Salt stress induces Kranz anatomy and expression of C4 photosynthetic enzymes in the amphibious sedge Eleocharis vivipara

Photosynthesis Research - Eleocharis vivipara Link is a unique amphibious leafless plant of the Cyperaceae. The terrestrial form develops culms with Kranz anatomy and C4-like traits, while the...     

Molecular evolution and genetic engineering of C4 photosynthetic enzymes

The majority of terrestrial plants, including many important crops such as rice, wheat, soybean, and potato, are classified as C(3) plants that assimilate atmospheric CO(2) directly through the C(3) photosynthetic pathway. C(4) plants, such as maize and sugarcane, evolved from C(3) plants, acquiring the C(4) photosynthetic pathway in addition to the C(3) pathway to achieve high photosynthetic performance and high water- and nitrogen-use efficiencies. Consequently, the transfer of C(4) traits to C(3) plants is one strategy being adopted for improving the photosynthetic performance of C(3) plants. The recent application of recombinant DNA technology has made considerable progress in the molecular engineering of photosynthetic genes in the past ten years. It has deepened understanding of the evolutionary scenario of the C(4) photosynthetic genes. The strategy, based on the evolutionary scenario, has enabled enzymes involved in the C(4) pathway to be expressed at high levels and in desired locations in the leaves of C(3) plants. Although overproduction of a single C(4) enzyme can alter the carbon metabolism of C(3) plants, it does not show any positive effects on photosynthesis. Transgenic C(3) plants overproducing multiple enzymes are now being produced for improving the photosynthetic performance of C(3) plants.     

Leaf anatomy as a constraint for photosynthetic acclimation: differential responses in leaf anatomy to increasing growth irradiance among three deciduous trees

Interspecific variation in the response to transfer from low to high growth irradiance with respect to anatomical and photosynthetic characteristics was studied in mature leaves of three tree species, Betula ermanii Cham., Acer rufinerve Sieb. et Zucc. and Fagus crenata Blume, which occur in different successional stages in temperate deciduous forests. Transfer from low to high irradiance increased the light-saturated rate of photosynthesis per unit leaf area ( P _max) significantly in B. ermanii and A. rufinerve , but not in F. crenata . Leaves of B. ermanii grown at low irradiance were relatively thick and had vacant spaces along the mesophyll cell surfaces which was not occupied by chloroplasts or other organelles. After transfer to high irradiance, chloroplasts enlarged to fill the space along with P _max without an increase in leaf thickness. Leaves of A. rufinerve were plastic in mesophyll cell surface area and in leaf thickness, both of which increased after the transfer to high irradiance, along with an increase in the amount of chloroplasts and in P _max. On the other hand, F. crenata had little mesophyll cell surface unoccupied by chloroplasts and leaf anatomy was not changed after the transfer. In all species, P _max was strongly correlated with chloroplast surface area adjacent to the exposed mesophyll surface across different growth irradiances. An increase in P _max was observed only when chloroplast volume also increased. We conclude that light acclimation potential is primarily determined by the availability of unoccupied cell surface into which chloroplasts expand, as well as by the plasticity of the mesophyll that allows an increase in its surface area.     

Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice

Four enzymes, namely, the maize C(4)-specific phosphoenolpyruvate carboxylase (PEPC), the maize C(4)-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C(3)-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO(2) assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO(2) assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO(2) assimilation, however, was more marked at higher CO(2) concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO(2) inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C(4)-like photosynthetic pathway into C(3) plants, are discussed.     

