Photosynthetic Features of Transgenic Rice Expressing Sorghum C 4 Type NADP-ME

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1 Acta Botanica Sinica 2004, 46 (7): Photosynthetic Features of Transgenic Rice Expressing Sorghum C 4 Type NADP-ME CHI Wei, ZHOU Jing-Song *, ZHANG Fang, WU Nai-Hu ** (Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, Beijing , China) Abstract: The gene encoding sorghum NADP malic enzyme, which plays a key role in C 4 photosynthetic pathway, was isolated by RT-PCR and cdna library screening. The bp cdna sequence obtained includes a bp open reading frame that encodes 636 amino acids and a terminating codon (GenBank accession number: AY274836). It was then introduced into Nongken 58, a rice variety, using an Agrobacteriummediated system. Southern hybridization, Northern hybridization and enzyme activity determination all confirmed the effective expression of sorghum (Sorghum vulgare L.) C 4 type NADP-ME in rice, with the enzyme activity being elevated 1 7 folds. However, no appreciable change was demonstrated in carbon assimilation of the transgenic rice though increased photoinhibition was noted under high light intensity. Key words: NADP malic enzyme (NADP-ME); sorghum; transgenic rice; physiology of photosynthesis According to differences in carbon assimilation pathways in photosynthesis, three photosynthetic types may be distinguished among land plants: C 3, C 4, and CAM. Compared with C 3 plants, the unique Kranz structure and C 4 pathway endow C 4 plants and CAM plants with higher efficiency in photosynthesis, water utilization and nutrient utilization (Hatch, 1987). These advantages of C 4 plants are especially valuable during stresses caused by high temperature, high light intensity and drought. It has therefore long been a much sought for objective to improve the photosynthetic properties of C 3 plants by introducing the photosynthetic traits of C 4 plants (CAM plants) into them. NADP-ME, a key enzyme in the C 4 pathway in the NADP- ME subtype of C 4 plants, was located in the chloroplasts of the bundle sheath cells. In the C 4 pathway, ambient CO 2 is first fixed by PEPCase in the form of malic acid in mesophyll cells of leaves, which is then transported to bundle sheath cells where it is decarboxylated by NADP-ME, releasing CO 2 again. The released CO 2 is further fixed by RuBisco into the C 3 pathway. Evidently, it is the decarboxylation of NADP-ME that helps enrich the CO 2 immersing RuBisco, thus lowering photorespiration and improving the photosynthetic efficiency. As pointed out by Ku et al. (1991), the activity of NAPD-ME shows negative correlation with the activity of photorespiration. It thus appears that transfer of NADP-ME gene into C 3 plants might be an effective way of lowering photorespiration and improving photosynthetic efficiency of C 3 plants. There are many reports on the successful transfer of photosynthetic enzymes of C 4 plants into C 3 plants and their high level expression in the latter by means of gene engineering techniques (Ku et al., 1999, Takeuchi et al., 2000, Furayama et al., 2001, Zhang et al., 2003). Yet inconsistent results have been obtained in studies aimed at elucidating the underlying physiological mechanisms. Recently, Takeuchi et al. (2000) tried to account for some of the physiological characteristics of transgenic rice expressing high level maize NADP-ME in terms of chloroplast development (Takeuchi et al., 2000). So far, however, no report has been published on systematic study of the photosynthetic physiology such as CO 2 exchange in transgenic plants expressing NADP-ME of the C 4 plant. In this work of ours, sorghum NADP-ME was cloned and introduced into rice using the transformation system mediated by Agrobacterium, and then systematic study on the photosynthetic physiology involved was carried out. It is hoped that better understanding of the physiology might prove helpful to the genetic approach for improving photosynthesis in C 3 plants. 1 Materials and Methods 1.1 Experimental materials Jinzhong 405, a variety of sorghum (Sorghum vulgare L.), was cultivated in greenhouse and, after 15 d of growth, seedlings were harvested for further experiments. The variety of rice (Oryza sativa L.) used for Received 22 Oct Accepted 15 Jan Supported by the State Key Basic Research and Development Plan of China (G ). * Current address: Research Center of Freshwater Fishery, National Institutes of Aquatic Products Research, Wuxi , China. ** Author for correspondence. Tel: +86 (0) ; <nhwu@genetics.ac.cn>.

