Ziyu Dai

C4 Photosynthesis (The CO2-Concentrating Mechanism and Photorespiration)

Despite previous reports of no apparent photorespiration in C4 plants based on measurements of gas exchange under 2 versus 21% O2 at varying [CO2], photosynthesis in maize (Zea mays) shows a dual response to varying [O2]. The maximum rate of photosynthesis in maize is dependent on O2 (approximately 10%). This O2 dependence is not related to stomatal conductance, because measurements were made at constant intercellular CO2 concentration (Ci); it may be linked to respiration or pseudocyclic electron flow. At a given Ci, increasing [O2] above 10% inhibits both the rate of photosynthesis, measured under high light, and the maximum quantum yield, measured under limiting light ([phi]CO2). The dual effect of O2 is masked if measurements are made under only 2 versus 21% O2. The inhibition of both photosynthesis and [phi]CO2 by O2 (measured above 10% O2) with decreasing Ci increases in a very similar manner, characteristically of O2 inhibition due to photorespiration. There is a sharp increase in O2 inhibition when the Ci decreases below 50 [mu]bar of CO2. Also, increasing temperature, which favors photorespiration, causes a decrease in [phi]CO2 under limiting CO2 and 40% O2. By comparing the degree of inhibition of photosynthesis in maize with that in the C3 species wheat (Triticum aestivum) at varying Ci, the effectiveness of C4 photosynthesis in concentrating CO2 in the leaf was evaluated. Under high light, 30[deg]C, and atmospheric levels of CO2 (340 [mu]bar), where there is little inhibition of photosynthesis in maize by O2, the estimated level of CO2 around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the bundle sheath compartment was 900 [mu]bar, which is about 3 times higher than the value around Rubisco in mesophyll cells of wheat. A high [CO2] is maintained in the bundle sheath compartment in maize until Ci decreases below approximately 100 [mu]bar. The results from these gas exchange measurements indicate that photorespiration occurs in maize but that the rate is low unless the intercellular [CO2] is severely limited by stress.

Light dependence of quantum yields of Photosystem II and CO2 fixation in C3 and C4 plants

The light dependence of quantum yields of Photosystem II (ΦII) and of CO2 fixation were determined in C3 and C4 plants under atmospheric conditions where photorespiration was minimal. Calculations were made of the apparent quantum yield for CO2 fixation by dividing the measured rate of photosynthesis by the absorbed light [A/I=ΦCO2 and of the true quantum yield by dividing the estimated true rate of photosynthesis by absorbed light [(A+Rl)/Ia=ΦCO2·], where RL is the rate of respiration in the light. The dependence of the ΦII/ΦCO2 and ΦII/ΦCO2* ratios on light intensity was then evaluated. In both C3 and C4 plants there was little change in the ratio of ΦII/ΦCO2 at light intensities equivalent to 10–100% of full sunlight, whereas there was a dramatic increase in the ratio at lower light intensities. Changes in the ratio of ΦII/ΦCO2 can occur because respiratory losses are not accounted for, due to changes in the partitioning of energy between photosystems or changes in the relationship between PS II activity and CO2 fixation. The apparent decrease in efficiency of utilization of energy derived from PS II for CO2 fixation under low light intensity may be due to respiratory loss of CO2. Using dark respiration as an estimate of RL, the calculated ΦII/ΦCO2* ratio was nearly constant from full sunlight down to approx 5% of full sunlight, which suggests a strong linkage between the true rate of CO2 fixation and PS II activity under varying light intensity. Measurements of photosynthesis rates and ΦII were made by illuminating upper versus lower leaf surfaces of representative C3 and C4 monocots and dicots. With the monocots, the rate of photosynthesis and the ratio of ΦII/ΦCO2 exhibited a very similar patterns with leaves illuminated from the adaxial versus the abaxial surface, which may be due to uniformity in anatomy and lack of differences in light acclimation between the two surfaces. With dicots, the abaxial surface had both lower rates of photosynthesis and lower ΦII values than the adaxial surface which may be due to differences in anatomy (spongy versus palisade mesophyll cells) and/or light acclimation between the two surfaces. However, in each species the response of ΦII/ΦCO2 to varying light intensity was similar between the two surfaces, indicating a comparable linkage between PS II activity and CO2 fixation.

