9年后贝加尔针茅草原群落物种组成、重要值、多样性及生物量等指标对不同施氮水平的响应。结果表明:(1)长期施氮导致植物群落物种组成发生了明显的变化,在高浓度氮素添加量下群落物种数最低,但可显著提高优势种在群落中的重要值(P<0.05)。(2)长期施氮使贝加尔针茅草原植物多样性指数不同程度地降低,使优势度指数增加,其中N300添加水平效果最为明显(P<0.05)。(3)长期施氮使草原植被生产力(地上、地下生物量)整体呈先上升后下降的趋势,地上生物量在N100水平时出现峰值,地下生物量在N50水平时出现最大值(P<0.05),且根系分布具有明显的表聚特征(0~10 cm)。(4)RDA分析表明,寸草苔和狭叶柴胡地上生物量与土壤pH呈显著正相关关系(P<0.05);展枝唐松草和二裂委陵菜地上生物量与土壤有机质含量呈显著正相关关系(P<0.05)。研究认为,长期不同施氮水平均可改变贝加尔针茅草原群落物种组成,降低物种多样性,对植物群落结构有着明显的影响。     

Nitrogen mineralization in native cultivated and abandoned fields in shortgrass steppe

We conducted a set of in situ incubations to evaluate patterns of N availability among dominant land uses in the shortgrass steppe region of Colorado, USA, and to assess recovery of soil fertility in abandoned fields. Replicated 30 d incubations were performed in 3 sets of native (never cultivated), abandoned (cultivated until 1937), and currently cultivated, fallow fields. Net N mineralization and the percentage of total N that was mineralized increased in the order: native, abandoned, cultivated. Higher soil water content in fallow fields is the most likely reason for greater mineralization in cultivated fields, while higher total organic C and C/N ratios in native and abandoned fields may explain differences in mineralization between these land uses. Recovery of soil organic matter in abandoned fields appears to involve accumulation of soil C and N under perennial plants, but probable methodological artifacts complicate evaluation of the role of individual plants in recovery of N availability. Higher N mineralization and turnover in cultivated fields may make them more susceptible to N losses; recovery of N cycling in abandoned fields appears to involve a return to slower N turnover and tighter N cycling similar to native shortgrass steppe.     

Cd污染及其与大气CO2浓度升高、N添加复合作用对大叶相思生长的影响

为了解Cd污染胁迫下树木对CO_2浓度升高、N添加及其复合作用的响应,应用开顶箱,探讨Cd及其与CO_2、N的复合作用对大叶相思(Acacia auriculiformis)基径、树高和生物量的影响。结果表明,Cd添加抑制大叶相思基径、树高和生物量的增长,并且具有时间滞后性;大气CO_2浓度升高、N添加及CO_2+N均有缓解Cd对植物生长抑制作用的趋势,其中, N添加更能促进大叶相思基径的生长,树高生长则对CO_2升高更为敏感;在Cd污染土壤中,N添加的缓解作用最显著。因此,氮肥管理是重金属污染土地修复初期促进植物修复的重要策略。     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Enhanced root system C-sink activity,water relations and aspects of nutrient acquisition in mycotrophic Bouteloua gracilis subjected to CO2 enrichment

In order to better elucidate fixed-C partitioning, nutrient acquisition and water relations of prairie grasses under elevated [CO₂], we grew the C₄ grass Bouteloua gracilis (H.B.K.) lag ex Steud. from seed in soil-packed, column-lysimeters in two growth chambers maintained at current ambient [CO₂] (350 μL L⁻¹) and twice enriched [CO₂] (700 μL L⁻¹). Once established, plants were deficit irrigated; growth chamber conditions were maintained at day/night temperatures of 25/16°C, relative humidities of 35%/90% and a 14-hour photoperiod to simulate summer conditions on the shortgrass steppe in eastern Colorado. After 11 weeks of growth, plants grown under CO₂ enrichment had produced 35% and 65% greater total and root biomass, respectively, and had twice the level of vesicular-arbuscular mycorrhizal (VAM) infection (19.8% versus 10.8%) as plants grown under current ambient [CO₂]. The CO₂-enriched plants also exhibited greater leaf water potentials and higher plant water use efficiencies. Plant N uptake was reduced by CO₂ enrichment, while P uptake appeared little influenced by CO₂ regime. Under the conditions of the experiment, CO₂ enrichment increased root biomass and VAM infection via stimulated growth and adjustments in C partitioning below-ground. The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged. The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Biomass production and nitrogen content of C3- and C4- grasses in pure and mixed culture with different nitrogen supply