2 transformation experiment was Nongken 58N. Both the transgenic rice (ME) and the untransformed controls (WT) were cultivated under controlled conditions until the booting stage when specimens were collected for determination of the physiological indices. 1.2 Cloning and sequencing of sorghum NADP-ME Based on the published conserved regions of the sequences encoding C 4 type NADP-ME, two primers were synthesized: primer 5: 5'-GAAGGTTTGGCTIGTGGACTC - 3' primer 3: 5'-GATGCTGGTGAAIGGTGGGAA-3' where I stands for hypoxanthine. Using cdna taken from green leaves of sorghum as templates and the above two primers, PCR amplification was performed in a reaction system of 20 µl. After initial denaturing at 94 for 5 min, the sample underwent the following steps for 30 cycles: denaturing at 94 for 1 min, annealing at 61 for 1 min, then extension at 72 for 1 min. After final extension at 72 for 10 min, the amplification product was cloned into the vector pgem- T-easy. It was then sent to Bo Ya Company (Shanghai, China) for sequencing. As shown by homology study, it was identified as a fragment of NADP-ME gene. mrna was extracted from 1 g of green leaves of sorghum, using the Quickprep Micro mrna isolation kit (Phamacia, Piscataway, New Jersy, USA). Then cdna was synthesized from the mrna obtained, using the TimeSaver cdna synthesis kit (Phamacia Piscataway, New Jersy, USA). Next, the cdna was ligated to the vector, λ ZAP (Stratagene, La Jolla, California, USA), and packed with the packing protein of Statagene to form a cdna library. The cdna library was screened, using a gene fragment of sorghum NADP-ME as probe. Eight positive clones were obtained from about plagues. The recombinant DNA isolated therefrom was then digested with EcoR to determine the length of the insert and clones with inserts longer than 2 kb were sequenced. Analysis of the nucleotide sequence obtained and its homology study were performed using GENTYX-WIN 3.1. The sequence of the chloroplast transfer peptide was analyzed using Chlop V1, a program downloaded from Internet. 1.3 Amplification of CAB promoter, construction of expression vector, and genetic transformation of rice Two primers were designed based on the sequence of the promoter of chloroplast a/b binding protein gene (CAB) of rice (GenBank accession number: E03236): Cab5: 5'- ACTCTAGAGATTGGGATTAAGGTAATG-3'; Cab3: 5'- ACGATATCGATGCAGTGAGCTGTGAGAG-3'. Next, PCR amplification was performed in a 20-µL reaction system using rice genomic DNA as template. After initial denaturing at 94 for 5 min, the sample underwent the following steps for 25 cycles: denaturing at 94 for 1 min, annealing at 55 for 1 min, then extension at 72 for 1 min. After final extension at 72 for 10 min; the product was cloned into pbluescript. Sequencing showed it to be basically the same as the CAB promoter as recorded in GenBank, with a similarity of 99%. A transgenesis vector was constructed on the basis of pcambia1301: at the polycloning sites were inserted CAB promoter, ME cdna and NOS terminator. Designated as p1301-me, the structure is shown in Fig.1. p1301-me was then introduced into the Agrobacterium tumefaciens strain EHA105 by freezing-thawing. The transformation of rice was performed by the method of Hiei et al. (1994). 1.4 Southern and Northern hybridization of transgenic rice Genomic DNA was extracted from rice using the method of CTAB. Fifteen µg of the extracted DNA, digested with BstX, were separated by electrophoresis in 0.7% agarose gel before being transferred to Hybond membrane for Southern hybridization. All the procedures, including prehybridization, were performed in routine manner. Total RNA was extracted from the leaves using guannidine isothiocyanate and was separated with electrophoresis in 1.2% denaturing gel. It was then transferred to Hybond nylon membrane for Northern hybridization. 1.5 Extraction of soluble proteins and determination of its enzyme activity Naught point two gram of rice leaves that had been exposed to full light (at 9:00 11:00, with an intensity of about µmol m 2 s 1 ) for 2 h was rapidly placed in a pre-cooled mortar and ground with 1.5 ml extracting buffer in ice bath. The buffer was composed of 50 mmol/l Tris- HCl (ph 7.0), 10 mmol/l MgCl 2, 1 mmol/l EDTA, 5 mmol/l DTT and 5% insoluble PVP. After full maceration, the Fig.1. The schematic diagram of transgenesis vector of p1301-me. 35s, promoter of cauliflower mosaic virus; GUS, β-glucuronidase gene Tnos, terminator of the nopine synthase gene; P CAB, promoter of chloroplast a/b binding protein gene; ME, cdna of sorghum NADP-ME, HPTII, hygromycin phosphotransferase gene; LB, left T-DNA border; RB, right T-DNA border.