C4 photosynthesis: the effects of leaf development on the CO2-concentrating mechanism and photorespiration in maize

The effect of O2 on photosynthesis was determined in maize (Zea mays) leaves at different developmental stages. The optimum level of O2 for maximum photosynthetic rates was lower in young and senescing tissues (2–5 kPa) than in mature tissue (9 kPa). Inhibition of photosynthesis by suboptimal levels of O2 may be due to a requirement for functional mitochondria or to cyclic/pseudocyclic photophosphorylation in chloroplasts; inhibition by supraoptimal levels of O2 is considered to be due to photorespiration. Analysis of a range of developmental stages (along the leaf blade and at different leaf ages and positions) showed that the degree of inhibition of photosynthesis by supraoptimal levels of O2 increased rapidly once the ribulose-1,5-bisphosphate carboxylase/oxygenase and chlorophyll contents were below a critical level and was similar to that of C3 plants. Tissue having a high sensitivity of photosynthesis to O2 may be less effective in concentrating CO2 in the bundle sheath cells due either to limited function of the C4 cycle or to higher bundle sheath conductance to CO2. An analysis based on the kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase was used to predict the maximum CO2 level concentrated in bundle sheath cells at a given degree of inhibition of photosynthesis by supraoptimal levels of O2.

Promoter elements controlling developmental and environmental regulation of a tobacco ribosomal protein gene L34

The rpL34 gene, which encodes a cytoplasmic ribosomal protein with a high homology to the rat 60S r-protein L34, was isolated from a genomic library of tobacco (Nicotiana tabacum L. cv. Xanthi-nc). A 1500 bp upstream promoter fragment was fused to the chloramphenicol acetyltransferase (CAT) reporter gene or β-glucuronidase (GUS) reporter gene and transferred into tobacco plants by the Agrobacterium-mediated leaf disk transformation method. Analysis of CAT activity in leaf tissues showed that mechanical wounding increased the rpL34 promoter activity about 5 times as compared to untreated controls and that the promoter activity was further enhanced by plant growth regulators, 2,4-dichlorophenoxyacetic acid and benzyladenine. Histochemical GUS staining patterns of the transgenic plants showed that the rpL34 promoter activity is high in actively growing tissues, including various meristems, floral organs, and developing fruits. A series of 5′ deletion analyses of the rpL34 promoter indicated that a 50 bp region located between −179 and −129 is essential for wound, auxin and cytokinin responses. Deletion of this region reduced the promoter activity to an undetectable level. Insertion of the 50 nucleotide sequence into a minimal promoter restored the promoter activity and the promoter strength was proportional to the copy number of the upstream sequence. The role of TATA and CAAT box regions was studied by a series of 3′ deletion analyses. A 3′ deletion up to −28 did not significantly affect the promoter strength. However deletion of the promoter up to 70 bp, which deleted the TATA box region, significantly reduced promoter activity. Further deletion of the promoter up to −104, eliminating the CAAT box region, abolished the promoter activity. These results suggest that the TATA box and CAAT box regions are also important for the rpL34 promoter activity in addition to the 50 bp upstream region.

Effects of growth regulators on the induction of crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum L.

The classical induction of Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum L. by water stress is observed within one week when fourto five-week-old plants (grown under a 16/8 h photoperiod at ca. 600 μmol quanta · m−2 · s−1) are irrigated with 350 mM NaCl. The induction of CAM was evaluated by measuring phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) and NADP-malic enzyme (NADP-ME, EC 4.1.1.82) activities and nocturnal increases in malate content and titratable acidity of leaf extracts, and the daily pattern of CO2 exchange and stomatal conductance during the 7-d induction period. Three growth regulators, abscisic acid (ABA), farnesol (an antitranspirant and analog of ABA), and benzylaminopurine (BAP), were found to substitute for NaCl for induction of CAM when fed to plants in nutrient media. Daily irrigation with solutions containing micromolar levels (optimum ca. 10 micromolar) of these growth regulators led to the induction of CAM similar to that by high salt. Application of the growth regulators, like NaCl, caused large increases in the activity of NADP-ME and the activity and level of PEPCase, which are components of the biochemical machinery required for CAM. Western immunoblotting showed that the increased activity of PEPCase on addition of ABA, farnesol and BAP was mainly due to increased levels of the CAM-specific isoforms. Also, dehydration of cut leaves over 8.5 h under light resulted in a severalfold increase in PEPCase activity. An equivalent increase in PEPCase activity in excised leaves was also obtained by feeding 150 mM NaCl, or micromolar levels of ABA or BAP via the petiole, which supports results obtained by feeding the growth regulators to roots. However, the increase in PEPCase activity was inhibited by feeding high levels of BAP to cut leaves prior to dehydration, indicating a more complex response to the cytokinin. Abscisic acid may have a role in induction of CAM in M. crystallinum under natural conditions as there is previous evidence that induction by NaCl causes an increase in the content of ABA, but not cytokinins, in leaves of this species.