Effect of growth temperature on photosynthetic capacity and respiration in three ecotypes of Eriophorum vaginatum

Ecotypic differentiation in the tussock‐forming sedge Eriophorum vaginatum has led to the development of populations that are locally adapted to climate in Alaska's moist tussock tundra. As a foundation species, E. vaginatum plays a central role in providing topographic and microclimatic variation essential to these ecosystems, but a changing climate could diminish the importance of this species. As Arctic temperatures have increased, there is evidence of adaptational lag in E. vaginatum, as locally adapted ecotypes now exhibit reduced population growth rates. Whether there is a physiological underpinning to adaptational lag is unknown. Accordingly, this possibility was investigated in reciprocal transplant gardens. Tussocks of E. vaginatum from sites separated by ~1° latitude (Coldfoot: 67°15′N, Toolik Lake: 68°37′, Sagwon: 69°25′) were transplanted into the Toolik Lake and Sagwon sites and exposed to either an ambient or an experimental warming treatment. Five tussocks pertreatment combination were measured at each garden to determine photosynthetic capacity (i.e., V_cmax and J_max) and dark respiration rate (R_d) at measurement temperatures of 15, 20, and 25°C. Photosynthetic enhancements or homeostasis were observed for all ecotypes at both gardens under increased growth temperature, indicating no negative effect of elevated temperature on photosynthetic capacity. Further, no evidence of thermal acclimation in R_d was observed for any ecotype, and there was little evidence of ecotypic variation in R_d. As such, no physiological contribution to adaptational lag was observed given the increase in growth temperature (up to ~2°C) provided by this study. Despite neutral to positive effects of increased growth temperature on photosynthesis in E. vaginatum, it appears to confer no lasting advantage to the species.     

A survey of photosynthetic carbon metabolism in 4 ecotypes of Phragmites australis in northwest China: Leaf anatomy, ultrastructure, and activities of ribulose 1,5-bisphosphate carboxylase, phosphoenolpyruvate carboxylase and glycollate oxidase

Four ecotypes of Phragmites australis from different habitats in northwest China were examined to compare their photosynthetic characteristics. In a swamp ecotype, the Δ ¹³C value of leaf materials was −34.0‰, and bundle sheath cells contained a small amount of organelles and round-shaped chloroplasts, as being similar to typical C₃ plants. In a dune ecotype, the Δ ¹³C value was −20.9‰ and bundle sheath cells contained oval-shaped chloroplasts with poorly-developed grana. In light and heavy salt meadow ecotypes, Δ ¹³C values were −30.6‰ and −35.6‰, respectively. The shape of bundle sheath chloroplasts in the light salt meadow ecotype was intermediate between those of the swamp and dune ecotypes. Abundance of bundle sheath organelles in the heavy salt meadow ecotype was intermediate. The swamp ecotype had photosynthetic enzyme activities typical of C₃ type plants, whereas the dune ecotype had an increased activity of phosphoenolpyruvate carboxylase (PEPC), a key C₄ enzyme, and a decreased ribulose 1,5-bisphosphate carboxylase (Rubisco) activity. The light salt meadow and heavy salt meadow ecotypes had substantial activities of PEPC, which indicates potential for C₄ photosynthesis. These data suggest that this species evolved the C₃-like ecotype in swamp environments and the C₄-like C₃-C₄ intermediate in dune desert environments, and C₃-like C₃-C₄ intermediates in salt environments.     

Relationship between leaf nitrogen and photosynthetic rate for three NAD-ME and three NADP-ME C4 grasses

Theoretical considerations have suggested that there may be differences in photosynthetic nitrogen use efficiency (PNUE) among plants that use different biochemical variants of C(4) photosynthesis. To test this hypothesis we examined the leaf nitrogen content and photosynthetic rates of six grass species (three of C(4) subtype NAD-ME and three of C(4) subtype NADP-ME) grown over a wide range of nitrogen supply. While there were significant differences among the species in various traits, there were no consistent differences between the C(4) subtypes in either leaf nitrogen content at a given level of nitrogen supply or in the leaf nitrogen-photosynthesis relationship. We suggest that species-level variation in photosynthetic nitrogen use efficiency among C(4) species is large enough to mask any differences that may be due to C(4) subtype.     

