Sakae Agarie

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.

Lotus japonicus nodulation is photomorphogenetically controlled by sensing the red/far red (R/FR) ratio through jasmonic acid (JA) signaling

Light is critical for supplying carbon to the energetically expensive, nitrogen-fixing symbiosis between legumes and rhizobia. Here, we show that phytochrome B (phyB) is part of the monitoring system to detect suboptimal light conditions, which normally suppress Lotus japonicus nodule development after Mesorhizobium loti inoculation. We found that the number of nodules produced by L. japonicus phyB mutants is significantly reduced compared with the number produced of WT Miyakojima MG20. To explore causes other than photoassimilate production, the possibility that local control by the root genotype occurred was investigated by grafting experiments. The results showed that the shoot and not the root genotype is responsible for root nodule formation. To explore systemic control mechanisms exclusive of photoassimilation, we moved WT MG20 plants from white light to conditions that differed in their ratios of low or high red/far red (R/FR) light. In low R/FR light, the number of MG20 root nodules dramatically decreased compared with plants grown in high R/FR, although photoassimilate content was higher for plants grown under low R/FR. Also, the expression of jasmonic acid (JA) -responsive genes decreased in both low R/FR light-grown WT and white light-grown phyB mutant plants, and it correlated with decreased jasmonoyl-isoleucine content in the phyB mutant. Moreover, both infection thread formation and root nodule formation were positively influenced by JA treatment of WT plants grown in low R/FR light and white light-grown phyB mutants. Together, these results indicate that root nodule formation is photomorphogenetically controlled by sensing the R/FR ratio through JA signaling.

Isolation and Characterization of Mutants of Common Ice Plant Deficient in Crassulacean Acid Metabolism

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.

Overexpression of C4 PEPC caused O2-insensitive photosynthesis in transgenic rice plants

We have introduced an intact maize gene for phosphoenolpyruvate carboxylase (PEPC) into C3 plants, rice (Oryza sativa cv. Kitaake). Most transgenic rice plants showed high-level expression of the maize gene. PEPC was two to three times more active in the leaves of some transgenic plants than in maize leaves. In transformants, the sensitivity of photosynthesis to O2 inhibition was reduced with the increased activity of the maize C4 PEPC. However, the alleviation of O2 inhibition was not due to an increase in the partial direct fixation of atmospheric CO2 via the enhanced maize PEPC, but rather due to the reduced stimulation of photosynthesis with a subatmospheric O2 level. Pi feeding to the leaves restored CO2 assimilation rate under the subatmospheric O2 condition, and consequently, the O2 inhibition in the transformants increased to a level comparable to that of the non-transformants. These results suggested that the O2-insensitive photosynthesis in the PEPC transformants was caused by a Pi limitation of photosynthesis. The activities of two key enzymes for sucrose synthesis, SPS and FBPase, and the sucrose and starch content were reduced in the leaves of the transformants. On the other hand, the dark respiration rate and the malate content in the leaves increased in the transformants. These results indicated that enhanced PEPC activity led to a decrease in the availability of Pi in chloroplast via a reduction of the activities of the key enzymes responsible for Pi recycling and it also caused an increased consumption of the substrate (triose-phosphate) in respiration, but not in sucrose biosynthesis.

Expression of C3 and C4 photosynthetic characteristics in the amphibious plant Eleocharis vivipara: structure and analysis of the expression of isogenes for pyruvate, orthophosphate dikinase

Eleocharis vivipara, a unique leafless amphibious sedge, adopts the C4 mode of photosynthesis under terrestrial conditions and the C3 mode under submerged aquatic conditions. To analyze the molecular basis of these responses to the contrasting environments, we isolated and characterized two full-length cDNAs for a key C4 enzyme, pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1). The isogenes for PPDK, designated ppdk1 and ppdk2, were highly homologous to one another but not identical. The PPDK1 protein, deduced from the nucleotide sequence of the cDNA, contained an extra domain at the amino terminus which, presumably, serves as a chloroplast transit peptide, while PPDK2 lacked this extra domain. It seems likely, therefore, that the ppdk1 and ppdk2 genes encode a chloroplastic and a cytosolic PPDK, respectively. Genomic Southern blot analysis revealed the existence of a small family of genes for PPDK in the genome of E. vivipara. Northern blot analysis indicate that both chloroplastic and cytosolic genes for PPDK are expressed simultaneously in the culms, a photosynthetic organ, of E. vivipara and that the pattern of expression of these genes differs between the growth forms.

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.

Physiological roles of betacyanin in a halophyte, Suaeda japonica Makino.

Abstract: Halophytes can be used as a crop on salinized agricultural land. Suaeda japonica Makino is an annual highly salt-tolerant plant, that inhabits salt marshes in the Ariake Sea, Japan. Accompanying growth, leaves of S. japonica change from green to red with accumulation of a red pigment, betacyanin. To elucidate the physiological roles of betacyanin in S. japonica, we tested the antioxidant capacity of purified betacyanin and leaf extracts containing different levels of betacyanin under oxidative-stress conditions. The assay with 1,1-diphenyl-2-picrylhydrazyl (DPPH) showed that the purified betacyanin had antioxidant activity. H2O2-induced protein oxidation of the leaf extracts was prevented by the addition of betacyanin. The antioxidant enzyme activity decreased in the red leaves, but the content of malondialdehyde (MDA), an oxidative stress marker in the red leaves was nearly the same as that in the green leaves. Betacyanin synthesis was induced under photoinhibition-inducible conditions of low temperature and high-intensity light. These results indicate that betacyanin serves as an antioxidant in S. japonica and that the leaf reddening of this species is a key adaptive strategy for coping with the harsh environmental conditions in salt marshes of the Ariake Sea.

