The Potential of Two Perennial C4 Grasses and a Perennial C4 Sedge as Ligno-cellulosic Fuel Crops in N.W. Europe. Crop Establishment and Yields in E. England

Three perennial C₄ rhizomatous species, Cyperus longus L., Spartinacynosuroides and Spartina pectinata Link, were examined as potentialrenewable energy crops. These species are unusual among C₄ plantsin showing natural distributions which extend into cool temperateregions. This study examined whether these species could beestablished in the cool temperate climate of eastern Englandand whether they could consistently attain the relatively highdry matter yields associated with C₄ plants of warmer regions.Clonally produced material was planted in 1986, on two siteswith contrasting soil types in Essex, eastern England. Plantingwas within a randomized-block design incorporating replicatedplots of each species, both with and without fertilizer. Survivorshipand stem demography were monitored at monthly intervals from1986 to Jun. 1989 for stem recruitment and to Dec. 1991 forstem density. Yields were determined from 1987 (the year followingestablishment) to 1993. Survivorship of the planted propagules over the first 12 monthswas 92% for S. pectinata , 96% for S. cynosuroides and 100%for C. longus. Recruitment of new stems peaked in Apr. of mostyears, although a significant number of new stems appeared asearly as Feb. Stem death peaked in Sep. or early Oct. and allabove-ground stems had died by mid-Nov. Stem density trendsindicated that 2-4 years were required to reach a steady-statedensity, depending upon species. The stem density of the twoSpartina species had reached more than 1000 m^-2 in 1989 althoughthat of S. pectinata fluctuated considerably in the subsequentyears. C. longus stem density rose to approx. 600 m^-2 by 1988and did not change significantly in the subsequent years. In the 6 years following establishment, annual yields averagedacross all fertilizer treatments and both sites were 1·0,1·1 and 1·3 kg m^-2 for C. longus, S. cynosuroidesand S. pectinata, respectively. The average annual yield ofall three species at the site with the heavier soil was 1·3kg m^-2. This was significantly greater than the 1·0 kgm^-2 on the lighter soil. Nitrogen addition did not significantlyincrease yield. Even in the absence of any nitrogen addition,the annual yield of S. pectinata averaged 1·2 kg m^-2over the 6 years, with no evidence of any decline with the increasingage of the stands.Copyright 1995, 1999 Academic Press Cyperus longus, Spartina cynosuroides, Spartina pectinata, energy crop, dry matter yield     

Modeling the Impact of Exotic Annual Brome Grasses on soil Organic Carbon Storage in a Northern Mixed-Grass Prairie

Annual brome grasses, Bromus japonicus and B. tectorum, are common invaders of northern mixed-grass prairie, and have been shown to alter the structure and function of prairie ecosystems, including plant biomass production and litter decomposition. To build on previous findings, our objective was to model the impact of annual brome grasses on soil organic carbon storage as a step towards forecasting ecological change. Specifically, we measured differences in carbon storage between patches dominated by annual bromes and perennial grasses, in addition to evaluating key plant functional characteristics that impact carbon storage. Using the CENTURY model, we simulated high- and low-brome vegetation based on differences in functional characteristics, allowing us to extrapolate the findings from the field study across a broader time scale. We sampled a prairie site in 1996 and 1997 to quantify differences between the high- and low-brome cover plots. High-brome plots averaged 40% brome cover, while the low-brome plots averaged 1% brome cover. We found differences in functional attributes for growth characteristics and litter quality, as well as minor differences in edaphic variables between the plots. Based on field measurements, more soil organic carbon was stored under high-brome vegetation than low-brome, but the differences were not statistically significant. Results from model simulations were consistent with field measurements, and suggested that this prairie ecosystem was not significantly impacted by the functional differences between high- and low-brome vegetation for the first 50 years after the brome invasion under historical management and climate. However, the model results also showed that the differences in soil organic carbon storage continue to diverge after 50 years and consequently could be significant in the future.     

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.     