Leaf anatomical characters in relation to the C3 and C4 photosynthetic pathway in Cyperus (Cyperaceae)

Leaf anatomical characters of twelve species from the genus Cyperus, a genus known to contain species with both C₃ and C₄ plants, have been investigated. We investigated and established the usefulness of all‐inclusive functional leaf anatomical characters for identifying the photosynthetic pathways of these species. The species investigated were C. articulatus L., C. compressus L., C. difformis L., C. dilatatus Schum. & Thonn., C. distans L., C. esculentus L., C. haspan L., C. imbricatus Retz., C. iria L., C. rotundus L., C. sphacelatus Rottb. and C. tenuiculmis (Boeck.) Hooper, collected from locations in southwestern Nigeria. Standard anatomical procedures for examining epidermal and cross sections of leaves were employed. Our data suggested that a combination of characters, such as the occurrence of Kranz tissue, maximum cell distant count, maximum lateral cell count, interveinal distance, and to some extent leaf and mesophyll thickness, provide a reliable basis for the assessment of the photosynthetic pathways of the investigated species as compared to the isolated characters used previously. The study indicate that C. difformis and C. haspan are C₃ species while the rest follow the C₄ photosynthetic pathway. A salient feature of this study is the identification of C. dilatatus as belonging to the C₄ group.     

Leaf anatomy and photosynthetic characteristics of succulent alpine plants growing at high elevations

Sempervivum monlanum and Saxifraga particulate are succulent alpine plants, with flat leaves, growing at up to 3300 m; Sedum atraium , with cylindrical leaves, is only present at the highest elevations. We have made a preliminary study of the photosynthetic mechanism (C, or CAM) of these species in relation to morphological and ultrastructural differences. The following physiological parameters were determined in plants collected at 3000 m: δ¹³C, night/day variations of titratable acidity and kinetic properties (Vmax, Km, S_0.5, n) of phosphoenolpyruvate carboxylase (EC 4.1.1.31). The results suggest that, even at the highest elevations, the photosynthetic system can be obligate CAM ( Sempervivum monlanum ), facultative C₃-CAM ( Sedum atratum ) or obligate C, ( Saxifraga paniculata ). Other parameters such as chlorophyll a/b ratio, succulence and succulence of mesophyll were measured and the results were consistent with the above mechanisms. Morphological analyses were performed by using LM, SEM and TEM. Sempervivum monlanum (CAM) leaves were found to differ from those of Saxifraga paniculata (C,) in having fewer thyreoids per granum, more plastoglobules per chloroplast, a higher plastoglobule relative density and a lower stomatal density. In Sedum atratum (facultative C₃-CAM) corresponding values were intermediate.     

A possible role for C4 photosynthetic enzymes in tolerance of Zea mays to NaCl

Treatment of 14-day-old maize cultivars (Hybrid351 and Giza2) with 250 mM NaCl significantly reduced shoot fresh and dry weights and protein content during the subsequent 12 days. The magnitude of reduction was more pronounced in Giza than Hybrid. Both cultivars contained converging levels of protein for the enzymes phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), pyruvate phosphate dikinase (PPDK) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) under normal conditions; however, NaCl led to increase these levels in Hybrid and decrease them in Giza. Moreover, NaCl significantly inhibited the activities of PEPC, MDH and PPDK in both cultivars during the first 2 days, thereafter the inhibition nullified only in Hybrid; nonetheless, Rubisco was the least affected enzyme in both cultivars. In addition, NaCl slightly increased V _max of PEPC, MDH and PPDK in Hybrid with no change in K _m; nevertheless V _max dropped in Giza with an increase in K _m of only PEPC and MDH. Also K _cat, K _cat/K _m and V _max/K _m of all enzymes were lower in treated Giza than in treated Hybrid. The increased V _max of all enzymes in only Hybrid by NaCl confirms that they were synthesised more in Hybrid than in Giza. However, the decreased V _max in Giza concomitant with the increased K _m points to an interference of salinity with synthesis of enzymes and their structural integrity. This would lead to a noncompetitive inhibition for the enzymes. These findings declare that maize tolerance to NaCl was larger in Hybrid compared to Giza due to a role for C4 enzymes.     

