Maurice S. B. Ku

High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants

Using an Agrobacterium-mediated transformation system, we have introduced the intact gene of maize phosphoenolpyruvate carboxylase (PEPC), which catalyzes the initial fixation of atmospheric CO2 in C4 plants into the C3 crop rice. Most transgenic rice plants showed high-level expression of the maize gene; the activities of PEPC in leaves of some transgenic plants were two- to threefold higher than those in maize, and the enzyme accounted for up to 12% of the total leaf soluble protein. RNA gel blot and Southern blot analyses showed that the level of expression of the maize PEPC in transgenic rice plants correlated with the amount of transcript and the copy number of the inserted maize gene. Physiologically, the transgenic plants exhibited reduced O2 inhibition of photosynthesis and photosynthetic rates comparable to those of untransformed plants. The results demonstrate a successful strategy for installing the key biochemical component of the C4 pathway of photosynthesis in C3 plants.

Evolution and Expression of C4 Photosynthesis Genes

Based on the differences in the mechanism of CO, assimilation, land plants can be divided into three major photosynthetic types, namely C,, C,, and Crassulacean acid metabolism (CAM) plants. Each photosynthetic type possesses a unique set of anatomical, physiological, and biochemical features that allow them to adapt to a specific ecological niche. C, plants perform well in temperate climates, whereas C, plants thrive in high-light, arid, and warm environments. CAM plants, characterized by CO, uptake in the night, adapt to extreme arid conditions. Taxonomical and phylogenetic studies suggest that CAM and C, plants were derived from C, plants and the transitions occurred many times in diverse taxa during the course of evolution (Moore, 1982). A drastic decline in atmospheric CO, leve1 during the late Cretaceous period (65-85 million years ago), a time of major expansion of the angiosperms, has been proposed to account for the increase of C, plants (Ehleringer et al., 1991). The major function of the C, pathway is thought to overcome the limitation of low CO,, which results in significant increases in photorespiration and thus reduces competitiveness. Although it is widely assumed that CAM evolved in response to selection for increased water-use efficiency, the occurrence of CAM in aquatic plants (Keeley and Morton, 1982), in which photosynthesis is often limited by low CO, rather than water, strongly argues that the driving force for its evolution may be low CO, as well. Both C, and CAM evolved a very similar photosynthetic biochemistry for concentrating CO, in the leaf, except that the C0,-concentrating steps are spatially separated in C, plants but temporally separated in CAM plants. C, photosynthesis and CAM occur in only one cell type, the mesophyll cells, whereas C, photosynthesis requires the coordination of two photosynthetic cell types, the mesophyll and bundle-sheath cells. Therefore, the genetic modifications required for achieving the C0,-concentrating mechanism are considered relatively small for CAM, as compared with those required for C, photosynthesis. Comparative phylogenetic studies also suggest that CAM evolved earlier than C, photosynthesis (Moore, 1982).

C3-C4 Intermediate Photosynthesis in Plants

Among higher plants, C3 species have high levels of photorespiration, which limit the rate of net carbon dioxide assimilation. C4 plants have evolved a mechanism that overcomes photorespiration. Some species have photorespiration levels intermediate to C3 and C4 species, as well as other intermediate anatomical, physiological, and biochemical characteristics. These C3-C4 intermediates provide excellent opportunities for studying how photorespiration is reduced, as well as the genetics and evolution of C4 photosynthesis. (Accepted for publication 11 April 1984)

5 – Biochemistry of C3–C4 Intermediates

Publisher Summary This chapter focuses on the biochemistry of C3–C4 intermediates. The reductive pentose phosphate pathway, or PCR cycle, is the means through which higher plants assimilate CO2. There is currently no evidence that any terrestrial plants have undergone a change in the properties of D-ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) that would increase its capacity to react with CO2 over O2. In general, a C3–C4 intermediate species can be defined as a species in which: (1) one or more of the features of the Kranz syndrome is “intermediate,” that is, the character is at some stage or level between that of a C3 and a C4 species, or (2) there is a mixture of fully expressed features of the Kranz syndrome combined with those of species lacking this syndrome. If the biochemical and anatomical features of the Kranz syndrome are developed to varying degrees, then a variety of atypical physiological responses in photosynthesis and photorespiration may occur.

