Molecular characterisation of a new mutant allele of the plastid phosphoglucomutase in Arabidopsis, and complementation of the mutant with the wild-type cDNA

Screening of transposon-associated mutants of Arabidopsis thaliana for altered starch metabolism resulted in the isolation of a mutant that did not accumulate starch in any tissue or at any developmental stage (starch-free mutant, stf1). Allelism tests with known mutants showed that stf1 represents a new mutant allele of the plastid isoform of the enzyme phosphoglucomutase (PGMp). The mutation was mapped to chromosome 5. An Arabidopsis EST that showed significant homology to the cytosolic isoform of phosphoglucomutase (PGM) from maize was able to complement the mutant phenotype. The Arabidopsis EST was transcribed and translated in vitro and the protein product was efficiently imported into isolated chloroplasts and processed to its mature form. The lack of starch biosynthesis in stf1 is accompanied by the accumulation of soluble sugars. The rate of CO₂ assimilation measured in individual leaves was substantially diminished only under conditions of high CO₂ and low O₂. Remarkably, stf1 exhibits an increase rather than a decrease in total leaf PGM activity, suggesting an induction of the cytosolic isoform(s) in the mutant. The substrate for PGM, glucose 6-phosphate, accumulated in stf1 during the day, resulting in 10-fold higher content than in the wild type at the end of the photoperiod. Received: 4 January 2000 / Accepted: 21 March 2000     

Influence of atmospheric oxygen on leaf structure and starch deposition in Arabidopsis thaliana

Plant culture in oxygen concentrations below ambient is known to stimulate vegetative growth, but apart from reports on increased leaf number and weight, little is known about development at subambient oxygen concentrations. Arabidopsis thaliana (L.) Heynh. (cv. Columbia) plants were grown full term in pre-mixed atmospheres with oxygen partial pressures of 2·5, 5·1, 10·1, 16·2, and 21·3 kPa O₂, 0·035 kPa CO₂ and the balance nitrogen under continuous light. Fully expanded leaves were harvested and processed for light and transmission electron microscopy or for starch quantification. Growth in subambient oxygen concentrations caused changes in leaf anatomy (increased thickness, stomatal density and starch content) that have also been described for plants grown under carbon dioxide enrichment. However, at the lowest oxygen treatment (2·5 kPa), developmental changes occurred that could not be explained by changes in carbon budget caused by suppressed photorespiration, resulting in very thick leaves and a dwarf morphology. This study establishes the leaf parameters that change during growth under low O₂, and identifies the lower concentration at which O₂ limitation on transport and biosynthetic pathways detrimentally affects leaf development.     

Arabidopsis thaliana carbonic anhydrase: cDNA sequence and effect of CO2 on mRNA levels

A full-length cDNA clone encoding carbonic anhydrase was isolated from an Arabidopsis thaliana (Columbia) leaf library. Comparison of the derived amino acid sequence obtained from this clone with those of pea and spinach reveals a considerable degree of identity. The carbonic anhydrase cDNA was used to probe the level of RNA encoding this protein in the leaves of plants grown in elevated CO₂ (660 ppm). We have found that under these conditions the steady-state level of carbonic anhydrase mRNA was increased in comparison with control plants grown in normal atmospheric concentrations of CO₂ (330 ppm). This raises the intruiging possibility that there exists in higher plants a mechanism for perceiving and responding to changes in environmental CO₂ concentrations at the genetic level.     

Oxygen and electron flow in C4 photosynthesis: Mehler reaction, photorespiration and CO2 concentration in the bundle sheath