Potential of the Common Ice Plant, Mesembryanthemum crystallinum as a New High-Functional Food as Evaluated by Polyol Accumulation

Abstract We measured the concentration of polyols (pinitol, ononitol, and myo-inositol), which are known to have health-promoting and/or disease-preventing functions, in the common ice plant (Mesembryanthemum crystallinum L.) cultured under salt- and drought-stressed treatments. In NaCl-treated plant the concentration of pinitol/ononitol increased with increasing NaCl concentration in culture solution. The maximal concentration was 3.6 mg g-1 FW, which was foundin the shoot top, followed by small side shoots (2.1 mg g-1 FW) of mature plants grown with 400 mM NaCl for 35 ds. The drought stress also accelerated the accumulation of pinitol/ ononitol. The maximal concentration was 1.2 mg g-1 FW, which was found in the shoot top of plants under the stress for 25 ds. The myo-inositol increased in salt-stressed plants at 3 ds after the start of the treatment and then decreased with the lapse of time during stress. The concentration of polyols in the ice plant was comparable to that in the other species reported to accumulate polyols at high levels. Radical scavenging activity evaluated by DPPH assay was increased two-fold by 400 mM NaCl treatment, which was twice as high as that in the leaves of lettuce (Lactuca sativa L.). These results indicated the high potential of the ice plant as a polyol-rich high-functional food.

How to express some C4 photosynthesis genes at high levels in rice.

To investigate the difference between Pdk genes that encode pyruvate, orthophosphate dikinase (PPDK), a Pdk gene homologous to the maize C4-type Pdk gene was isolated from a C3 plant, rice, and compared with the maize gene. The primary structures of the genes are essentially the same, except that the rice gene has two additional introns. A transient expression assay of Pdk promoters using maize mesophyll protoplasts showed that the mode of expression of the maize and rice genes differs only in the expression activity of the promoter for the chloroplast-type PPDK: the maize gene was expressed fourfold higher than the rice gene. It was also found that a chimeric gene containing the maize Pdk promoter and a reporter gene led to high expression of the reporter gene in transgenic rice. Based on the above observations, the intact genes from maize encoding enzymes for C4 photosynthesis were introduced into rice to increase the activity of the C4 enzymes. As expected, the introduction of the maize gene led to high expression of C4 enzymes in transgenic rice. The activities of phospoenolpyruvate carboxylase (PEPC) and PPDK increased up to 110- and 40-fold more, respectively, than those of nontransgenic rice. High expression of C4 enzymes did not result solely from the high expression activity of the maize gene, since the introduction of a maize PPDK cDNA fused to the maize Pdk promoter or rice Cab promoter did not lead to high expression of PPDK. In some transgenic rice plants carrying the intact maize gene, the level of PPDK protein amounted to 35% of total leaf-soluble protein. The high expression of each C4 enzyme altered metabolism slightly but did not seem to increase the photosynthetic efficiency of transgenic rice leaves.

Effect of urea-type cytokinins on the adventitious shoots regeneration from cotyledonary node explant in the common ice plant, Mesembryanthemum crystallinum

Abstract Mesembryanthemum Crystallinum (Common Ice Plant) Was Used As A Model Plant To Study The Regulatory Properties of Crassulacean Acid Metabolism (Cam) and Tolerance To Abiotic Stresses. Although Transformation Is A Useful Genetic Approach, It Has Not Been Established in This Species Due To Recalcitrancy For Regeneration. To Establish An Efficient Procedure For Regeneration of M. Crystallinum, We Examined The Effects of Urea-Type Cytokinins, Thidiazuron (Tdz) and Forchlorofenuron (Cppu) On The Adventitious Shoot induction. Adventitious Shoots Were Generated Only From Explants Obtained From The Cotyledonary Node, Not From Explants Obtained From The Cotyledon, Hypocotyl and Roots. Urea-Type Cytokinins, Tdz and Cppu Were More Effective For The induction and The Morphogenesis of Adventitious Shoots Than Adenine-Type Cytokinin, 6-Benzyladenopurine (Ba). We Have Found That The 2.5 Mg L-1 Tdz induced The Largest Number of Multiple Shoots and The Highest Frequency of Adventitious Shoot induction From Single Explant. in Addition, Fewer Hyperhydric Shoots Were Produced On The Medium Containing Tdz Than in That Containing Ba and Cppu in The Presence of 1.0 Mg L-1 Naa. The Regenerated Shoots Rooted On The Ms Medium Within One Month, and The Rooting Was Promoted By Replacing The Agar Medium With Vermiculite Or Florialite. The Fertile Plant With Normal Morphological Properties Was Harvested For Four Months After Sowing. Using The Improved Regeneration Procedure With Tdz, We Successfully introduced A Kanamycin-Resistant Gene (Nptii-Hph) into The Cotyledonary Node Mediated By Agrobacterium Tumefaciens. These Results indicated That This Regeneration Procedure Using Cotyledonary Node Explants and Tdz Could Be Useful For The Genetic Engineering of M. Crystallinum.