The Effects of Exotic Grasses on Litter Decomposition in a Hawaiian Woodland: The Importance of Indirect Effects

Exotic grasses and grass-fueled fires have altered plant species composition in the seasonal submontane woodlands of Hawaii Volcanoes National Park. These changes have altered both structural and functional aspects of the plant community, which could, in turn, have consequences for litter decomposition and nitrogen (N) dynamics. In grass-invaded unburned woodland, grass removal plots within the woodland, and woodland converted to grassland by fire, we compared whole-system fluxes and the contributions of individual species to annual aboveground fine litterfall and litterfall N, and litter mass and net N loss. We assessed the direct contribution of grass biomass to decomposition and N dynamics, and we determined how grasses affected decomposition processes indirectly via effects on native species and alteration of the litter layer microenvironment. Grasses contributed 35% of the total annual aboveground fine litterfall in the invaded woodland. However, total litterfall mass and N were not different between the invaded woodland and the grass removal treatment because of compensation by the native tree Metrosideros polymorpha, which increased litter production by 37% ± 5% when grasses were removed. The 0.3 g N m^–2/y^–1 contained in this production increase was equal to the N contained in grass litter. Litter production and litterfall N was lowest in the grassland due to the loss of native litter inputs. Decomposition of litterfall on an area basis was highest in the grass-invaded woodland. We attributed this effect to increased inherent decomposability of native litter in the presence of grasses because (a) the microenvironment of the three vegetation treatments had little effect on decomposition of common litter types and (b) M. polymorpha litter produced in the invaded woodland decomposed faster than that produced in the grass removal plots due to higher lignin concentrations in the latter than in the former. Area-weighted decomposition was lowest in the grassland due to the absence of native litter inputs. Across all treatments, most litter types immobilized N throughout the incubation, and litter net N loss on an area basis was not different among treatments. Our results support the idea that the effects of a plant species or growth form on decomposition cannot be determined in isolation from the rest of the community or from the direct effects of litter quality and quantity alone. In this dry woodland, exotic grasses significantly altered decomposition processes through indirect effects on the quantity and quality of litter produced by native species.     

Responses of wild C4 and C3 grass (Poaceae) species to elevated atmospheric CO2 concentration: a meta-analytic test of current theories and perceptions

C4 plants contribute ≈ 20% of global gross primary productivity, and uncertainties regarding their responses to rising atmospheric CO₂ concentrations may limit predictions of future global change impacts on C4-dominated ecosystems. These uncertainties have not yet been considered rigorously due to expectations of C4 low responsiveness based on photosynthetic theory and early experiments. We carried out a literature review (1980–97) and meta-analysis in order to identify emerging patterns of C4 grass responses to elevated CO₂, as compared with those of C3 grasses. The focus was on nondomesticated Poaceae alone, to the exclusion of C4 dicotyledonous and C4 crop species. This provides a clear test, controlled for genotypic variability at family level, of differences between the CO₂-responsiveness of these functional types. Eleven responses were considered, ranging from physiological behaviour at the leaf level to carbon allocation patterns at the whole plant level. Results were also assessed in the context of environmental stress conditions (light, temperature, water and nutrient stress), and experimental growing conditions (pot size, experimental duration and fumigation method). Both C4 and C3 species increased total biomass significantly in elevated CO₂, by 33% and 44%, respectively. Differing tendencies between types in shoot structural response were revealed: C3 species showed a greater increase in tillering, whereas C4 species showed a greater increase in leaf area in elevated CO₂. At the leaf level, significant stomatal closure and increased leaf water use efficiency were confirmed in both types, and higher carbon assimilation rates were found in both C3 and C4 species (33% and 25%, respectively). Environmental stress did not alter the C4 CO₂-response, except for the loss of a significant positive CO₂-response for above-ground biomass and leaf area under water stress. In C3 species, stimulation of carbon assimilation rate was reduced by stress (overall), and nutrient stress tended to reduce the mean biomass response to elevated CO₂. Leaf carbohydrate status increased and leaf nitrogen concentration decreased significantly in elevated CO₂ only in C3 species. We conclude that the relative responses of the C4 and C3 photosynthetic types to elevated CO₂ concur only to some extent with expectations based on photosynthetic theory. The significant positive responses of C4 grass species at both the leaf and the whole plant level demand a re-evaluation of the assumption of low responsiveness in C4 plants at both levels, and not only with regard to water relations. The combined shoot structural and water use efficiency responses of these functional types will have consequential implications for the water balance of important catchments and range-lands throughout the world, especially in semiarid subtropical and temperate regions. It may be premature to predict that C4 grass species will lose their competitive advantage over C3 grass species in elevated CO₂.     