3 CHI Wei et al.: Photosynthetic Features of Transgenic Rice Expressing Sorghum C 4 Type NADP-ME extract was centrifuged at g and 4 for 10 min and the supernatant was gathered for activity determination. Activities of the enzymes were determined respectively by the following methods: PEPCase by that of Gonzalez et al. (1984); NADP-ME and NADP-MDH by those of Li et al. (1987) and Chen et al. (1981) ; PPDK by that of Hatch et al. (1975); and Rubisco by Kung et al. (1980). 1.6 Determination of substrate contents for photosynthesis The contents of malate, pyruvate and phosphoenolpyruvate (PEP) were determined by the method of Sakae et al. (2002). 1.7 Measurement of CO 2 exchange and chlorophyll fluorescence Photosynthetic rates of rice leaves in booting stage under various light intensities were measured using a portable photosyntometer, LI-6400 (LI-COR Co., USA). After adaptation to darkness for 30 min, parameters of chlorophyll fluorescence were measured using a portable fluorometer, FMS2 (Hansatech Co., UK). 2 Results 2.1 Amplification of fragments of sorghum NADP-ME and cloning of full-length cdna A pair of primers were designed based on a relatively conserved region of NADP-ME of C 4 plants and each had an hypoxanthine incorporated. Since hypoxanthine may pair with anyone of the three nucleotides, T, C, or A, its incorporation ensures both the specificity and conservativeness of the primers, thus enhancing the efficiency of amplification. PCR amplification of a single chain of the cdna retrotranscribed from leaf RNA using this pair of primers produced a fragment about 500 bp long, within the range of the molecular weight as expected. The amplified fragment was cloned into pgem-t-easy vector and subsequent sequence showed it had 532 bp and a 91% similarity with maize NADP-ME. This confirmed our result as in the cdna fragment of sorghum NADP-ME. Next, screening of cdna library with the fragment obtained as probe produced a positive clone about 2.1 kb. The insert of this clone was subcloned into pbluescript SK and sequencing showed the insert to be bp in length. Analysis using GENETYX Win 3.1 revealed an opening reading frame of bp which encodes 636 amino acids and a terminator (GenBank accession number: AY274836). 2.2 Structural analysis of sorghum NADP-ME Using software GENETYX Win 3.1, homology study of the amino acid sequence encoded by sorghum NADP-ME as compared with those encoded by NADP-MEs of other plants revealed a 89% similarity (the highest) to that of the C 4 monocot maize, and a 40% similarity (the lowest) to the non-c 4 type NADP-ME of the C 3 plant Arabidopsis. It was interesting that it was more similar to non-c 4 type NADP- ME of C 4 monocot plant (maize) than to C 4 type NADP-ME of C 4 dicot plants (Flaveria trinervia). A similar finding has also been reported in other C 4 type photosynthetic enzymes (Toh et al., 1994). In terms of plant evolution, this seems to indicate that the divergence of plants into C 3 and C 4 photosynthetic types occurred earlier than that into dicots and monocots. At the N terminal of C 4 type NADP-ME, there is a transit peptide sequence that serves to transport the precursor of ME into chloroplast where it is further processed into the mature enzyme. Structural prediction of the transit peptide was performed by importing the amino acid sequence of sorghum C 4 type NADP-ME into Chlop V.1, an analytical software. A transit sequence was indeed found at the N terminal and two splicing sites at the 62nd and 80th amino acids (Fig.2). The predicted molecular weight of the mature protein is somewhere between 61 and 63 kd, basically the same as the value determined for C 4 type NADP-ME in other plants. As a rule, each NADP-ME molecule combines four NADPs in performing its biochemical function. In addition, NADP-ME combines NAD as well as NADP so that the former may work as a competitive inhibitor for the latter. As may be noted in the amino acid sequence predicted, highly conserved sequences are found between the positions and (Fig.2) as compared with those reported for NADP and NAD combining sites (Rothermel and Nelson, 1989). 2.3 Expression of NADP-ME from sorghum in transgenic rice plants Altogether 33 plants of transgenic rice transformed with sorghum C 4 type NADP-ME were obtained using Agrobacterium tumefaciens as mediator. Determination of NADP-ME activity in leaves of transgenic rice revealed elevation to various extents; 1 7 folds increase was noted in comparison with the control. Examples of transgenic rice plants ME1.2, ME5.2, ME6.0 and ME6.7 are shown in Fig.3A, with enzyme activity elevated by 1.2, 5.2, 6.0 and 6.7 folds respectively. Further molecular-level examination was performed on the transgenic rice. Southern hybridization of BstX -digested genomic DNA of the transgenic rice was carried out using a 1.3-kb BstX -digested fragment of the cdna of sorghum NADP-ME as probe (Fig.3B). As expected, a hybridization band was found at the position of 1.3 kb in each