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C4 photosynthesis was investigated in five C3-C4 intermediate species, one C3 species, and one C4 species in the genus Flaveria, using 14CO2 pulse-12CO2 chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating 14CO2 into the C4 acids malate and aspartate, following an 8-s pulse. The proportion of 14C label in these C4 products ranged from 50–55% to 20–26% in the C3-C4 intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, 14CO2 into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C4 photosynthesis among the intermediate species based on the apparent rate of conversion of 14C label from the C4 cycle to the C3 cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C4-cycle products and an increase in percentage label in C3-cycle products during chase periods with 12CO2, although the rate of change was slower than in the C4 species, F. palmeri. In these C3-C4 intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C3-C4 intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C4-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C4 and C3 cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C3 species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C4-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O2, the absorbed quantum yields for CO2 uptake (in mol CO2·[mol quanta]-1) averaged 0.053 in F. cronquistii (C3), 0.051 in F. trinervia (Spreng.) Mohr (C4), 0.052 in F. ramosissima (C3-C4), 0.051 in F. anomala (C3-C4), 0.050 in F. linearis (C3-C4), 0.046 in F. floridana (C3-C4), and 0.044 in F. pubescens (C3-C4). In 2% O2 an enhancement of the quantum yield was observed in all of the C3-C4 intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O2 were intermediate in value to the C3 and C4 species, indicating a co-function of the C3 and C4 cycles in CO2 assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O2 presumably reflect an ineffcient transfer of carbon from the C4 to the C3 cycle. The response of the quantum yield to four increasing O2 concentrations (2–35%) showed lower levels of O2 inhibition in the C3-C4 intermediate F. ramosissima, relative to the C3 species. This indicates that the co-function of the C3 and C4 cycles in this intermediate species leads to an increased CO2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O2.

Analysis of inhibition of photosynthesis due to water stress in the C3 species Hordeum vulgare and Vicia faba: Electron transport, CO2 fixation and carboxylation capacity

A C3 monocot, Hordeum vulgare and C3 dicot, Vicia faba, were studied to evaluate the mechanism of inhibition of photosynthesis due to water stress. The net rate of CO2 fixation (A) and transpiration (E) were measured by gas exchange, while the true rate of O2 evolution (JO2) was calculated from chlorophyll fluorescence analysis through the stress cycle (10 to 11 days). With the development of water stress, the decrease in A was more pronounced than the decrease in JO2 resulting in an increased ratio of Photosystem II activity per CO2 fixed which is indicative of an increase in photorespiration due to a decrease in supply of CO2 to Rubisco. Analyses of changes in the JO2A ratios versus that of CO2 limited photosynthesis in well watered plants, and RuBP pool/RuBP binding sites on Rubisco and RuBP activity, indicate a decreased supply of CO2 to Rubisco under both mild and severe stress is primarily responsible for the decrease in CO2 fixation. In the early stages of stress, the decrease in Ci (intercellular CO2) due to stomatal closure can account for the decrease in photosynthesis. Under more severe stress, CO2 supply to Rubisco, calculated from analysis of electron flow and CO2 exchange, continued to decrease. However, Ci, calculated from analysis of transpiration and CO2 exchange, either remained constant or increased which may be due to either a decrease in mesophyll conductance or an overestimation of Ci by this method due to patchiness in conductance of CO2 to the intercellular space. When plants were rewatered after photosynthesis had dropped to 10–30% of the original rate, both species showed near full recovery within two to four days.