The activities of PEP carboxylase and the C4 acid decarboxylases are little changed by drought stress in three C4 grasses of different subtypes

The C(4) photosynthetic pathway involves the assimilation of CO(2) by phosphoenolpyruvate carboxylase (PEPC) and the subsequent decarboxylation of C(4) acids. The enzymes of the CO(2) concentrating mechanism could be affected under water deficit and limit C(4) photosynthesis. Three different C(4) grasses were submitted to gradually induced drought stress conditions: Paspalum dilatatum (NADP-malic enzyme, NADP-ME), Cynodon dactylon (NAD-malic enzyme, NAD-ME) and Zoysia japonica (PEP carboxykinase, PEPCK). Moderate leaf dehydration affected the activity and regulation of PEPC in a similar manner in the three grasses but had species-specific effects on the C(4) acid decarboxylases, NADP-ME, NAD-ME and PEPCK, although changes in the C(4) enzyme activities were small. In all three species, the PEPC phosphorylation state, judged by the inhibitory effect of L: -malate on PEPC activity, increased with water deficit and could promote increased assimilation of CO(2) by the enzyme under stress conditions. Appreciable activity of PEPCK was observed in all three species suggesting that this enzyme may act as a supplementary decarboxylase to NADP-ME and NAD-ME in addition to its role in other metabolic pathways.     

Relative nutritional quality of C3 and C4 grasses for a graminivorous lepidopteran,Paratrytone melane (Hesperiidae)

Summary We tested the hypothesis that C₄ grasses are inferior to C₃ grasses as host plants for herbivorous insects by measuring the relative performance of larvae of a graminivorous lepidopteran, Paratrytone melane (Hesperiidae), fed C₃ and C₄ grasses. Relative growth rates and final weights were higher in larvae fed a C₃ grass in Experiment I. However, in two additional experiments, relative growth rates and final weights were not significantly different in larvae fed C₃ and C₄ grasses. We examined two factors which are believed to cause C₄ grasses to be of lower nutritional value than C₃ grasses: foliar nutrient levels and nutrient digestibility. In general, foliar nutrient levels were higher in C₃ grasses. In Experiment I, protein and soluble carbohydrates were digested from a C₃ and a C₄ grass with equivalent efficiencies. Therefore, differences in larval performance are best explained by higher nutrient levels in the C₃ grass in this experiment. In Experiment II, soluble carbohydrates were digested with similar efficiencies from C₃ and C₄ grasses but protein was digested with greater efficiency from the C₃ grasses. We conclude (1) that the bundle sheath anatomy of C₄ grasses is not a barrier to soluble carbohydrate digestion and does not have a nutritionally significant effect on protein digestion and (2) that P. melane may consume C₄ grasses at compensatory rates.     