Oxygen sensitivity of photosynthesis and photorespiration in different photosynthetic types in the genus Flaveria

Two major indicators were used to access the degree of photorespiration in various photosynthetic types of Flaveria species (C3, C3-C4, C4-like, and C4): the O2 inhibition of photosynthesis measured above the O2 partial pressure which gives a maximum rate, and O2- and light-dependent whole-chain electron flow measured at the CO2 compensation point (Γ). The optimum level of O2 for maximum photosynthetic rates under atmospheric levels of CO2 (34 Pa) was lower in C3 and C3-C4 species (ca. 2 kPa) than in C4-like and C4 species (ca. 9 kPa). Increasing O2 partial pressures from the optimum for photosynthesis up to normal atmospheric levels (ca. 20 kPa) caused an inhibition of photosynthesis which was more severe under lower CO2. This inhibition was calculated as the O2 inhibition index (ΘA, the percentage inhibition of photosynthesis per kPa increase in O2). From measurements of 18 Flaveria species at atmospheric CO2, the ΘA values decreased from C3 (1.9–2.1) to C3-C4 (1.2–1.6), C4-like (0.6–0.8) and C4 species (0.3–0.4), indicating a progressive decrease in apparent photorespiration in this series. With increasing irradiance at Γ under atmospheric levels of O2, and increasing O2 partial pressure at 300 μmol quanta·m−2·s−1, there was a similar increase in the rate of O2 evolution associated with whole-chain electron flow (Jo2, calculated from chlorophyll fluorescence analysis) in the C3 and C3-C4 species compared to a much lower rate in the C4-like and C4 species. The results indicate that there is substantial O2-dependent electron flow in C3 and C3-C4 species, reflecting a high level of photorespiration compared to that in C4-like and C4 species. Consistent with these results, there was a significant decrease in Γ from C3 (6–6.2 Pa) to C3-C4 (1.0–3.0 Pa), to C4-like and C4 species (0.3–0.8 Pa), indicating a progressive decrease in apparent photorespiration. However, C3 and C3-C4 species examined had high intrinsic levels of photorespiration with the latter maintaining low apparent rates of photorespiration and lower Γ values, primarily by refixing photorespired CO2. The C4-like and C4Flaveria species had low, but measurable, levels of photorespiration via selective localization of ribulose-1,5-bisphosphate carboxylase in bundle sheath cells and operation of a CO2 pump via the C4 pathway.

Factors Affecting the Induction of Crassulacean Acid Metabolism in Mesembryanthemum crystallinum

Some succulent plants initially perform C3 photosynthesis but subsequently shift to CAM after developmental or environmental changes (e.g. photoperiod, water stress, day/night temperature). Species which have a strong dependency on the environment for expression of CAM are facultative, in contrast to obligate CAM plants which tend to function in the CAM mode under all conditions (Cockburn 1985; Winter 1985). Associated with this transition in facultative species is nocturnal opening of stomata with concomitant CO2 uptake, and diurnal fluctuations of tissue acidity and malate content. Facultative CAM plants may maximize their growth by assimilating carbon via the C3 pathway when environmental conditions are less stressful, and utilize the CAM mode when environmental conditions cause more potential for water stress.

Induction of Nopaline Synthase Promoter Activity by H2O2 Has No Direct Correlation with Salicylic Acid

Transgenic tobacco (Nicotiana tabacum L.) plants carrying a fusion between the nopaline synthase (nos) promoter and chloramphenicol acetyltransferase (CAT) reporter gene (cat) were tested for their response to treatment with H2O2. The nos promoter-driven CAT activity increased significantly by addition of H2O2, reaching the maximum level at 15 mM. Kinetic analysis for CAT activity showed that induction by H2O2 was similar to that of methyl jasmonate (MJ), but was much slower than induction by salicylic acid (SA). Time-course experiments for mRNA level also revealed that the response to H2O2 treatment was similar to that of MJ. The nos promoter displayed a rapid and transient induction of mRNA with SA treatment, with the maximum levels occurring at 3 h, whereas the levels induced by H2O2 or MJ treatment increased continuously during the 11-h experimental period. The antioxidants N-acetyl-L-cysteine and catechol did not alter the SA effect. The responses of the nos promoter to H2O2, MJ, and wounding were significantly reduced by deletions of the CAAT box region and the sequence between -112 and -101. However, these deletions did not significantly alter the SA response. This suggests that H2O2 may have a different mechanism from that of SA for inducing nos promoter activity.

Functional role of CAAT box element of the nopaline synthase (nos) promoter

The nopaline synthase (nos) promoter is active in a wide range of plant tissues and regulated by various environmental stimuli. It was previously found that the CAAT box region is important for nos promoter activity. In the present study, the location of the CAAT box element was determined by site specific mutation analysis. Point mutations within the conserved CAAT box element significantly reduced the promoter response in transgenic tobacco plants and calli to wounding, H2O2, methyl jasmonate, and 2,4-D, but not to salicylic acid. However, mutations immediately upstream from the CAAT box did not affect these responses. These results suggest that the CAAT box element is important in responding to certain stimuli.