Leaf anatomy and subgeneric affiliations of C3 and C4 species of Suaeda (Chenopodiaceae) in North America

The halophytic genus Suaeda (Chenopodiaceae) includes species with the C3 and C4 photosynthetic pathways. North American species of this genus were investigated to determine whether C3 and C4 leaf anatomy are consistent within the two sections of Suaeda, Chenopodina and Limbogermen, present on this continent. All species from section Chenopodina were found to possess C3 anatomy, whereas all species from section Limbogermen were found to be C4 species. Characteristics of leaf anatomy and chloroplast ultrastructure are similar to those reported from C3 and C4 species, respectively, from the Eastern Hemisphere. All species from section Limbogermen have the suaedoid type of leaf anatomy, characterized by differentiation of the mesophyll into palisade parenchyma and a chlorenchymatous sheath surrounding central water-storage tissue, as well as leaf carbon isotope ratios (_13C) of above -20. All species from section Chenopodina have austrobassioid leaf anatomy without a chlorenchymatous sheath and _13C values of below -20. According to our literature review, the photosynthetic pathway has now been reported for about half (44) of the Suaeda species worldwide. The C3 and C4 photosynthetic syndromes are with few exceptions distributed along sectional or subsectional lines. These findings throw new light on the infrageneric taxonomy of this genus.     

Engineering C4 photosynthetic regulatory networks

C4 photosynthesis is a complex metabolic pathway responsible for carbon fixation in major feed, food and bioenergy crops. Although many enzymes driving this pathway have been identified, regulatory mechanisms underlying this system remain elusive. C4 photosynthesis contributes to photosynthetic efficiency in major bioenergy crops such as sugarcane, Miscanthus, switchgrass, maize and sorghum, and international efforts are underway to engineer C4 photosynthesis into C3 crops. A fundamental understanding of the C4 network is thus needed. New experimental and informatics methods can facilitate the accumulation and analysis of high-throughput data to define components of the C4 system. The use of new model plants, closely related to C4 crops, will also contribute to our understanding of the mechanisms that regulate this complex and important pathway.     

Leaf development and photosynthetic properties of three tropical tree species with delayed greening

Leaf developmental patterns were characterized for three tropical tree species with delayed greening. Changes in the pigment contents, photosynthetic capacity, stomata development, photosystem 2 efficiency, rate of energy dissipation, and the activity of partial protective enzymes were followed in developing leaves in an attempt to elucidate the relative importance of various photoprotective mechanisms during leaf ontogeny. Big leaves of Anthocephalus chinensis, a fast-growing light demanding species, expanded following an exponential pattern, while relatively small leaves of two shade-tolerant species Litsea pierrei and Litsea dilleniifolia followed a sigmoidal pattern. The juvenile leaves of A. chinensis and L. pierrei contained anthocyanin located below the upper epidermis, while L. dilleniifolia did not contain anthocyanin. Leaves of A. chinensis required about 12 d for full leaf expansion (FLE) and photosynthetic development was delayed 4 d, while L. pierrei and L. dilleniifolia required 18 or 25 d for FLE and photosynthetic development was delayed 10 or 15 d, respectively. During the leaf development the increase in maximum net photosynthetic rate was significantly related to changes in stomatal conductance and the leaf maturation period was positively related to the steady-state leaf dry mass per area for the three studied species. Dark respiration rate of leaves at developing stages was greater, and pre-dawn initial photochemical efficiency was lower than that of mature leaves. Young leaves displayed greater energy dissipation than mature leaves, but nevertheless, the diurnal photoinhibition of young L. dilleniifolia leaves was higher than that of mature leaves. The young red leaves of A. chinensis and L. pierrei with high anthocyanin contents and similar diurnal photoinhibition contained more protective enzymes (superoxide dismutase, ascorbate peroxidase) than mature leaves. Consequently, red leaves may have higher antioxidant ability.     