The photosynthetic linear electron transport rate in excess of that used for CO₂ reduction was evaluated in Sorghum bicolor Moench. [NADP-malic enzyme (ME)-type C₄ plant], Amaranthus cruentus L. (NAD-ME-type C₄ plant) and Helianthus annuus L. (C₃ plant) leaves at different CO₂ and O₂ concentrations. The electron transport rate (J _F) was calculated from fluorescence using the light partitioning factor (relative PSII cross-section) determined under conditions where excess electron transport was assumed to be negligible: low light intensities, 500 μmol CO₂ · mol⁻¹ and 2% O₂. Under high light intensities there was a large excess of J _F/4 at 10–100% O₂ in the C₃ plant due to photorespiration, but very little in sorghum and somewhat more in amaranth, showing that photorespiration is suppressed, more in the NADP-ME- and less in the NAD-ME-type species. It is concluded that when C₄ photosynthesis is limited by supply of atmospheric CO₂ to the C₄ cycle, the C₃ cycle becomes limited by regeneration of ribulose 1,5-bisphosphate (RuBP) which in turn limits RuBP oxygenase activity and photorespiration. The rate of excess electron transport over that consumed for CO₂ fixation in C₄ plants was very sensitive to the presence of O₂ in the gas phase, rapidly increasing between 0.01 and 0.1% O₂, and at 2% O₂ it was about two-thirds of that at 21% O₂. This shows the importance of the Mehler O₂ reduction as an electron sink, compared with photorespiration in C₄ plants. However, the rate of the Mehler reaction is still too low to fully account for the extra ATP which is needed in C₄ photosynthesis. Received: 8 November 1997 / Accepted: 26 December 1997     

Nutrition and the ozone sensitivity of birch (Betula pendula)

 Cuttings of a single birch clone (Betula pendula) were grown in field fumigation chambers throughout the growing season in either filtered air (control) or 90/40 nl O₃ l^–1 (day/night). Both regimes were split into plants under high and low nutrient supply (macro- and micronutrients). The stomatal density of leaves was increased by ozone but was lowered at high nutrition, while the inner air space was hardly affected by the treatments. Ozone induced macroscopic leaf injury regardless of nutrition, but leaf shedding was delayed in the low-fertilized plants, despite O₃ uptake being similar to that in high-fertilized plants. The leaf turn-over was enhanced in the O₃-exposed high-fertilized plants, but length growth and leaf formation of stems were not affected by ozone in either nutrient regime. Leaves of high-fertilized plants showed O₃-caused decline in photosynthetic capacity, water-use efficiency, apparent carbon uptake efficiency and quantum yield earlier as compared with low-fertilized plants, whereas chlorophyll fluorescence (F_V/F_M) and leaf nitrogen concentration were rather stable. CO₂ uptake rate and rubisco activity of young leaves compensated for the O₃ injury in the ageing leaves of the low-fertilized plants. In 8-week-old leaves, however, the O₃-induced decline in CO₂ uptake did not differ between the nutrient regimes and was associated with increased dark respiration rather than changed photorespiration. The balance between CO₂ supply and demand was lost, as was stomatal limitation on CO₂ uptake. High nutrition did not help leaves to maintain a high photosynthetic capacity and life span under O₃ stress. Received: 6 July 1996 / Accepted: 4 June 1997     

A study of photorespiratory ammonia production in the C4 plant Amaranthus edulis, using mutants with altered photosynthetic capacities

Previous studies have indicated that the rate of photorespiration in C₄ plants is low or negligible. In this study, wild-type and mutant leaves of the C₄ plant Amaranthus edulis were treated with the glutamine synthetase inhibitor, phosphinothricin and the glycine decarboxylase inhibitor, aminoacetonitrile, at different concentrations of CO₂. The time course of ammonia accumulation in leaves of the wild type was compared with a mutant lacking phosphoenolpyruvate carboxylase activity (EC 4.1.1.31), and with three different mutants that accumulated glycine. The increase in the concentration of ammonia in the leaves, stimulated by the treatments was used as a measurement of the rate of photorespiration in C₄ plants. The application of glutamine and glycine maintained the rate of photorespiratory ammonia production for a longer period in the wild type, and increased the rate in a mutant lacking phosphoenolpyruvate carboxylase suggesting that there was a lack of amino donors in these plants. The calculated rate of photorespiration in Amaranthus edulis wild-type leaves when the supply of amino donors was enough to maintain the photorespiratory nitrogen flow, accounted for approximately 6% of the total net photosynthetic CO₂ assimilation rate. In a mutant lacking phosphoenolpyruvate carboxylase, however, this rate increased to 48%, when glutamine was fed to the leaf, a value higher than that found in some C₃ plants. In mutants of Amaranthus edulis that accumulated glycine, the rate of photorespiration was reduced to 3% of the total net CO₂ assimilation rate. The rate of ammonia produced during photorespiration was 60% of the total produced by all metabolic reactions in the leaves. The data suggests that photorespiration is an active process in C₄ plants, which can play an important role in photosynthetic metabolism in these plants.     

