Comparative ecophysiology of C3 and C4 plants

Abstract. In this review we relate the physiological significance of C₄ photosynthesis to plant performance in nature. We begin with an examination of the physiological consequences of the C₄ pathway on photosynthesis, then discuss the ecophysiological performance of C₄ plants in contrasting environments. We then compare the performance of C₃ and C₄ plants when they occur together in similar habitats, and finally discuss the distribution of C₄ photosynthesis with respect to the physical environment, phylogeny, and life form.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

Internode length in Pisum. Estimation of GA1 levels in genotypes Le, le and led

The levels of GA₁, 3-epiGA₁ and GA₈ in genotypes Le, le and le^d of Pisum sativum L. were determined by gas chromatography-selected ion monitoring (GC-SIM) after feeds of [³H, ¹³C]-GA₂₀ to each genotype. The levels of endogenous and [¹³C]-labelled metabolites were determined by reverse isotope dilution with unlabelled GA₁, 3-epiGA₁ and GA₈. The results demonstrate a quantitative relationship between the level of GA₁ and the extent of elongation both on a per plant and a per g fresh weight basis. These results are consistent with previous findings in peas and other species possessing a predominant early 13-hydroxylation pathway for GA biosynthesis.
The levels of 3-epiGA₁ also decreased in the genotypic sequence Le, le, le^d although not as rapidly as for the level of GA₁. This may suggest that the alleles at the le locus also influence the formation of 3-epiGA₁.     

C4 photosynthesis at low temperatures

Abstract. C₄ plants grown in optimum conditions are, by comparison to C₃, capable of higher maximum dry-matter yields and greater efficiencies of water and nitrogen use, yet they are rare outside the subtropics. Both latitudinal and altitudinal limits of C₄ distributions correlate most closely with a mean minimum temperature of 8-10°C during the period of active growth. The possibility that the C₄ process is inherently incapable of functioning at low temperatures is examined. The reversible effects of chilling on the quantum efficiency of C₄ photosynthesis and the functioning of the individual steps in the C₄ cycle are examined. Chilling also produces an irreversible loss of capacity to assimilate CO₂ which is directly proportional to the light received during chilling. It is suggested that the reversible reduction in capacity to assimilate CO₂ and the lack of an alternative pathway for the utilization of lightgenerated reducing power may make C₄ species more prone to chilling-dependent photoinhibition. Laboratory studies and limited field observations suggest that this damage would be most likely to occur during photosynthetic induction at the temperatures and light levels encountered on clear, cool mornings during the spring and early summer in cool climates. Even those C₄ species occurring naturally in cool climates do not appear fully capable of tolerating these conditions; indeed their growth patterns suggest that they may be adapted by avoiding 'rather than enduring' such conditions.     

Gibberellins in relation to flowering in Polianthes tuberosa

Endogenous gibberellins (GAs) in corms of Polianthes tuberosa L. (cv. Double) were isolated and identified by high performance liquid chromatography, bioassay and combined capillary gas chromatography-mass spectrometry (GC-MS). Gibberellins A₁, A₁₉, A₂₀ and A₅₃ were quantified at the vegetative, early floral initiation and flower development stages. The identification of 13-hydroxylated GAs indicates the presence of the early 13-hydroxylation pathway in P. tuberosa corms. An increase in GA₁ and GA₂₀, and a decrease in GA₁₉ levels, coincided with the transition from the vegetative phase to the stages of early floral initiation and flower development. GA₅₃ stayed at constant levels at the 3 different growth stages. The absence of GA₁ in vegetative corms and its presence in corms at early floral initiation and flower development stages suggest that GA₁ is a causal factor in inducing floral initiation in P. tuberosa . When GA₁, GA₃, GA₄, GA₂₀ and GA₃₂ were applied to corms at the vegetative stage (plants about 5 cm in height), floral initiation was promoted by all of the GAs used, GA₃₂ being the most active. In contrast with the other GAs, GA₃₂ had no effect on stem elongation. Therefore, it is suggested that hydroxylated C-19 GAs play an important role in flower induction in P. tuberosa .     

