Photosynthetic down-regulation in N2-fixing alfalfa under elevated CO2 alters rubisco content and decreases nodule metabolism via nitrogenase and tricarboxylic acid cycle

Although responsiveness of N₂-fixing plants to elevated CO₂ conditions have been analyzed in previous studies, important uncertainties remain in relation to the effect enhanced CO₂ in nodule proteomic profile and its implication in leaf responsiveness. The aim of our study was to deepen our understanding of the relationship between leaf and nodule metabolism of N₂-fixing alfalfa plants after long-term exposure to elevated CO₂. After 30-day exposure to elevated CO₂, plants showed photosynthetic down-regulation with reductions in the light-saturated rate of CO₂ assimilation (A _sat) and the maximum rate of rubisco carboxylation (Vc_max). Under elevated CO₂ conditions, the rubisco availability limited potential photosynthesis by around 12 %, which represented the majority of the observed fall in Vc_max. Photosynthetic down-regulation has been associated with decreased N availability even if those plants are capable to assimilate N₂. Diminishment in shoot N demand (as reflected by the lower rubisco and leaf N content) suggests that the lower aboveground N requirements affected negatively nodule performance. In this condition, specific nodule activity was reduced due to an effect on nodule metabolism that manifested as a lower amount of nitrogenase reductase. Moreover, the nodule proteomic approach also revealed that nodule functioning was altered simultaneously in various enzyme quantity apart from nitrogenase. At elevated CO₂, the tricarboxylic acid cycle was also altered with a reduced amount of isocitrate synthase protein. The nodule proteome analysis also revealed the relaxation of the antioxidant system as shown by a decline in the amount of catalase and isoflavone reductase protein.     

The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants

We investigated the effects of an autumn sowing of contrasting cover crops (oats, rye and a combination of oats and rye) on soil aggregate stability, mycorrhizal colonization, phosphorus uptake and yield of sweet corn planted the following summer. Rye is a common cover crop in the middle Atlantic region of the United States of America. It grows slowly in the autumn, survives the winter, grows rapidly in the spring and flowers in the summer. Thus, herbicide is commonly used to kill rye prior to planting spring crops. Oats, in contrast, grows rapidly in the autumn but is killed by frost during the winter. Thus, with oats, potentially less herbicide is needed to prepare the field for spring planting. When compared to fallow, oats was as effective as rye in increasing mycorrhizal colonization of sweet corn, density of mycorrhizal hyphae, and soil aggregate stability. An oats cover crop may thus be a viable alternative to rye. The combination of cover crops (rye and oats), however, was significantly better than single species of cover crops in terms of sweet corn mycorrhizal colonization, P uptake and yield of sweet corn.     

Natural 15N abundance of presumed N2-fixing and non-N2-fixing plants from selected ecosystems

Summary The ¹⁵N/¹⁴N ratios of plant and soil samples from Northern California ecosystems were determined by mass spectrometry. The ¹⁵N abundance of 176 plant foliar samples averaged 0.0008 atom % ¹⁵N excess relative to atmospheric N₂ and ranged from-0.0028 to 0.0064 atom % ¹⁵N excess relative to atmospheric N₂. Foliage from reported N₂-fixing species had significantly lower mean ¹⁵N abundance (relative to atmospheric N₂ and total soil N) and significantly higher N concentration (% N dry wt.) than did presumed non-N₂-fixing plants growing on the same sites. The mean difference between N₂-fixing species and other plants was 0.0007 atom % ¹⁵N. N₂-fixing species had lower ¹⁵N abundance than the other plants on most sites examined despite large differences between sites in vegetation, soil, and climate. The mean ¹⁵N abundance of N₂-fixing plants varied little between sites and was close to that of atmospheric N₂. The ¹⁵N abundance of presumed non-N₂-fixing species was highest at coastal sites and may reflect an input of marine spray N having relatively high ¹⁵N abundance. The ¹⁵N abundance of N₂-fixing species was not related to growth form but was for other plants. Annual herbaceous plants had highest ¹⁵N abundance followed in decreasing order by perennial herbs, shrubs, and trees. Several terrestrial ferns (Pteridaceae) had ¹⁵N abundances comparable to N₂-fixing legumes suggesting N₂-fixation by these ferns. On sites where the ¹⁵N abundance of soil N differs from that of the atmosphere, N₂-fixing plants can be identified by the natural ¹⁵N abundance of their foliage. This approach can be useful in detecting and perhaps measuring N₂-fixation on sites where direct recovery of nodules is not possible.     