Leaf development,gas exchange characteristics,and photorespiratory activity in maize seedlings

Five decades ago, a novel mode of CO₂ assimilation that was later described as C₄-photosynthesis was discovered on mature leaves of maize (Zea mays L.) plants. Here we show that 3- to 5-day-old developing maize leaves recapitulate the evolutionary advance from the ancient, inefficient C₃ mode of photosynthesis to the C₄ pathway, a mechanism for overcoming the wasteful process of photorespiration. Chlorophyll fluorescence measurements documented that photorespiration was high in 3-day-old juvenile primary leaves with non-specialized C₃-like leaf anatomy and low in 5-day-old organs with the typical “Kranz-anatomy” of C₄ leaves. Photosynthetic gas (CO₂)-exchange measurements on 5-day-old leaves revealed the characteristic features of C₄ photosynthesis, with a CO₂ compensation point close to zero and little inhibition of photosynthesis by the normal oxygen concentration in the air. This indicates a very low photorespiratory activity in contrast to control experiments conducted with mature C₃ sunflower (Helianthus annuus L.) leaves, which display a high rate of photorespiration.     

Control of photosynthesis in barley plants with reduced activities of glycine decarboxylase

A mutant (LaPr 87/30) of barley (Hordeum vulgare L.) deficient in glycine decarboxylase (GDC; EC 2.1.2.10) was crossed with wild-type plants to generate heterozygous plants with reduced GDC activities. Plants of the F₂ generation were grown in air and analysed for reductions in GDC proteins and GDC activity. The leaves of heterozygous plants contained reduced amounts of H-protein, and when the content of H-protein was lower than 60% of the wild-type, the P-protein was also reduced. The contents of the other two proteins of the GDC complex, T-protein and L-protein were not affected. Glycine decarboxylase activities, measured as the decarboxylation of [1-¹⁴C]glycine by intact mitochondria released from protoplasts, were between 47% and 63% of the wild-type activity in heterozygous plants and between 86% and 100% in plants with normal contents of H-protein. The enzyme activity was linearly correlated with the relative content of H-protein. Plants with reduced GDC activities developed normally and did not show major pleiotropic effects. In air, the reduction in GDC activity had no effect on the leaf metabolite content or photosynthesis, but under conditions of enhanced photorespiration (low CO₂ and high light), glycine accumulated and the rates of photosynthesis decreased compared to the wild-type. The accumulation of glycine did not lead to a depletion of amino donors or to the accumulation of glyoxylate. The lower rates of photosynthesis were probably caused by an impaired recycling of carbon in the photorespiratory pathway. It is concluded that GDC has no control over CO₂ assimilation under normal growth conditions, but appreciable control by GDC becomes apparent under conditions leading to higher rates of photorespiration. Received: 24 November 1996 / Accepted: 23 January 1997     

Comparison of leaf water use efficiency of oak and sycamore in the canopy over two growing seasons

The seasonal trends in water use efficiency of sun and shade leaves of mature oak (Quercus robur) and sycamore (Acer pseudoplatanus) trees were assessed in the upper canopy of an English woodland. Intrinsic water use efficiency (net CO₂ assimilation rate/leaf conductance, A/g) was measured by gas exchange and inferred from C isotope discrimination (δ¹³C) methods. Shade leaves had consistently lower δ¹³C than sun leaves (by 1–2‰), the difference being larger in sycamore. Buds had distinct sun and shade isotopic signatures before bud break and received an influx of ¹³C-rich C before becoming net autotrophs. After leaf full expansion, δ¹³C declined by 1–2‰ gradually through the season, emphasising the importance of imported carbon in the interpretation of leaf δ¹³C values in perennial species. There was no significant difference between the two species in the value of intrinsic water use efficiency for either sun or shade leaves. For sun leaves, season-long A/g calculated from δ¹³C (72–78 μmol CO₂ [mol H₂O]⁻¹) was 10–16% higher than that obtained from gas exchange and in situ estimates of leaf boundary layer conductance. For shade leaves, the gas exchange–derived values were low, only 10–18% of the δ¹³C-derived values. This is ascribed to difficulties in obtaining a comprehensive sample of gas exchange measurements in the rapidly changing light environment.     

Effects of temperature,oxygen concentration,leaf age and seasonal variations on the CO2 compensation point of Lolium perenne L.