Distribution of C3 and C4 grasses along an altitudinal gradient in Central Argentina

The distribution pattern of C₃ and C₄ grasses was studied in eight sites located between 350 m and 2100 m along an altitudinal gradient in Central Argentina. Of 139 taxa fifty-nine are C₃ and eighty C₄. Species of the C₃ tribes (Stipeae, Poeae, Meliceae, Aveneae, Bromeae and Triticeae) and C₃ Paniceae species increase in number at higher elevations; only one C₃ species was found below 650 m. C₄ Aristideae, Pappophoreae, Eragrostideae, Cynodonteae, Andropogoneae and Paniceae increase at lower altitudes. The floristic crossover point is at about 1500 m; the ground cover cross-over point is at about 1000 m. Analysis of the relationships between % C₄ species along the gradient and nine climatic and environmental variables showed the highest correlation with July mean temperature, but all temperature variables show highly significant correlations with % C₄. Correlation with annual rainfall is lower but also significant. These results are consistent with previous research showing the relative importance of C₄ grasses as temperature increases. C₃ species make a high contribution to relative grass coverage below the C₃/C₄ floristic crossover point but are rare below 1000 m.     

Effects of the triazole plant growth retardant BAS 111¨W on gibberellin levels in oilseed rape, Brassica napus

Three-week-old shoots of the spring oilseed rape cv. Petranova ( Brassica napus L. ssp. napus ) were found by combined gas chromatography-mass spectrometry to contain GA₁, GA₈, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₂₉, 3-epi-GA₁ and a previously uncharacterised C₁₉ dicarboxylic acid that is probably structurally related to GA₂₄. Shoots of the winter cultivar Belinda, harvested at the early flowering stage, contained the same GAs with the exception of the C₁₉ dicarboxylic acid and, in addition, GA₃₄ and GA₅₁ were identified. All material contained higher levels of GA₂₀ than of GA₁; the ratio of GA₁ to GA₂₀ was highest in shoots containing the largest proportion of young immature tissues. Soil treatment of cv. Petranova seedlings with the growth retardant BAS 111¨W [1-phenoxy-5,5-dimethyl-3-(1,2,4-triazol-1-yl)-hexan-4-ol] caused 80% reduction in height 18 days after treatment and the levels of all GAs were 20% or less that of control plants. Foliar treatment at the same dosage reduced height by 50% and caused an 85% or greater reduction in the concentrations of the GA₁ precursors GA₂₀, GA₁₉ and GA₄₄. However, the levels of GA₁, GA₈ and GA₂₉ were affected to a much smaller extent. Foliar application of BAS 111¨W to cv. Belinda 1 month after sowing resulted in only a 20% height reduction at flowering, but no uniform decrease in the concentrations of endogenous GAs at this stage.     

Re-evaluation of proposed C4 photosynthetic characteristics in the genus Larix

It has been suggested previously that Japanese larch ( Larix kaempferi ) exhibits characteristics of C₄ photosynthesis. To further evaluate this suggestion, stable carbon isotope ratios were determined for leaf and bark tissue of Larix gmelini, L. kaempferi, L. laricina, L. Iyallii, L. occidentalis , and L. sibirica. All δ¹³C values were more negative than –22‰. Short-term labeling with ¹⁴CO₂ showed that phosphoglyceric acid and other phosphorylated compounds were the first products of photosynthesis in L. sibirica. Both of these results strongly suggest that the initial fixation of atmospheric CO₂ in these six Larix species is accomplished solely via the C₃ photosynthetic pathway.     

The ectopic overexpression of a citrus gibberellin 20-oxidase enhances the non-13-hydroxylation pathway of gibberellin biosynthesis and induces an extremely elongated phenotype in tobacco