Photosynthetic acclimation of maize to growth under elevated levels of carbon dioxide

Abstract. The effects of elevated CO2 concentrations on the photochemistry, biochemistry and physiology of C4 photosynthesis were studied in maize (Zea mays L.). Plants were grown at ambient (350 μL L−1) or ca. 3 times ambient (1100 μL L−1) CO2 levels under high light conditions in a greenhouse for 30 d. Relative to plants grown at ambient CO2 levels, plants grown under elevated CO2 accumulated ca. 20% more biomass and 23% more leaf area. When measured at the CO2 concentration of growth, mature leaves of high-CO2-grown plants had higher light-saturated rates of photosynthesis (ca. 15%), lower stomatal conductance (71%), higher water-use efficiency (225%) and higher dark respiration rates (100%). High-CO2-grown plants had lower carboxylation efficiencies (23%), measured under limiting CO2, and lower leaf protein contents (22%). Activities of a number of C3 and C4 cycle enzymes decreased on a leaf-area basis in the high-CO2-grown plants by 5–30%, with NADP-malate dehydrogenase exhibiting the greatest decrease. In contrast, activities of fructose 1,6-bisphosphatase and ADP-glucose pyrophosphorylase increased significantly under elevated CO2 condition (8% and 36%, respectively). These data show that the C4 plant maize may benefit from elevated CO2 through acclimation in the capacities of certain photosynthetic enzymes. The increased capacity to synthesize sucrose and starch, and to utilize these end-products of photosynthesis to produce extra energy by respiration, may contribute to the enhanced growth of maize under elevated CO2.

Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes

The photosynthetic characteristics of four transgenic rice lines over-expressing rice NADP-malic enzyme (ME), and maize phosphoenolpyruvate carboxylase (PC), pyruvate,orthophosphate dikinase (PK), and PC+PK (CK) were investigated using outdoor-grown plants. Relative to untransformed wild-type (WT) rice, PC transgenic rice exhibited high PC activity (25-fold increase) and enhanced activity of carbonic anhydrase (more than two-fold increase), while the activity of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) and its kinetic property were not significantly altered. The PC transgenic plants also showed a higher light intensity for saturation of photosynthesis, higher photosynthetic CO2 uptake rate and carboxylation efficiency, and slightly reduced CO2 compensation point. In addition, chlorophyll a fluorescence analysis indicates that PC transgenic plants are more tolerant to photo-oxidative stress, due to a higher capacity to quench excess light energy via photochemical and non-photochemical means. Furthermore, PC and CK transgenic rice produced 22–24% more grains than WT plants. Taken together, these results suggest that expression of maize C4 photosynthesis enzymes in rice, a C3 plant, can improve its photosynthetic capacity with enhanced tolerance to photo-oxidation.

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.

Effects of UV-B radiation on growth, photosynthesis, UV-B-absorbing compounds and NADP-malic enzyme in bean (Phaseolus vulgaris L.) grown under different nitrogen conditions

The effects of UV-B radiation on growth, photosynthesis, UV-B-absorbing compounds and NADP-malic enzyme have been examined in different cultivars of Phaseolous vulgaris L. grown under 1 and 12 mM nitrogen. Low nitrogen nutrition reduces chlorophyll and soluble protein contents in the leaves and thus the photosynthesis rate and dry-matter accumulation. Chlorophyll, soluble protein and Rubisco contents and photosynthesis rate are not significantly altered by ambient levels of UV-B radiation (17 microW m-2, 290-320 nm, 4 h/day for one week). Comparative studies show that under high nitrogen, UV-B radiation slightly enhances leaf expansion and dry-matter accumulation in cultivar Pinto, but inhibits these parameters in Vilmorin. These results suggest that the UV-B effect on growth is mediated through leaf expansion, which is particularly sensitive to UV-B, and that Pinto is more tolerant than Vilmorin. The effect of UV-B radiation on UV-B-absorbing compounds and on NADP-malic enzyme (NADP-ME) activity is also examined. Both UV-B radiation and low-nitrogen nutrition enhance the content of UV-B-absorbing compounds, and among the three cultivars used, Pinto exhibits the highest increases and Arroz the lowest. The same trend is observed for the specific activity and content of NADP-ME. On a leaf-area basis, the amount of UV-B-absorbing compounds is highly correlated with the enzyme activity (r2 = 0.83), suggesting that NADP-ME plays a key role in biosynthesis of these compounds. Furthermore, the higher sensitivity of Vilmorin than Pinto to UV-B radiation appears to be related to the activity of NADP-ME and the capacity of the plants to accumulate UV-B-absorbing compounds.