Elevated CO2 alleviates the effects of low P on the growth of N2-fixing Acacia auriculiformis and Acacia mangium

Nodulated seedlings of Acacia auriculiformis Cunn. ex Benth and Acacia mangium Willd were grown with different phosphorus (P) regimes for 90 days, and half of them were exposed to elevated CO₂ (800 μl l⁻¹) during the last 30 days. Under ambient CO₂, plant growth and the amount of N fixed symbiotically in N₂-fixing seedlings decreased with the decrease of supplied P; this relationship did not occur under elevated CO₂. The increase in plant biomass by elevated CO₂ at low P was accompanied by the increase in internal P use efficiency, the amount of N fixed symbiotically and N use efficiency. Elevated CO₂ recovered the low P-induced reduction in leaf dry matter per unit area or unit fresh weight, but it had no effect on the low P-induced increase in partitioning dry matter to roots. These results suggest that elevated CO₂ alleviates the low P effect mainly by increasing the use efficiency of internal P for plant growth and symbiotic N₂ fixation, and the source-sink relationship is possibly an important driving force for this effect of elevated CO₂ in A. auriculiformis and A. mangium.     

Effect of elevated temperature and water availability on CO2 exchange and nitrogen fixation of nodulated alfalfa plants

The aim of this study was to investigate the effects of predicted temperature increases and drought conditions of Mediterranean environments on N₂-fixing alfalfa plants. One-month-old plants inoculated with Sinorhizobium meliloti strain 102F78 were grown in growth chambers under different temperature (25/15 or 28/18 °C, day/night) and water availability (control or drought) regimes. Elevated temperature and drought reduced plant dry mass and leaf area, especially when both stresses were combined. The inhibitory effect of elevated temperature on plant growth was a consequence of decreased CO₂ and N₂ fixation rates. A photosynthetic decrease resulted from the inhibition of rubisco activity, probably associated with a lower activation state. An absence of differences in photosynthesis in relation to water availability suggests that drought decreased plant growth due to its negative effect on leaf area. Rising temperature and drought affected the nitrogen content negatively, although effects differed. Elevated temperature inhibited nodule activity drastically, whereas the inhibitory effect resulting from drought centred on nodule dry mass (DM) production. Plants exposed to a combination of elevated temperature and drought were the most negatively affected.     

Molecular basis of the establishment and functioning of a N2-fixing root nodule

Compatible interactions between rhizobia and their leguminous host plant(s) culminate in the formation of a new plant organ, the root nodule. Within this structure, the bacteria reduce N₂ to NH₃ which is then assimilated by the plant. The formation of a N₂-fixing nodule requires a continuous process of two-way signalling and cellular recognition between the prokaryote and the plant. Such a process involves the sequential activation and/or repression of host plant- and bacteria-encoded genes. Finally, functioning of a legume-nodule necessitates not only the adaptation of plant and bacterial carbon, nitrogen and oxygen metabolism to an environment allowing N₂-fixation to occur, but also requires a tight co-ordination and integration of these plant and bacterial metabolic processes.     

Nitrogenase activity and N2 fixation are stimulated by elevated CO2 in a tropical N2-fixing tree