Transgenic plants of Nicotiana tabacum overexpressing a gibberellin (GA) 20-oxidase cDNA ( CcGA20ox1 ) from citrus, under the control of the 35S promoter, were taller (up to twice) and had larger inflorescences and longer flower peduncles than those of control plants. Hypocotyls of transgenic seedlings were also longer (up to 4 times), and neither the seedlings nor the growing plants elongated further after application of GA₃. Hypocotyl and stem lengths were reduced by application of paclobutrazol, and this inhibition was reversed by exogenous GA₃. The ectopic overexpression of CcGA20ox1 enhanced the non-13-hydroxylation pathway of GA biosynthesis leading to GA₄, apparently at the expense of the early-13-hydroxylation pathway. The level of GA₄ (the active GA from the non-13-hydroxylation pathway) in the shoot of transgenic plants was 3–4 times higher than in control plants, whereas that of GA₁, formed via the early-13-hydroxylation pathway (the main GA biosynthesis pathway in tobacco), decreased or was not affected. GA₄ applied to the culture medium or to the expanding leaves was found to be at least equally active as GA₁ on stimulating hypocotyl and stem elongation of tobacco plants. The results suggest that the tall phenotype of tobacco transgenic plants was due to their higher content of GA₄, and that the GA response was saturated by the presence of the transgene.     

Gibberellins and photoperiodic control of shoot elongation in Salix

Effects of exogenous gibberellins GA₅₃, GA₄₄, GA₁₉, GA₂₀ and GA₁ on photoperiodically controlled shoot elongation in seedlings of Salix pentandra L. were studied. Gibberellins GA₂₀ and GA₁ induced shoot elongation under short days (SD) and could substitute for a transfer to long day (LD), while gibberellins A₅₃, A₄₄ and A₁₉ were inactive. In seedlings exposed to a prolonged SD-treatment (30 days) there was a significant positive interaction between a transfer to LD and a treatment with GA₂₀ and GA₁ on shoot elongation. In addition, GA₁₉ enhanced the growth promotive effect of LD in these seedlings. The results are compatible with the suggestion that conversion of GA₁₉ to GA₂₀ is blocked under SD. This effect is supposed to be an early process leading to the cessation of shoot elongation under SD. Responsiveness of the seedlings to LD and to a GA-treatment gradually decreased with an increasing length of exposure to SD.     

C4 photosynthesis in Cyperus longus L., a species occurring in temperate climates

Abstract. Cyperus longus L. , which has a widespread but disjunct distribution throughout Europe and extends northwards into Britain, was found to be a C₄ species based upon its Kranz leaf anatomy, low CO₂ compensation point and the labelling of malate as an early product of ¹⁴CO₂ fixation. The photosynthetic characteristics of C. longus are similar to many other C₄ species with a high maximum rate of photosynthesis (> 1.5 mg CO₂ m ⁻² s ⁻¹) and a relatively high temperature optimum (30–35°C), but unlike many C₄ species the rate of photosynthesis does not decline rapidly below the optimum temperature and a substantial rate (0.6 mgCO₂ m⁻²s⁻¹)occursat 15°C. Leaf extension is very slow at 15°C and shows a curvilinear response to temperatures between 15 and 25°C. Leaves extend at a rate of almost 4 cm d⁻¹ at 25°C.     

Elevated pyruvate,orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene

Changes in carbon metabolism and δ¹³C value of transgenic potato plants with a maize pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) gene are reported. PPDK catalyzes the formation of phospho enol pyruvate (PEP), the initial acceptor of CO₂ in the C₄ photosynthetic pathway. PPDK activities in the leases of transgenic potatoes were up to 5.4‐fold higher than those of control potato plants (wild‐type and treated control plants). In the transgenic potato plants, PPDK activity in leaves was negatively correlated with pyruvate content (r²= 0.81), and was positively correlated with malate content (r²= 0.88). A significant increase in the δ¹³C value was observed in the transgenic potato plants, suggesting a certain contribution of PEP carboxylase as the initial acceptor of atmospheric CO₂. These data suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C₄‐type carbon metabolism. However, since parameters associated with CO₂ gas exchange were not affected, the altered carbon metabolism had only a small effect on the total photosynthetic characteristics of the transgenic plants.     