4 Fig.2. Nucleotide sequence and predicted amino acid sequence of cdna of sorghum C 4 type NADP-ME. Underlined sequence, predicted combining site for NADP; boxed sequence, predicted combining site for NAD; arrows, predicted splicing sites for chloroplast transit peptide; asterisk, stop codon.

5 CHI Wei et al.: Photosynthetic Features of Transgenic Rice Expressing Sorghum C 4 Type NADP-ME Fig.2. (continued)

6 Fig.3. Expression analysis of transgenic rice plants. A. Activity of NADP-ME in transgenic rice. B. Southern Hybridization. C. Northern Hybridization. 1, transgenic plant ME 1.2; 2, ME 5.2; 3, ME 6.0; 4, ME 6.7; WT, control; S, sorghum; P, position of cdna of sorghum NADP-ME as revealed by hybridization. of the transgenic plant while nothing was detected in the corresponding position of the control. This confirms the successful integration of sorghum NADP-ME into rice genome. But two further hybridization bands were found in both the control and transgenic plants, indicating the presence of other genes homologous to NADP-ME in rice. In order to make clear if there is any relation between the level of expression of ME in rice and the level of transcription, Northern hybridization was done to analyze the expression of NADP-ME in rice. And correlation is found between NADP-ME activity and transcription level as can be seen from Fig.3C. 2.4 Light regulation of NADP-ME activity in transgenic rice In C 4 plants, the expression of NADP-ME is under the regulation of light. It has been demonstrated that rice CAB promoter can direct the mesophyll-specific and light-regulated expression in leaves of C 3 plants (Sakamoto et al., 1991). This has led us to adopt the CAB promoter as the promoter for the target gene to ensure the specific expression of NADP-ME in rice. To make clear if the activity of NADP-ME in the transgenic rice is light-regulated, the activity of NADP-ME under light and in darkness was separately measured and compared with each other. As shown in Fig.4, the activity of NADP-ME in rice rapidly declines 10 h after being transferred into darkness. Yet the activation levels (the ratio of activity under light as compared with in darkness) of NADP-ME in sorghum and the transgenic rice were different. The ratios of activity were in sorghum, 1.2 in WT, 6.0 in ME5.2 and 8.5 in ME6.7 respectively. This may reflect regulatory differences between the CAB promoter in rice and the promoter of the sorghum NADP-ME itself. On the other hand, NADP-ME is expressed specifically in bundle sheath cells in C 4 plant while, in the transgenic plants, they are expressed in mesophyll cells. Consequently, the differences between the physiological and biochemical microenvironments of the bundle sheath cells and those of mesophyll, with regard to, for example, the concentration of substrates, the ph values, and the mount of regulatory factors of enzyme activity, may all contribute to the differences at activation levels. As it has been demonstrated, changes of activity of certain enzymes in C 4 cycle may exert influences on other enzymes of C 4 cycle through feedback mechanism of the metabolites (Hausler et al., 2001). We thus measured the activity of other photosynthetic enzymes in the C 4 cycle and that of Rubisco, the rate-limiting enzyme of C 3 cycle, in transgenic rice. As compared with the control, no appreciable changes were noted in PEPCase, NADP-MDH and PPDK in the transgenic plant, nor was any found in Rubisco (data not shown), both of these suggested that the transformation with NADP-ME did not lead to enhanced Fig.4. Effect of light on activity of NADP-ME in transgenic rice. Normally growing transgenic rice was first exposed to light for 8 h (light intensity: 600 µmol m 2 s 1 ) before growing in darkness for another 10 h. Activity of NADP-ME in rice leaves under each condition was measured. The data are Mean SE (n = 5). S, sorghum; ME5.2, ME 6.7, transgenic rice; WT, control.