 Seeds of Gliricidia sepium, a fast-growing woody legume native to seasonal tropical forests of Central America, were inoculated with N₂-fixing Rhizobium bacteria and grown in environmentally controlled glasshouses for 67–71 days under ambient CO₂ (35 Pa) and elevated CO₂ (70 Pa) conditions. Seedlings were watered with an N-free, but otherwise complete, nutrient solution such that bacterial N₂ fixation was the only source of N available to the plant. The primary objective of our study was to quantify the effect of CO₂ enrichment on the kinetics of photosynthate transport to nodules and determine its subsequent effect on N₂ fixation. Photosynthetic rates and carbon storage in leaves were higher in elevated CO₂ plants indicating that more carbon was available for transport to nodules. A ¹⁴CO₂ pulse-chase experiment demonstrated that photosynthetically fixed carbon was supplied by leaves to nodules at a faster rate when plants were grown in elevated CO₂. Greater rates of carbon supply to nodules did not affect nodule mass per plant, but did increase specific nitrogenase activity (SNA) and total nitrogenase activity (TNA) resulting in greater N₂ fixation. In fact, a 23% increase in the rate of carbon supplied to nodules coincided with a 23% increase in SNA for plants grown in elevated CO₂, suggesting a direct correlation between carbon supply and nitrogenase activity. The improvement in plant N status produced much larger plants when grown in elevated CO₂. These results suggest that Gliricidia, and possibly other N₂-fixing trees, may show an early and positive growth response to elevated CO₂, even in severely N-deficient soils, due to increased nitrogenase activity. Received: 27 February 1996 / Accepted: 19 June 1996     

Ecological consequences of the expansion of N2-fixing plants in cold biomes

Research in warm-climate biomes has shown that invasion by symbiotic dinitrogen (N₂)-fixing plants can transform ecosystems in ways analogous to the transformations observed as a consequence of anthropogenic, atmospheric nitrogen (N) deposition: declines in biodiversity, soil acidification, and alterations to carbon and nutrient cycling, including increased N losses through nitrate leaching and emissions of the powerful greenhouse gas nitrous oxide (N₂O). Here, we used literature review and case study approaches to assess the evidence for similar transformations in cold-climate ecosystems of the boreal, subarctic and upper montane-temperate life zones. Our assessment focuses on the plant genera Lupinus and Alnus, which have become invasive largely as a consequence of deliberate introductions and/or reduced land management. These cold biomes are commonly located in remote areas with low anthropogenic N inputs, and the environmental impacts of N₂-fixer invasion appear to be as severe as those from anthropogenic N deposition in highly N polluted areas. Hence, inputs of N from N₂ fixation can affect ecosystems as dramatically or even more strongly than N inputs from atmospheric deposition, and biomes in cold climates represent no exception with regard to the risk of being invaded by N₂-fixing species. In particular, the cold biomes studied here show both a strong potential to be transformed by N₂-fixing plants and a rapid subsequent saturation in the ecosystem’s capacity to retain N. Therefore, analogous to increases in N deposition, N₂-fixing plant invasions must be deemed significant threats to biodiversity and to environmental quality.     

Estimation of N2 fixation based on differences in the natural abundance of 15N among freshwater N2-fixing and non-N2-fixing algae

The hypothesis that small mammal burrows can increase the amount of water infiltrating into the soil profile was tested. The amount of water added to the soil profile from spring recharge in areas adjacent to ground squirrel (Spermophilus townsendii and S. elegans) burrows was compared to nearby areas without burrows. Recharge amounts in burrow areas were significantly higher than nonburrow areas. An average of 21% more of the winter precipitation infiltrated into the soil near burrows. The amount of recharge was also found to be positively related to burrow density. Burrows also affected the distribution of the recharge by adding significantly more water to the deeper portions (>50 cm) of the soil profile.     

Contrasting growth response of an N2-fixing and non-fixing forb to elevated CO2: dependence on soil N supply