Role of gibberellins in parthenocarpic fruit development induced by the genetic system pat-3/pat-4 in tomato

The role of gibberellins (GAs) in the induction of parthenocarpic fruit-set and growth by the pat-3/pat-4 genetic system in tomato ( Lycopersicon esculentum Mill.) was investigated using wild type (WT; Cuarenteno) and a near-isogenic line derived from the German line RP75/59 (the source of pat-3/pat-4 parthenocarpy). Unpollinated WT ovaries degenerated but GA₃ application induced parthenocarpic fruit growth. On the contrary, parthenocarpic growth of pat-3/pat-4 fruits, which occurs in the absence of pollination and hormone treatment, was not affected by applied GA₃. Unpollinated pat-3/pat-4 fruit growth was negated by paclobutrazol, an inhibitor of ent -kaurene oxidase, and this inhibitory effect was negated by GA₃. The quantification of the main GAs of the early 13-hydroxylation pathway (GA₁, GA₈, GA₁₉, GA₂₀, GA₂₉ and GA₄₄) in unpollinated ovaries at 3 developmental stages (flower bud, FB; pre-anthesis, PR; and anthesis, AN), by gas chromatography-selected ion monitoring, showed that the concentration of most of them was higher in pat-3/pat-4 than in WT ovaries at PR and AN stages. The concentration of GA₁, suggested previously to be the active GA in tomate, was 2–4 times higher. Unpollinated pat-3/pat-4 ovaries at FB, PR and AN stages also contained relatively high amounts (5–12 ng g⁻¹) of GA₃, a GA found at less than 0.5 ng g⁻¹ in WT ovaries. It is concluded that the mutations pat-3/pat-4 may induce natural facultative parthenocarpy capacity in tomato by increasing the concentration of GA₁ and GA₃ in the ovaries before pollination.     

Optimal leaf area indices in C3 and C4 mono- and dicotyledonous species at low and high nitrogen availability

From an analytical model it was shown that for a given total amount of nitrogen in the canopy, there exists an optimal leaf area index (LAI), and therefore an optimal average leaf introgen content, at which canopy photosynthesis is maximal. If the LAI is increased above this optimum, increased light interception will not compensate for reduction in photosynthetic capacity of the canopy resulting from reduced leaf nitrogen contents. It was further derived from the model that the value of the optimal LAI increases with the photosynthetic nitrogen use efficiency (PNUE) and decreases with the canopy extinction coefficient for light (K_L) and incident photon flux density (PFD) at the top of the canopy. These hypotheses were tested on dense stands of species with different photosynthetic modes and different architectures. A garden experiment was carried out with the C₄ monocot sorghum ( Sorghum bicolor [L.] Moensch cv. Pioneer), the C₃ monocot rice ( Oryza sativa L. cv. Araure 4), the C₄ dicot amaranth ( Amaranthus cruentus L. cv. K113) and the C₃ dicot soybean ( Glycine max [L.] Merr. cv. Williams) at two levels of nitrogen availability.
The C₄ species had higher PNUEs than the C₃ species while the dicots formed stands with higher extinction coefficients for light and had lower PNUEs than the monocots. The C₄ and monocot species were found to have formed more leaf area per unit leaf nitrogen (i.e., had lower leaf nitrogen contents) than the C₃ and dicot species, respectively. These results indicate that the PNUE and the extinction coefficient for light are important factors determining the amount of leaf area produced per unit nitrogen as was predicted by the model.     

C2H2 and Ar induce rapid declines in nitrogenase activity and CO2 evolution in nodules of Datisca glomerata

Preliminary studies have indicated that after addition of C₂H₂ there is a rapid decline in nitrogenase activity in the nodules of Datisca glomerata . The present work was undertaken to determine whether (1) there is also a decline in respiration and (2) the decline is associated with the cessation of ammonia production. The rates of C₂H₄ and CO₂ evolution by nodulated root systems of Datisca were measured as a function of time after exposure to C₂H₂. The peak rate of C₂H₄ evolution occurred at 30 s after C₂H₂ exposure, while the rate of CO₂ evolution started to decline at 60 s after exposure to C₂H₂. Incubation of nodules in a gas mixture containing Ar also caused a decline in CO₂ evolution. Further, pretreatment with Ar eliminated most of the C₂H₂-induced decline in nitrogenase activity and CO₂ evolution. These C₂H₂- and Ar-induced declines in Datisca nodules are more rapid than those reported in any other nodules. They are evidence that continued ammonia formation is essential for maintenance of normal nitrogenase activity in Datisca nodules.     