Water relations of native and introduced C4 grasses in a neotropical savanna

Introduced African grasses are invading Neotropical savannas and displacing the native herbaceous community. This work, which is part of a program to understand the success of the African grasses, specifically investigates whether introduced and native grasses differ in their water relations. The water relations of the native Trachypogon plumosus and the successful invader Hyparrhenia rufa were studied in the field during two consecutive years in the seasonal savannas of Venezuela. The two C₄ grasses differed clearly in their responses to water stress. H. rufa consistently had higher stomatal conductance, transpiration rate, leaf water and osmotic potential and osmotic adjustment than the native T. plumosus. Also, leaf senescence occurred much earlier during the dry season in H. rufa. Both grasses showed a combination of water stress evasion and tolerance mechanisms such as stomatal sensitivity to atmospheric or soil water stress, decreased transpiring area and osmotic adjustment. Evasion mechanisms are more conspicuous in H. rufa whereas T. plumosus is more drought tolerant and uses water more conservatively. The evasion mechanisms and oportunistic use of water by H. rufa, characteristic of invading species, contribute to, but only partially explain, the success of this grass in the Neotropical savannas where it displaces native plants from sites with better water and nutrient status. Conversely, the higher water stress tolerance of t. plumosus is consistent with its capacity to resist invasion by alien grasses on shallow soils and sites with poorer nutrient and water status.     

Dynamics of energy and nutrient concentration and construction cost in a native and two alien C4 grasses from two neotropical savannas

In Venezuela, the alien grasses Melinis minutiflora Beauv. and Hyparrhenia rufa (Nees.) Stapf tend to displace the native savanna plant community dominated by Trachypogon plumosus (Humb. and Bonpl.) Nees. This occurs in either relatively wetter and fertile highland savannas or in drier and less fertile lowland savannas. Although the native and aliens are perennial C₄ grasses, higher net assimilation leaf biomass per plant and germination rate of the latter are some causes for their higher growth rates and for their competitive success. The objective of this study is to compare seasonal tissue energy, N, P and K concentrations and the calculated construction costs (CC) between the native grass and either one of the alien grasses from lowland and highland savannas. We predict that, in order to out-compete native plants, alien grasses should be more efficient in resource use as evidenced by lower tissue energy and nutrient concentrations and CC.Tissue energy and nutrient concentration were measured throughout the year and compared between M. minutiflora and the co-occurring local population of T. plumosus in a highland savanna and between H. rufa and its neighbor local population of T. plumosus in a lowland savanna. CC was calculated from energy, N and ash concentrations considering ammonium as the sole N source. Differences between co-occurring species, T. plumosus populations, seasons, and organs were analyzed with ANOVA.Highland and lowland grasses differed in concentration and allocation of energy and nutrients whereas the differences between alien and native grasses were specific for each pair considered. Highland grasses had higher energy, N, P and CC than lowland grasses. These variables were always lowest in the culms. In the more stressed lowland site, tissue energy and nutrient concentrations decreased significantly during the dry season except in the roots of both grasses which had the highest energy and nutrients concentrations during the drought. This seasonal response was more marked in the local lowland population of T. plumosus in which maximum CC alternated seasonally between leaves and roots. Energy and nutrient concentrations and CC were the lowest in H. rufa. In the lowland savannas, the higher efficiency of resource use in the invader grass contributes to its higher competitive success through increased growth rate. In the highlands, overall tissue energy concentration and CC, but not N nor P concentration, were lower in the fast growing M. minutiflora but seasonal differences were lacking. The higher leaf CC in T. plumosus can be attributed to the higher proportion of sclerenchyma tissue which is more expensive to construct. Considering CC, both fast growing alien grasses are more efficient in resource use than the co-occurring native grass. However, the role of CC explaining the competitive success of the former, through higher growth rates, is more evident in the more stressful environment of the lowland savanna.     

The Potential of Thiarubrine C as a Nematicidal Agent against Plant- parasitic Nematodes

Thiarubrine C, a polyacetylenic 1,2-dithiin isolated from the roots of Rudbeckia hirta (Asteraceae), exhibited strong nematicidal activity in in vitro and growth chamber assays. Thiarubrine C was toxic, in the absence of light, to the plant-parasitic nematodes Meloidogyne incognita and Pratylenchus penetrans at LC₅₀s of 12.4 ppm and 23.5 ppm, respectively. A minimum exposure time between 12 and 24 hours was the critical period for nematode mortality due to thiarubrine C. Although thiarubrine C was not totally dependent on light for toxicity, activity was enhanced in the presence of light, especially with the microbivorous nematode, Teratorhabditis dentifera. Upon exposure of M. incognita juveniles to 20 ppm thiarubrine C for 1 hour, infection of tomato plants was greatly reduced compared to untreated checks. Thiarubrine C was also effective in reducing plant infection when mixed with soil 24 hours prior to or at planting, unlike other related compounds such as δ-terthienyl.     