With the ability to symbiotically fix atmospheric N₂, legumes may lack the N-limitations thought to constrain plant response to elevated concentrations of atmospheric CO₂. The growth and photosynthetic responses of two perennial grassland species were compared to test the hypotheses that (1) the CO₂ response of wild species is limited at low N availability, (2) legumes respond to a greater extent than non-fixing forbs to elevated CO₂, and (3) elevated CO₂ stimulates symbiotic N₂ fixation, resulting in an increased amount of N derived from the atmosphere. This study investigated the effects of atmospheric CO₂ concentration (365 and 700 mol mol^–1) and N addition on whole plant growth and C and N acquisition in an N₂-fixing legume (Lupinus perennis) and a non-fixing forb (Achillea millefolium) in controlled-chamber environments. To evaluate the effects of a wide range of N availability on the CO₂ response, we incorporated six levels of soil N addition starting with native field soil inherently low in N (field soil + 0, 4, 8, 12, 16, or 20 g N m^–2 yr^–1). Whole plant growth, leaf net photosynthetic rates (A), and the proportion of N derived from N₂ fixation were determined in plants grown from seed over one growing season. Both species increased growth with CO₂enrichment, but this response was mediated by N supply only for the non-fixer, Achillea. Its response depended on mineral N supply as growth enhancements under elevated CO₂ increased from 0% in low N soil to +25% at the higher levels of N addition. In contrast, Lupinus plants had 80% greater biomass under elevated CO₂ regardless of N treatment. Although partial photosynthetic acclimation to CO₂ enrichment occurred, both species maintained comparably higher A in elevated compared to ambient CO₂ (+38%). N addition facilitated increased A in Achillea, however, in neither species did additional N availability affect the acclimation response of A to CO₂. Elevated CO₂ increased plant total N yield by 57% in Lupinus but had no effect on Achillea. The increased N in Lupinus came from symbiotic N₂ fixation, which resulted in a 47% greater proportion of N derived from fixation relative to other sources of N. These results suggest that compared to non-fixing forbs, N₂-fixers exhibit positive photosynthetic and growth responses to increased atmospheric CO₂ that are independent of soil N supply. The enhanced amount of N derived from N₂ fixation under elevated CO₂ presumably helps meet the increased N demand in N₂-fixing species. This response may lead to modified roles of N₂-fixers and N₂-fixer/non-fixer species interactions in grassland communities, especially those that are inherently N-poor, under projected rising atmospheric CO₂.     

CO 2 -fixing enzymes in a marine psychophile

A psychrophilic marine Pseudomonas was found to contain phosphoenolpyruvate (PEP) carboxylase and an adenosine triphosphate-linked PEP carboxykinase. Some properties of these CO(2)-fixing enzymes were compared with those homologous enzymes from the terrestrial mesophile Enterobacter cloacae. The PEP carboxylases from both organisms were activated by acetyl-coenzyme A (CoA) and inhibited by l-aspartate. The enzyme from Pseudomonas was less dependent on the presence of the activator, but maximal activation was attained at acetyl-CoA concentrations much lower (50 mum) than those required to saturate the enzyme from E. cloacae. In both cases the main effect of acetyl-CoA was to decrease the K(m) value for PEP. The activity of PEP carboxylase from Pseudomonas was only slightly inhibited by NaCl, KCl, or NH(4)Cl up to 100 mm, whereas the enzyme from E. cloacae was inhibited by about 70% under similar experimental conditions. Both PEP carboxylase and PEP carboxykinase from Pseudomonas showed considerably lower thermal stability than their counterparts from E. cloacae. Our results suggest that the CO(2)-fixing enzymes from a marine Pseudomonas and E. cloacae are similar in nature and regulation, but they differ in properties related to the peculiar conditions of the marine environment.     

A N2-fixing endophytic Burkholderia sp. associated with maize plants cultivated in Mexico

In the frame of a survey of potentially endophytic N2-fixing Burkholderia associated with maize in Mexico, its country of origin, the soil of an indigenous maize field near Oaxaca was studied. Under laboratory conditions, plant seedlings of two ancient maize varieties were used as a trap to select endophyte candidates from the soil sample. Among the N2 fixers isolated from inside plant tissues and able to grow on PCAT medium, the most abundant isolates belonged to genus Burkholderia (API 20NE, rrs sequences). Representative isolates obtained from roots and shoots of different plants appeared identical (rrs and nifH RFLP), showing that they were closely related. In addition, their 16S rDNA sequences differed from described Burkholderia species and, phylogenetically, they constituted a separate deep-branching new lineage in genus Burkholderia. This indicated that these isolates probably constituted a new species. An inoculation experiment confirmed that these N2-fixing Burkholderia isolates could densely colonize the plant tissues of maize. More isolates of this group were subsequently obtained from field-grown maize and teosinte plants. It was hypothesized that strains of this species had developed a sort of primitive symbiosis with one of their host plants, teosinte, which persisted during the domestication of teosinte into maize.     