Seasonal changes of polyamines in habitat adaptation of different ecotypes of reed plants

The leaves of four reed ecotypes (Phragmites communis Trinius) growing in the desert regions of northwest China were investigated for levels of polyamines and activity of arginine decarboxylase (ADC; EC 4.1.1.19) during the growing season of 5 months. The polyamines in the leaves of all reed ecotypes consisted of putrescine, spermidine and spermine. The polyamine levels of the leaves were lower in the swamp reed than in the terrestrial reed ecotypes. Leaf polyamine levels decreased in all ecotypes over the course of the season. Compared to the swamp reed, the terrestrial reed ecotypes maintained higher ADC activity and a predominance of spermine, resulting in a lower ratio of putrescine to spermidine and spermine. It seems that the adaptation of reed plants to drought and saline habitats may be correlated with putrescine synthesis via the ADC pathway, and with a successful conversion of putrescine to spermidine and spermine.     

Differential expression of photosynthesis-related genes of reed ecotypes in response to drought and saline habitats

Three ecotypes of reed (Phragmites communis Trinius), swamp reed (SR), dune reed (DR), and heavy salt meadow reed (HSMR), growing in desert regions of northwest China were simultaneously investigated in their natural state for gas exchange patterns and the expression of three photosynthesis-related genes, cab (the gene for the light-harvesting chlorophyll a/b binding protein, LHC), psbA (the gene for the reaction centre D1 protein of photosystem 2, PS2), and 16S rDNA (the gene for plastid 16S rRNA). Stomatal conductance (gs) and intercellular CO2 concentration (ci) were markedly lower in the two terrestrial ecotypes (DR and HSMR) as compared to SR, paralleling a similar observed depression in net photosynthetic rate (PN). However, DR with the lowest measured gs and ci still exhibited a higher PN compared to HSMR. These results suggest that both stomatal and non-stomatal factors account for the comparatively low carbon assimilation in the terrestrial ecotypes. An increase in the expression of photosynthesis-related genes was observed in DR compared to SR, whereas the reverse situation was true in HSMR. The expression of photosynthesis-related genes may contribute to reed plants' photosynthetic capacity per leaf area under natural water deficits, but the levels of photosynthesis-related gene expression are not directly correlated with reed plants' general ability for survival and adaptation under water deficient conditions.     

Evolution of C4 photosynthetic genes and overexpression of maize C4 genes in rice

C3 plants including many agronomically important crops exhibit a lower photosynthetic efficiency due to inhibition of photosynthesis by O₂ and the associated photorespiration. C4 plants had evolved the C4 pathway to overcome low CO₂ and photorespiration. This review first focuses on the generation of a system for high level expression of the C4-specific gene for pyruvate, orthophosphate dikinase (Pdk), one of the key enzyme in C4 photosynthesis. Based on the results with transgenic rice plants, we have demonstrated that the regulatory system controlling thePdk expression in maize is not unique to C4 plants but rice (C3 plant) posses a similar system. Second, we discussed the possibility of the high level expression of maize C4-specific genes in transgenic rice plants. Introduction of the maize intact phosphoenolpyruvate carboxylase gene (Ppc) caused 30–100 fold higher PEPC activities than non-transgenic rice. These results demonstrated that intact C4-type genes are available for high level expression of C4 enzymes in rice plants. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Leaf area expansion and net photosynthetic rate of three bromegrass (Bromus) species

This study measured individual leaf area expansion rate and leaf net photosynthetic rate (P _N) of meadow bromegrass (Bromus riparius Rehm.), smooth bromegrass (Bromus inermis Leyss.) and hybrid bromegrass (B. riparius × B. inermis). Smooth bromegrass expanded individual leaf area 1.5 times faster than meadow bromegrass and hybrid bromegrass. P _N was highest in smooth bromegrass, intermediate in hybrid bromegrass, and lowest in meadow bromegrass. Rapid growth of meadow bromegrass following defoliation compared to smooth bromegrass and hybrid bromegrass could not be explained by higher rates of these measured characteristics.