Summary Two C₃ grasses (Hordeum vulgare L., Avena sativa L.) and two C₄ grasses (Panicum miliaceum L., Panicum crus-galli L.) were cultivated in standard soil in the open air in pure cultures and in various mixed cultures at low and high nitrogen fertilization levels. After three months the dry weight, length and nitrogen content of the aboveground and below-ground parts of the plants and the shoot/root ratios were determined. Hordeum vulgare was the most successful species irrespective of the nitrogen fertilization level, and also exhibited in most cases the highest nitrogen concentrations. Panicum miliaceum, on the other hand, was the species least able to compete. The production of biomass was reduced in cultures growing under nitrogen starvation conditions, this phenomenon being more pronounced with respect to the C₄ than to the C₃ species. The decrease in the production of biomass at low N conditions was most drastic with Panicum crus-galli, the species with the lowest nitrogen content and thus assumed to be best adapted to nitrogen starvation conditions. In cultures growing at low nitrogen fertilization levels the shoot/root ratios of all species.shifted in favour of an increasing root proportion. The extent of this shift, however, differed from species to species.     

Elevated CO<Subscript>2</Subscript> Effects on Peatland Plant Community Carbon Dynamics and DOC Production

Northern peatlands are important stores of carbon and reservoirs of biodiversity that are vulnerable to global change. However, the carbon dynamics of individual peatland plant species is poorly understood, despite the potential for rising atmospheric CO₂ to affect the vegetation’s contribution to overall ecosystem carbon function. Here, we examined the effects of 3 years exposure to elevated CO₂ (eCO₂) on (a) peatland plant community composition and biomass, and (b) plant carbon dynamics and the production of dissolved organic carbon (DOC) using a ¹³CO₂ pulse–chase approach. Results showed that under eCO₂, Sphagnum spp. cover declined by 39% (P < 0.05) and Juncus effusus L. cover increased by 40% (P < 0.001). There was a concurrent increase in above- and belowground plant biomass of 115% (P < 0.01) and 96% (P < 0.01), respectively. Vascular species assimilated and turned over more ¹³CO₂-derived carbon than Sphagnum spp. (49% greater turnover of assimilated ¹³C in J. effusus and F. ovina L. leaf tissues compared with Sphagnum, P < 0.01). Elevated CO₂ also produced a 66% rise in DOC concentrations (P < 0.001) and an order of magnitude more ‘new’ exudate ¹³DOC than control samples (24 h after ¹³CO₂ pulse-labelling 2.5 ± 0.5 and 0.2 ± 0.1% in eCO₂ and control leachate, respectively, P < 0.05). We attribute the observed increase in DOC concentrations under eCO₂ to the switch from predominantly Sphagnum spp. to vascular species (namely J. effusus), leading to enhanced exudation and decomposition (litter and peat). The potential for reduced peatland carbon accretion, increased DOC exports and positive feedback to climate change are discussed.     

Structural characterization of photosynthetic cells in an amphibious sedge, Eleocharis vivipara, in relation to C3 and C4 metabolism

Eleocharis vivipara Link is a unique amphibious leafless sedge. The terrestrial form has Kranz anatomy and the biochemical traits of C₄ plants while the submerged form develops structural and biochemical traits similar to those of C₃ plants. The structural features of the culms, which are the photosynthetic organs, of the two forms were examined and compared. The culms of the terrestrial form have mesophyll cells and three bundle sheaths which consist of three kinds of cell, namely, the innermost Kranz cells that contain large numbers of organelles, the middle mestome sheath cells that lack chloroplasts, and the outermost parenchyma sheath cells that contain chloroplasts. The culms of the submerged form had a tendency towards reduction in numbers and size of Kranz cells and vascular bundles, as compared to the terrestrial form, and they had spherical mesophyll cells that were tightly packed without intercellular spaces inside the epidermis. The submerged form had a higher ratio of cross-sectional area of mesophyll cells plus parenchyma sheath cells to that of Kranz cells than the terrestrial form. The difference was mainly due to a decrease in the number and the size of the Kranz cells and to a marked increase in the size of the mesophyll cells and the parenchyma sheath cells in the submerged form, as compared to the terrestrial form. The Kranz cells of the terrestrial form had basically the structural characteristics of plants of the NAD-malic enzyme type, with the exception of the intracellular location of organelles. The Kranz cells of the submerged form included only a few organelles, and the percentage of organelles partitioned to the Kranz cells was significantly smaller in the submerged form than in the terrestrial form. In addition, the size of chloroplasts of the Kranz cells was 60–70% of that of the terrestrial form. These structural differences between the two forms may be related to the functional differences in their mechanisms of photosynthesis.Abbreviations KC Kranz cell - MC mesophyll cell - PSC parenchyma sheath cell - NAD-ME NAD-malic enzyme - VB vascular bundle This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology).     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