Water-use efficiency of one C3 and two C4 grasses in response to varying soil moisture and herbage-removal levels in a seasonally dry tropical region

In a seasonally dry tropical region the water use efficiency (WUE) of three grasses (C₃ winter annualPolypogon monspeliensis, C₄ perennialDichanthium annulatum and C₄ warm seasonal annualEchinochloa colonum) was evaluated during summer and winter under nine experimental conditions (3 soil moisture×3 herbage removal). Generally leaf water status and transpiration rate decreased with soil moisture stress and increased with clipping intensity. During winter the transpiration rate of Dichanthium was much lower than that of Polypogon and its own rate in summer. Both soil moisture stress and clipping intensity increased the WUE in all instances. Despite differences in photosynthetic type, growing season and life form, these grasses exhibited broadly similar positive relationships, across nine treatments for WUE: soil moisture stress, and water consumption: production. The range of WUE (g. mm^–1) calculated on TNP through the nine treatments was: summer—Dichanthium 2.9–10.0, Echinochloa 2.0–6.7; winter—Dichanthium 4.3–36.3, Polypogon 1.9–12.0.     

Screening Marine Fungi for Inhibitors of the C4 Plant Enzyme Pyruvate Phosphate Dikinase: Unguinol as a Potential Novel Herbicide Candidate

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C₄ plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 ± 0.8 μM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C₄ plant but no effect on a model C₃ plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.     

Wordbirds: Developing a Web-based Data Collection System for the Global Monitoring of Bird Distribution and Abundance

There is an urgent need to develop simple and effective methods for monitoring bird populations that are cheap to deploy in resource-poor countries. This paper describes a newly developed system, provisionally referred to as, Wordbirds, that will provide a platform for the collection, storage and retrieval of new and existing data from bird observations recorded worldwide. This Internet-based global network of databases will capture field lists and ad hoc sightings routinely gathered by individuals observing birds recreationally and professionally. Huge numbers of lists are collected annually and could provide information on population trends spanning many years. By collecting these records, a valuable resource will be secured with the potential to map and monitor bird distributions and estimate trends in species abundance. An erratum to this article is available at .     

The occurrence of both C3 and C4 photosynthetic characteristics in a single Zea mays plant

The activities of the carboxylating enzymes ribulose-1,5-biphosphate (RuBP) carboxylase and phosphoenolpyruvate (PEP) carboxylase in leaves of three-week old Zea mays plants grown under phytotron conditions were found to vary according to leaf position. In the lower leaves the activity of PEP carboxylase was lower than that of RuBP carboxylase, while the upper leaves exhibited high levels of PEP carboxylase. Carbon dioxide compensation points and net photosynthetic rates also differed in the lower and upper leaves. Differences in the fine structure of the lowermost and uppermost leaves are shown. The existence of both the C₃ and C₄ photosynthetic pathways in the same plant, in this and other species, is discussed.Abbreviations PEP phosphoenolpyruvate - RuBP ribulose-1,5-biphosphate     

Contrasting physiological responsiveness of establishing trees and a C4 grass to rainfall events,intensified summer drought,and warming in oak savanna