Various factors affect the CO₂ compensation point of detached leaves of Lolium perenne L. These include oxygen concentration, temperature, leaf age, and season (spring and summer). Analysis of the results using the model of G.D. Farquhar, S. von Caemmerer and J.A. Berry (1980) Planta 149, 78–90, indicates that some of the CO₂ evolved by leaves in the light is derived from sources other than photorespiration. It is suggested that the operation of the tricarboxylic acid in the light can account for most of this CO₂.Azcón-Bieto—experimental work was done in Barcelona, the further analyses white at the A.N.U.     

The CURLY LEAF gene controls both division and elongation of cells during the expansion of the leaf blade in Arabidopsis thaliana

The CURLY LEAF (CLF ) gene in Arabidopsis thaliana (L.) Heynh. is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems To clarify the function of CLF in organ development, we characterized clf mutants using an anatomical and genetic approach. The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions. A decrease both in the extent of cell elongation and in the number of cells was evident in the clf mutant leaves, suggesting that the CLF gene might be involved in the division and elongation of cells during leaf morphogenesis. An analysis of the development of clf mutant leaves revealed that the period during which cell division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type. Two phases in the elongation of cells were also recognized from this analysis. From analysis of an angustifolia clf double mutant, we found that the two phases of elongation of leaf cells were regulated independently by each gene. Thus, the CLF gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia. Received: 19 February 1998 / Accepted: 24 March 1998     

Ontogenetic patterns of leaf CO2 exchange, morphology and chemistry in Betula pendula trees

In order to explore ontogenetic variation in leaf-level physiological traits of Betula pendula trees, we measured changes in mass- (A _mass) and area-based (A _area) net photosynthesis under light-saturated conditions, mass- (RS_mass) and area-based (RS_area) leaf respiration, relative growth rate, leaf mass per area (LMA), total nonstructural carbohydrates (TNC), and macro- and micronutrient concentrations. Expanding leaves maintained high rates of A _area, but due to high growth respiration rates, net CO₂ fixation occurred only at irradiances >200 μmol photons m^–2 s^–1. We found that full structural leaf development is not a necessary prerequisite for maintaining positive CO₂ balance in young birch leaves. Maximum rates of A _area were realized in late June and early July, whereas the highest values of A _mass occurred in May and steadily declined thereafter. The maintenance respiration rate averaged ≈8 nmol CO₂ g^–1 s^–1, whereas growth respiration varied between 0 and 65 nmol CO₂ g^–1 s^–1. After reaching its lowest point in mid-June, leaf respiration increased gradually until the end of the growing season. Mass and area-based dark respiration were significantly positively correlated with LMA at stages of leaf maturity, and senescence. Concentrations of P and K decreased during leaf development and stabilized or increased during maturity, and concentrations of immobile elements such as Ca, Mn and B increased throughout the growing season. Identification of interrelations between leaf development, CO₂ exchange, TNC and leaf nutrients allowed us to define factors related to ontogenetic variation in leaf-level physiological traits and can be helpful in establishing periods appropriate for sampling birch leaves for diagnostic purposes such as assessment of plant and site productivity or effects of biotic or abiotic factors. Received: 29 December 1998 / Accepted: 26 July 1999     

The isolation and characterisation of a mutant of the C4 plant Amaranthus edulis deficient in NAD-malic enzyme activity

A mutant of Amaranthus edulis (Speg.) lacking activity of the C₄ leaf form of NAD-malic enzyme (ME; EC 1.1.1.39) has been isolated. Homozygous mutant (5% wild-type ME activity) and heterozygous (50% wild-type ME activity) F₂ plants were shown to contain both the α and β NAD-ME subunits in similar amounts to those detected in the wild-type leaves. The rate of photosynthetic CO₂ assimilation was reduced in the homozygous mutant to 5% of that observed for the wild-type leaves. Other C₄ enzymes were not down-regulated in the mutant plants. There was little difference in photosynthetic rate of the heterozygous plants compared to the wild-type, suggesting that NAD-ME exerts little control over the rate of C₄ photosynthesis, and that in the wild-type the enzyme has a very low control coefficient. The activity loss in the heterozygote may therefore be compensated by regulatory mechanisms that increase the activity of the enzyme in vivo. Data for bundle-sheath strands indicated that although the homozygous mutants were able to oxidise malate via the Krebs cycle, they were unable to convert malate to pyruvate and alanine via NAD-ME. Received: 2 April 1998 / Accepted: 7 May 1998     