Elevated CO2 and the mineral content of herbaceous and woody plants

The CO₂ enrichment effects (300–650 µmol mol^-1) on mineral concentration (N, P, K, Ca, Mg, Mn, Fe, Zn), absolute total mineral contents per individual and of whole stands of four herbaceous (Trifolium repens L.,Trifolium pratense L.,Lolium perenne L.,Festuca pratensis HUDS.) and two woody species (Acer pseudo-platanus L.,Fagus sylvatica L.) were investigated.In general, the mineral concentration of the plant tissues decreased (all six species: N>Ca>K>Mg) with the exception of P. Mn and Fe were only determined for the tree species. Both decreased in concentration (Mn>Fe). Zn was only analysed forTrifolium pratense andFestuca pratensis and decreased significantly in the grass.Despite of decreases in concentrations of as much as 20% in some cases there were increases in absolute amounts per individual and, therefore, in the whole vegetation up to 25% because of the enhanced dry matter accumulation at elevated CO₂ supply.Dedicated to Prof. Dr. R. Bornkamm, TU-Berlin, on behalf of his 60th birthday     

Association between N2-fixing bacteria and pearl millet plants: Responses,mechanisms and persistence

Responses to inoculation with N₂-fixing bacteria were studied in relation to genotypic differences in pearl millet, effect of nitrogen levels, and FYM additions in India. In some experiments, inoculation increased mean grain yield up to 33% over the uninoculated control, whereas in the remaining 11 experiments there was no significant increase. Increased grain yields, >10% over the uninoculated controls were observed in 46% of the experiments withAzospirillum lipoferum (18.7% average increase) and withAzotobacter chroococcum (13.6% average increase). Yield increases were nil or reduced in three experiments withAzos. lipoferum and four experiments withAztb. chroococcum. In two experiments continued inoculation for two or three years resulted in increased grain, plant biomass yield, and N uptake. Interactions of bacterial cultures with cultivars or years were not observed. The counts of the inoculated strains increased two to three-fold when inoculation was continued for three years. Repeated inoculations increased the mean cumulative N uptake from season 1 to season 3 by 19 kg ha^–1. Repeated inoculations withAztb. chroococcum andAzos. lipoferum increased mean grain yield of a succeeding crop by 14.4% and 9.8%, respectively, over the uninoculated control. Inoculation increased the efficiency of N-assimilation by pearl millet. Marginal increase in nitrogenase activity, associated with the inoculated plants was observed during later stages of plant growth. Increased leaf nitrate reductase activity (NRA) was observed after inoculation with these bacteria. The responses to inoculation are mainly attributable to increased plant N assimilation which could be the effect of growth promoting substances secreated by the bacteria; and thus the contribution from BNF may be small.CRISAT, journal article 732.     

Underwater photosynthesis and respiration in leaves of submerged wetland plants: gas films improve CO2 and O2 exchange.

Many wetland plants have gas films on submerged leaf surfaces. We tested the hypotheses that leaf gas films enhance CO(2) uptake for net photosynthesis (P(N)) during light periods, and enhance O(2) uptake for respiration during dark periods. Leaves of four wetland species that form gas films, and two species that do not, were used. Gas films were also experimentally removed by brushing with 0.05% (v/v) Triton X. Net O(2) production in light, or O(2) consumption in darkness, was measured at various CO(2) and O(2) concentrations. When gas films were removed, O(2) uptake in darkness was already diffusion-limited at 20.6 kPa (critical O(2) pressure for respiration, COP(R)>/= 284 mmol O(2) m(-3)), whereas for some leaves with gas films, O(2) uptake declined only at approx. 4 kPa (COP(R) 54 mmol O(2) m(-3)). Gas films also improved CO(2) uptake so that, during light periods, underwater P(N) was enhanced up to sixfold. Gas films on submerged leaves enable continued gas exchange via stomata and thus bypassing of cuticle resistance, enhancing exchange of O(2) and CO(2) with the surrounding water, and therefore underwater P(N) and respiration.     