7 CHI Wei et al.: Photosynthetic Features of Transgenic Rice Expressing Sorghum C 4 Type NADP-ME operation of C 4 cycle and any remarkable effect on carbon assimilation. 2.5 Determination of the substrates and products of NADP-ME in transgenic rice Although determination of the activity of NADP-ME in transgenic rice has demonstrated the expression of sorghum C 4 type NADP-ME in rice, the test was conducted in vitro and consequently it provides no adequate evidence for the true nature of the expression product in vivo. As was found by Furayama et al. (2001), despite the high level of expression of C 4 type PPDK in rice, most of the PPDK molecules produced were in the form of inactive zymogens. It thus appeared that the contents of substrate and products of NADP-ME might better reflect the effective expression of NADP-ME in vivo, which we indeed measured in leaves of the transgenic rice. As compared with the control, the malic acid content dropped by about 30% and the pyruvic acid content rose by about 20%, thus proved that the sorghum NADP-ME protein is in active form in the transgenic rice (Fig.5). No appreciable change was noted in the content of another key enzyme in C 4 cycle, which was consistent with the observation that no change of activity Fig.5. Contents of malic acid, pyruvic acid and phosphoenolpyruvic acid in leaves of transgenic rice. The data are M SE (n = 5). * indicates significant difference (P < 0.05) as compared with control. of PEPcase and PPDK was detected. 2.6 CO 2 exchange and chlorophyll fluorescence in transgenic rice The introduction of C 4 photosynthetic enzymes into C 3 plants was intended to improve the efficiency of solar energy utilization. What then was the actual result of introduction of sorghum NADP-ME into rice? We measured the net photosynthetic rates of transgenic rice expressing NADP-ME under various light conditions. As shown in Fig. 6A, no appreciable differences of net photosynthetic rates were found between the transgenic rice and the control. With the increase of light intensity, light saturation was reached at µmol m 2 s 1. PS photochemical efficiency (Fv/Fm) is an important parameter characterizing the status of photochemical reaction. As shown in Fig.6B, greater decline of Fv /Fm was found under high light intensity at noon for the transgenic rice (ME5.2 and ME6.7) than for the control (WT), suggesting greater susceptibility of the former to photoinhibition. 3 Discussion Because C 3 plants do not possess the Kranz anatomy, for a long time it remains doubtful that an effective C 4 cycles could be established in C 3 plants simply by transferring C 4 photosynthetic enzyme genes into C 3 plants. However, it was recently discovered that some aquatic angiosperms, such as Hydrilla verticillate ( Magnin et al., 1997) and Egeria densa ( Casati et al., 2000), possess a primitive type of C 4 photosynthesis without Kranz anatomy. Moreover, it was reported lately that the expression of C 4 type PEPC in rice can improve its photosynthetic capacity with enhanced tolerance to photo-oxidation (Chi et al., 2001; Huang et al., 2001; Zhang et al., 2003). The primitive CO 2 concentration mechanisms in transgenic rice expressing PEPC gene and those expressing PCK gene were also detected (Suzuki et al., 2000; Jiao et al., 2003). These results further strengthened the possibility of improving C 3 photosynthetic performance by gene engineering. In this study, NADP-ME, another key enzyme involved in C 4 photosynthesis was introduced into a C 3 plants, rice and the photosynthetic characteristics of transgenic plants were analyzed in detail. To our surprise, although high level expression of NADP- ME gene in rice was achieved in our study, no marked improvement in photosynthetic properties was detected judged from CO 2 assimilation. It may suggest that the introduction of C 4 NADP-ME alone into C 3 plants could contribute little to the improvement of C 3 photosynthetic performance. Therefore, the introduction of other C 4 -related enzymes in addition to NADP-ME, such as PEPCase