Immunocytochemical localization of enzymes involved in the C3 and C4 pathways in the photosynthetic cells of an amphibious sedge, Eleocharis vivipara

Eleocharis vivipara link, an amphibious leafless sedge, develops traits of C₄ photosynthesis and Kranz anatomy in the terrestrial form but develops C₃-like traits with non-Kranz anatomy when submerged. The cellular localization of C₃ and C₄ enzymes in the photosynthetic cells of the two forms was investigated by immunogold labeling and electron microscopy. The terrestrial form has mesophyll cells and three kinds of bundle sheath cell, namely, parenchyma sheath cells, non-chlorophyllous mestome sheath cells, and Kranz cells. Phosphoenol-pyruvate carboxylase (PEPCase) was present in the cytosol of both the mesophyll cells and the parenchyma sheath cells, with higher-density labeling in the latter, but not in the Kranz cells. Pyruvate, Pi dikinase (PPDK) was found at high levels in the chloroplasts of both the mesophyll cells and the parenchyma sheath cells with some-what stronger labeling in the latter. This enzyme was also absent from the Kranz cells. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was found in the chloroplasts of all types of photosynthetic cell, but labeling was significantly less intense in the parenchyma sheath cells than in other types of cell. The submerged form also has three types of photosynthetic cell, as well as non-chlorophyllous mestome sheath cells, but it lacks the traits of Kranz anatomy as a consequence of modification of the cells. Rubisco was densely distributed in the chloroplasts of all the photosynthetic cells. However, PEPCase and PPDK were found in both the mesophyll cells and the parenchyma sheath cells but at lower levels than in the terrestrial form. These data reveal that the terrestrial form has a unique pattern of cellular localization of C₃ and C₄ enzymes, and they suggest that this pattern and the changes in the extent of accumulation of the various enzymes are the main factors responsible for the difference in photosynthetic traits between the two forms.Abbreviations CAM crassulacean acid metabolism - MC meso phyll cell - PSC parenchyma sheath cell - KC Kranz cell - PEP-Case phosphoenolpyruvate carboxylase - PPDK pyruvate, Pi dikinase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - LS large subunit - RuBP ribulose-1,5-bisphosphate This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology). The author is grateful to Drs M. Matsuoka and S. Muto for providing the antisera and Dr. M. Samejima for his advice at the early stages of this study.     

C3−C4 Intermediate species in the genus Flaveria: leaf anatomy,ultrastructure, and the effect of O2 on the CO2 compensation concentration

Leaf anatomical, ultrastructural, and CO₂-exchange analyses of three closely related species of Flaveria indicate that they are C₃–C₄ intermediate plants. The leaf mesophyll of F. floridana J.R. Johnston, F. linearis Lag., and F. chloraefolia A. Gray is typical of that in dicotyledonous C₃ plants, but the bundle sheath cells contain granal, starch-containing chloroplasts. In F. floridana and F. chloraefolia, the chloroplasts and numerous associated mitochondria are arranged largely centripetally, as in the closely related C₄ species, F. brownii A.M. Powell. In F. linearis, fewer mitochondria are present and the chloroplasts are more evenly distributed throughout the bundle sheath cytosol. There is no correlation between the bundle sheath ultrastructure and CO₂ compensation concentration. () values of these C₃–C₄ intermediate Flaveria species. At 21% O₂ and 25°C, for F. chloraefolia, F. linearis, and F. floridana is 23–26, 14–19, and 8–10 l CO₂ l^-1, respectively. The O₂ dependence of is the greatest for F. chloraefolia and F. linearis (similar to that for C₃–C₄ intermediate Panicum and Moricandia species) and the least for F. floridana, whose O₂ response is identical to that for F. brownii from 1.5 to 21% O₂, but greater at higher pO₂. The variation in leaf anatomy, bundle sheath ultrastructure, and O₂ dependence of among these Flaveria species may indicate an active evolution in the pathway of photosynthetic carbon metabolism within this genus.Abbreviations CO₂ compensation concentration - IRGA infrared gas analysis Published as Paper No. 7068, Journal Series, Nebraska Agricultural Experiment Station