Photosynthetic CO2-fixing activity in dark-grown spruce seedlings

Bicarbonate-dependent O₂-evolving activity in dark-grown cotyledonsof Picea abies was measured with an oxygen electrode with differentpreillumination times. The activity showed a slight linear increasewith increasing preillumination time. On the other hand, O₂-evolvingactivity (Hill activity) of chloroplasts prepared from preilluminateddark-grown cotyledons exhibited a characteristic change of asteep rise followed by a gradual increase with increasing preilluminationtime. The results obtained were discussed in connection withthe light activation of the latent, inactive O₂-evolving centerin dark-grown cotyledons. (Received December 8, 1978; )     

Elevated CO2 and nutrient addition after soil N cycling and N trace gas fluxes with early season wet-up in a California annual grassland

We examined the effects of growth carbon dioxide (CO₂)concentration and soil nutrient availability on nitrogen (N)transformations and N trace gas fluxes in California grasslandmicrocosms during early-season wet-up, a time when rates of Ntransformation and N trace gas flux are high. After plant senescenceand summer drought, we simulated the first fall rains and examined Ncycling. Growth at elevated CO₂ increased root productionand root carbon:nitrogen ratio. Under nutrient enrichment, elevatedCO₂ increased microbial N immobilization during wet-up,leading to a 43% reduction in gross nitrification anda 55% reduction in NO emission from soil. ElevatedCO₂ increased microbial N immobilization at ambientnutrients, but did not alter nitrification or NO emission. ElevatedCO₂ did not alter soil emission of N₂O ateither nutrient level. Addition of NPK fertilizer (1:1:1) stimulatedN mineralization and nitrification, leading to increased N₂Oand NO emission from soil. The results of our study support a mechanisticmodel in which elevated CO₂ alters soil N cycling and NOemission: increased root production and increased C:N ratio in elevatedCO₂ stimulate N immobilization, thereby decreasingnitrification and associated NO emission when nutrients are abundant.This model is consistent with our basic understanding of how C availabilityinfluences soil N cycling and thus may apply to many terrestrial ecosystems.     

Novel technique for component monitoring of CO2 exchange in plants

A novel technique designed for component monitoring of CO₂ exchange in plants is described. The system is based on application of self-clamping leaf chambers connected to an open gas-exchange measuring system and on automatic recording of CO₂ concentration. This technique was implemented in a commercially available instrument, PTM-48A Photosynthesis Monitor, which provides for long-term measurements of gas exchange and for discrimination of its separate components. Furthermore, many other plant functions can be monitored during plant growth and development under laboratory, greenhouse, and field conditions.     

Growth of White Clover, Dependent on N2 Fixation, in Elevated CO2 and Temperature

Clonal plants of white clover (Trifolium repens L ), whollydependent on N₂ fixation, were grown for 6 weeks in controlledenvironments providing either (C680 regime) 23/18 °C day/nighttemperatures and a CO₂, concentration of 680 µmol mol^–1,or (C340 regime) 20/15 °C day/night temperatures and a CO₂,concentration of 340 µmol mol^–1 During the firsthalf of the experimental period the C680 plants grew fasterthan their C340 counterparts so that by week 3 they were twicethe weight this 2 1 superiority in weight persisted until theend of the experiment The faster initial growth of the C680plants was based on an approx 70 % increase in leaf numbersand an approx 30 % increase in their individual area Initially,specific leaf area (cm2 g^–1 leaf) was lower in C680 thanin C340 leaves but became similar in the latter half of theexperiment Shoot organ weights, including petioles and stolons,reflected the C680 plant's better growth in terms of photosyntheticsurface Throughout, C680 plants invested less of their weightin root than C340 plants and this disparity increased with timeAcetylene reduction assays showed that nitrogenase activityper unit nodule weight was the same in both C680 and C340 plantsBoth groups of plants invested about the same fraction of totalweight in nodules Nitrogen contents of plant tissues were similarirrespective of growth regime, but C680 expanded leaves containedslightly less nitrogen and their stolons slightly more nitrogenthan their C340 counterparts However, C680 leaves containedmore non-structural carbohydrate Young, unshaded C680 leavespossessed larger palisade cells, packed more tightly withinthe leaf, than equivalent C340 leaves The reason for the C680regime's loss of superiority in relative growth rate duringthe second half of the experiment was not clear, but more accumulationof non-structural carbohydrate, constriction of root growthand increased self-shading appear to be the most likely causes Trifolium repens, white clover, elevated CO₂, elevated temperature, growth, N₂ fixation, leaf structure