Climate warming and drought may alter tree establishment in savannas through differential responses of tree seedlings and grass to intermittent rainfall events. We investigated leaf gas exchange responses of dominant post oak savanna tree (Quercus stellata and Juniperus virginiana) and grass (Schizachyrium scoparium, C₄ grass) species to summer rainfall events under an ambient and intensified summer drought scenario in factorial combination with warming (ambient, +1.5 °C) in both monoculture and tree‐grass mixtures. The three species differed in drought resistance and response of leaf gas exchange to rainfall events throughout the summer. S. scoparium experienced the greatest decrease in A_area (?56% and ?66% under normal and intensified drought, respectively) over the summer, followed by Q. stellata (?44%, ?64%), while J. virginiana showed increased A_area under normal drought (+13%) and a small decrease in A_area when exposed to intensified summer drought (?10%). Following individual rainfall events, mean increases in A_area were 90% for S. scoparium, 26% for J. virginiana and 22% for Q. stellata. The responsiveness of A_area of S. scoparium to rainfall events initially increased with the onset of drought, but decreased dramatically as summer drought progressed. For Q. stellata, A_area recovery decreased as drought progressed and with warming. In contrast, J. virginiana showed minimal fluctuations in A_area following rainfall events, in spite of declining water potential, and warming enhanced recovery. J. virginiana will likely gain an advantage over Q. stellata during establishment under future climatic scenarios. Additionally, the competitive advantage of C₄ grasses may be reduced relative to trees, because grasses will likely exist below a critical water stress threshold more often in a warmer, drier climate. Recognition of unique species responses to critical global change drivers in the presence of competition will improve predictions of grass–tree interactions and tree establishment in savannas in response to climate change.     

The grass leaf developmental gradient as a platform for a systems understanding of the anatomical specialization of C(4) leaves

C(4) photosynthesis relies on spatial and quantitative specializations of common features of leaf anatomy, including venation pattern, bundle sheath cell and chloroplast differentiation, plasmodesmatal abundance, and secondary cell wall enhancement. It has thus far been challenging to dissect the molecular basis for these C(4)-specific alterations in spatial and quantitative patterns of regulation. The target downstream networks of genes and protein interactions that produce these fundamental anatomical features in both C(4) and C(3) species are poorly understood. The developing leaves of monocot grasses provide a base-to-tip gradient of developmental stages that can provide the platform for comprehensive molecular and anatomical data that can yield a better understanding both of the regulators and the targets that produce C(4) patterns, through a variety of gene discovery and systems analysis strategies.     

The occurrence of C4 photosynthesis in Yunnan province, a tropical region in South-western China

Floristic composition, morphological functional types, and altitudinal distribution pattern for C₄ species were studied in Yunnan province, South-western China. 159 species, in 6 families and 60 genera, were identified with C₄ photosynthesis. 93 % of these C₄ species were found in Monocotyledoneae, e.g. Cyperaceae (18 species), Gramineae (129 species), and Commelinaceae (1 species), the other 7 % was in Dicotyledoneae, e.g. Amaranthaceae (5 species), Portulacaceae (4 species), and Chenopodiaceae (2 species). Hence C₄ plants mainly occurred in very few families in the tropical region. Compared with those in semi-arid grasslands and arid deserts in North China, more C₄ grasses and much less Chenopodiaceae C₄ species occurred in the tropical region. This indicates the physiological responses of C₄ plants from the two families are very different. Chenopodiaceae C₄ species may be more fit semi-arid and arid environments, while C₄ grasses are more fit the moist tropical conditions. There was a strong relationship between C₄ distribution and altitude in the tropical region. Altitudinal distribution pattern for C₄ species in the region was consistent with altitude, climate, and habitats.     

Fluorescence microscopy and radiolabeling of C3 and C4 chloroplasts using diisothiocyanatostilbene disulfonic acid as a marker for the phosphate translocator

The usefulness of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C₃ plant) and maize (Zea mays L.) (C₄ plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [³H₂]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, M_r, 30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [³H₂]DIDS. However, when broken chloroplasts were incubated with [³H₂]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [³H₂]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the 30-kDa protein — the presumptive phosphate translocator — in C₃ and C₄ chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the 30-kDa protein in the inner membrane of the envelope.Abbreviations CBB Coomassie brilliant blue - DIC differential interference contrast optics - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - [³H₂]DIDS 1,2-ditritio-1,2-(2,2-disulfo-4,4-diisothiocyano)diphenylethane - kDa kilodalton - M_r relative molecular mass - PGA 3-phosphoglycerate - Pitranslocator phosphate translocator - SDS sodium dodecyl sulfate