Growth, cell walls, and UDP-Glc dehydrogenase activity of Arabidopsis thaliana grown in elevated carbon dioxide

The impact of elevated CO₂ (1000 μmol/mol) was assessed on the common weed,Arabidopsis thaliana (Landsberg erecta), which is used as a model plant system. Elevated CO₂ stimulated relative growth rate (RGR) and leaf area gain ofArabidopsis beginning from the cotyledon stage and continuing through the juvenile stage. This early advantage in growth enabled the plants grown in elevated CO₂ to gain more DW despite similar RGRs throughout the latter stages of development. The greater accumulation of DW in leaves grown in elevated CO₂ resulted in a lower specific leaf area (SLA). However, the amount of cell wall investment per unit of leaf area, specific “wall” area (SWA), was similar indicating that elevated CO₂ did not affect the distribution of cell carbon to the cell wall of leaves beyond that needed for cell and leaf expansion. Furthermore, cell wall composition changed with time due to developmental changes and was not affected by elevated CO₂. Associated with the increase in RGR by elevated CO₂ was a concomitant increase in the activity of UDP-Glc dehydrogenase (E.C. 1.1.1.22), a key enzyme in the nucleotide-sugar interconversion pathway necessary for biosynthesis of many cell-wall polysaccharides.     

Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display

We used the differential display technique on total RNAs from roots of Arabidopsis thaliana (L.) Heynh. plants which had or had not been induced for 2 h by nitrate. One isolated cDNA clone, designated Nrt2:1At, was found to code for a putative high-affinity nitrate transporter. Two genomic sequences homologous to Nrt2:1At were found to be localized on the same fragment of chromosome 1 in the Arabidopsis genome. Expression analyses of both low- and high-affinity nitrate transporter genes, respectively Nrt1:1At (previously named Chl1) and Nrt2:1At, were carried out on plants grown under different nitrogen regimes. In this paper, we show that both genes are induced by very low levels of nitrate (50 μM KNO₃). However, stronger induction was observed with Nrt2:1At than with Nrt1:1At. Moreover, these two genes, although both over-expressed in a nitrate-reductase-deficient mutant, were differently regulated when N-sufficient wild-type or mutant plants were transferred to an N-free medium. Indeed, the steady-state amounts of Nrt1:1At mRNA declined whereas the amount of Nrt2:1At mRNA increased, probably reflecting the de-repression of the high-affinity transport system during N-starvation. Received: 4 May 1998 / Accepted: 26 August 1998     

Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees

The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R _Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves). Sun leaves exhibited higher photosynthetic rates P _N on a leaf area basis (mean of 9.1–10.1 μmol CO₂ m⁻² s⁻¹) and Chl basis, which correlated well with the higher values of stomatal conductance G _s (range 105–180 mmol m⁻² s⁻¹), as compared to shade leaves (G _s range 25–77 mmol m⁻² s⁻¹; P _N: 3.2–3.7 μmol CO₂ m⁻² s⁻¹). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R _Fd, which possessed higher values in sun leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In addition, via R _Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves.     

Interactive effects of elevated CO2 and temperature on the anatomical characteristics of leaves in eleven species

The anatomical features of leaves in 11 species of plants grown in a temperature gradient and a temperature + CO₂ gradient were studied. The palisade parenchyma thickness, the spongy parenchyma thickness and the total leaf thickness were measured and analyzed to investigate the effects of elevated temperature and CO₂ on the anatomical characteristics of the leaves. Our results show that with the increase of temperature, the leaf thickness of C₄ species increased while the leaf thickness of C₃ species showed no constant changes. With increased CO₂, seven out of nine C₃ species exhibited increased total leaf thickness. In C₄ species, leaf thickness decreased. As for the trend on the multi-grades, the plants exhibited linear or non-linear changes. With the increase of temperature or both temperature and CO₂ for the 11 species investigated, leaf thickness varied greatly in different plants (species) and even in different branches on the same plant. These results demonstrated that the effect of increasing CO₂ and temperature on the anatomical features of the leaves were species-specific. Since plant structures are correlated with plant functions, the changes in leaf anatomical characteristics in elevated temperature and CO₂ may lead to functional differences. Translated from Acta Ecologica Sinica, 2006, 26(2): 326–333 [译自: 生态学报]