8 Fig.6. CO 2 exchange and chlorophyll fluorescence characteristics of transgenic rice plants. A. CO 2 assimilation. B. Daily change of chlorophyll fluorescence. The data are Means SE (n = 5). Photosynthesis rates were determined under the following conditions: 340 µmol CO 2 m 2 s 1, 21% O 2 and 30 Pn, net photosynthesis rate; PFD, photons flux density. * indicates significant difference (P < 0.05) as compared with the control. and PPDK, may be necessary to drive the C 4 pathway in C 3 plants. Further studies on this point should also shed light on the coordination of parameters needed for C 4 photosynthesis. A recent report suggests that NADP malic enzyme could be detrimental in the development of normal chloroplasts when expressed at high levels (20 70 folds increases) in a C 3 plant (Takeuchi et al., 2000). For the plants analyzed in this study, which showed 1 7 folds increased activity, there was no change in phenotype under normal growth condition except for a slight reduction of chlorophyll content (data not shown). This seems to indicate that the physiological function varies with the level of expression of its gene. However, the photoinhibition was enhanced under intense light at noon for transgenic rice in our study. In general, photoinhibition happens whenever the reducing power and ATP produced by photosynthesis exceed the total consumption of Calvin cycle, photorespiration and other physiological processes. In the case of transgenic rice, therefore, a great amount of NADPH is accumulated in chloroplasts due to overproduction by the fully activated NADP-ME under the intense light at noon, thus leading to photoinhibition. Moreover, the accumulated NADPH in chloroplasts keeps the components of the photosynthetic electron transfer chain in a highly reduced state, leading to production of great amount of singlet oxygen at the terminal of the chain, which may attack the active center of PS, further aggravating the inhibition (Vass and Styring, 1993). Besides, malic acid, the substrate for NADP-ME, plays an important role in maintaining ph within the cell, regulating Mg 2+ concentration, and protecting the integrity of membranes, which are closely related to photoprotection mechanisms including xanthophyll cycle (Edwards and Andreo, 1992; Casati et al., 1997). Thus the down-regulation of photoprotection mechanisms caused by lowering of malic acid concentration might be another reason for the enhanced photoinhibition in transgenic rice expressing NADP-ME gene. Further physiological data are required to support those hypotheses. References: Casati P, Spampinato C P, Andreo C S Characterization and physiological function of NADP- malic enzyme from wheat. Plant Cell Physiol, 38: Casati P, Lara M, Andreo C S Induction of a C 4 -like mechanism of CO 2 fixation in Egeria densa, a submerged aquatic species. Plant Physiol, 123: Chen J-Z, Chen D-L,Wu M-X, Shi J-N Comparision of some characteristics of NADP malic enzymes from sorghum and wheat leaves. Acta Phytophysiol Sin, 7: (in Chinese with English sbstract) Chi W, Jiao D-M, Huang X-Q, Li X, Kuang T-Y, Ku M S B Photosynthetic characteristics of transgenic rice plants overexpressing maize phosphoenopyruvate carboxylase. Acta Bot Sin, 43: Edwards G E, Andreo C S NADP-malic enzyme from plants. Photochemistry, 31: Furayama H, Tsuchida H, Agarie S, Nomura M Significant accumulation of C 4 -specific pyruvate, orthophosphate dikinase in a C 3 plant, rice. Plant Physiol, 127: Gonzalez D H, Iglesias A A, Andreo C S On the regulation

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