Metabolism of tritiated and deuterated gibberellins A1, A4 and A9 in Sitka spruce (Picea sitchensis) shoots during the period of cone-bud differentiation

A mixture of tritiated and deuterated gibberellins (GAs) was injected into elongating shoots of Sitka spruce [ Picea sitchensis (Bong.) Carr.] grafts grown under environmental conditions that were either inductive (heat and drought, HD) or non-inductive (cool and wet, CW) for flowering. The metabolites were purified by high performance liquid chromatography (HPLC), detected by liquid scintillation counting of aliquots of collected fractions and identified by gas chromatography–mass spectrometry (GC-MS). Deuterated GA₉ was converted to deuterated GA₄, deuterated GA₃₄, and deuterated GA₁ in both treatments. Deuterated GA₄ was metabolized to deuterated GA₃₄ and deuterated GA₁ in the CW material, but only deuterated GA₁ was detected in the HD material. The amount of detected metabolites was higher in the HD material, caused by a higher rate of metabolism and/or smaller losses of the metabolites during sample purification. GA₁ was converted to a polar unidentified metabolite in both treatments, but to a higher degree in the CW treatment.     

Photosynthesis in Salsola Species (Chenopodiaceae) from Southern Africa Relative to their C4 Syndrome Origin and their African-Asian Arid Zone Migration Pathways

Abstract: Over 60 Salsola species of Chenopodiaceae from South Africa were studied for their photosynthesis type, using δ¹³C analysis and light microscopy of leaf anatomy. These species cover about 70 % of the total list of Southern African Salsola species and grow naturally in South and Southwest African desert regions. All species are shrubby forms and belong to the single subsection Caroxylon. Only C₄ photosynthesis was found in the Salsola species determined with ¹³C/¹²C carbon isotope discrimination values that ranged from - 11.04 to - 14.03 % (PDB), plus the presence of a Kranz type assimilation tissue anatomy. The apparent absence of C₃ in Salsola in South and Southwest Africa and the known presence of C₃ and C₃ - C₄ intermediate photosynthesis in Caroxylon, Salsola species in Asia strongly indicate that the genus Salsola originated in Asia and later migrated to South Africa.     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.     

Stimulation of shoot elongation in Salix pentandra by gibberellin A9; activity appears to be dependent upon hydroxylation to GAl via GA20

Cessation of shoot elongation in seedlings of Salix pentandra L. is induced by short photoperiod. Gibbereliin A₉ (GA₉) applied either to the apical bud or injected into a mature leaf, induced shoot elongation under a short photoperiod of 12 h, and GA₉ could completely substitute for a transfer to a long photoperiod. When [³H]GA₉ or [²H₂]GA₉ was injected into a leaf, no [³H]GA₉ was detected in the elongating apex and only traces of [³H]GA₉ were found in the shoot above the treated leaf. By the use of gas chromatography-mass spectrometry (GC-MS), [²H₂]GA₂₀ was identified as the main metabolite of [²H₂]GA₉ in both the shoot and the treated leaf. In addition, [²H₂]GA₁ and [²H₂]GA₂₉ were also identified as metabolites of [²H₂]GA₉. These results are consistent with the hypothesis that exogenous GA, promotes shoot elongation in Salix through its metabolism to GA₂₀ and GA,.     

Comparison of the specific activity of ribulose-1,5-bis-phosphate carboxylase-oxygenase from some C3 and C4 plants

The specific activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco, EC 4.1.1.39) was measured from the crude extracts of five C₃ plants consisting of wheat ( Triticum aestivum L. cv. Maris Mink), spinach ( Spinacia oleracea L.), pea ( Pisum sativum L. cv. Greenfeast), pumpkin ( Cucurbita pepo L. cv. Jättiläismeloni) and Ceratodon purpureus (Hedw.) Brid., and two C₄ plants, maize ( Zea mays L. ETA F₁) and sugar sorghum [ Sorghum saccharatum (L. emend, L.) Moench]. The amount of Rubisco in the crude extracts was estimated by polyacrylamide gel electro-phoresis with the Coomassie Brilliant Blue staining procedure. The amounts of the dye bound to the purified Rubisco of different higher plants were similar. The method gave a linear response for both purified enzyme and crude extracts, and the results agreed with those observed by immunochemical methods. The addition of positive effectors such as inorganic phosphate was necessary to obtain maximal activity in the crude extracts of all the studied plants except in that of maize. No significant differences in the specific carboxylase activity at 25°C were found between the C₃ and C₄ plants.