Differences in Expression of the RBCS Multigene Family and Rubisco Protein Content in Various Rice Plant Tissues at Different

Growth Stages
Yuji Suzuki, Kaori Nakabayashi, Ryuichi Yoshizawa, Tadahiko Mae and Amane Makino
Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan

Short Communication

Four out of ve members of the RBCS multigene family (OsRBCS2OsRBCS5) were highly expressed in leaf blades of rice (Oryza sativa L.) irrespective of plant growth stage, whereas accumulation of all RBCS mRNAs in leaf sheaths, roots and developing spikelets was quite low. A highly positive correlation was observed between total RBCS and RBCL mRNA levels and Rubisco content at their maxima, irrespective of tissue and growth stage. The results indicate that the total RBCS mRNA level may be a primary determinant for maximal Rubisco protein content and that Rubisco gene expression is well coordinated through the whole life of rice. Keywords: Oryza sativa L. RBCL, RBCS multigene family Rubisco. Abbreviations: DAS, days after sowing. Rubisco (EC 4.1.1.39) is a key enzyme in photosynthesis and the most abundant leaf protein. Rubisco catalyzes two competing reactions, the xation of CO2 in photosynthesis and the production of 2-phosphoglycolate in the photorespiratory pathway, and is a rate-limiting factor for both photosynthesis and photorespiration under conditions of saturating light and the present atmospheric CO2 and O2 levels (Evans 1986, Makino et al. 1988). Rubisco accounts for 1530% of total leaf N content in C3 species (Evans 1989, Makino et al. 1992). In higher plants, Rubisco is composed of eight small subunits, coded for by a nuclear multigene family (RBCS) (for a review, see Dean et al. 1989), and eight large subunits, coded for by a single gene (RBCL) in the plastome. The RBCS multigene family consists of 222 members, depending on the species (Rodermel 1999, Sasanuma 2001). The abundance of RBCS multigene family transcripts has been examined in different tissues and/or during tissue development in a

number of species including petunia (Dean et al. 1985), pea (Fluhr et al. 1986), tomato (Sugita and Gruissem 1987) and Lemna gibba (Silverthorne et al. 1990, Silverthorne and Tobin 1990). In these studies, mRNAs of RBCS genes accumulated to high levels in leaves. However, their relative ratios within a leaf were different depending on the species. For example, two or three out of ve or six expressed genes accounted for 6075% of total RBCS mRNA. In other tissues, these mRNA levels were generally low, with some variation in relative ratios among RBCS mRNAs. We previously data-mined ve rice RBCS genes from the database of full-length cDNA clones of rice (http://cdna01. dna.affrc.go.jp/cDNA) (Kikuchi et al. 2003) and designated them as OsRBCS1, 2, 3, 4 and 5 (Suzuki et al. 2007). However, their expression patterns are not well understood except the high expression levels of OsRBCS2OsRBCS5 in leaf blades (Suzuki et al. 2007, Suzuki et al. 2009). In the present study, the expression of the rice RBCS multigene family was further characterized in greater detail. RBCS mRNA levels, RBCL mRNA levels and Rubisco contents were determined in leaf blades, leaf sheaths, roots and developing spikelets, and their changes were evaluated at the seedling, vegetative and reproductive stages. Furthermore, quantitative relationships among those parameters were analyzed. Fig. 1 shows Rubisco and total N contents, the ratio of Rubisco-N to total N, and total RNA contents. The uppermost fully expanded leaf blades were used for the determination of Rubisco and total N content, since Rubisco content reaches a maximum at this leaf stage. The leaf sheath data were derived from the uppermost fully expanded leaf sheaths. Root samples were harvested about 5 cm from the root tip. Spikelets were collected 3 d after panicle emergence [97 days after sowing (DAS)] since the Rubisco content was maximal at this time (data not shown). Rubisco contents were highest in leaf blades, whereas those in the sheaths and

Corresponding

author: E-mail, ysuzuki@biochem.tohoku.ac.jp; Fax, + 81-22-717-8765.

Plant Cell Physiol. 50(10): 18511855 (2009) doi:10.1093/pcp/pcp120, available online at www.pcp.oxfordjournals.org The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Plant Cell Physiol. 50(10): 18511855 (2009) doi:10.1093/pcp/pcp120 The Author 2009.

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Fig. 1 Rubisco (A) and total N (B) contents, ratios of Rubisco-N to total N (C) and total RNA content (D) in leaf blades, leaf sheaths, roots and spikelets of rice at different plant growth stages. Data are presented as means SE (n = 3). Statistical analysis was carried out by ANOVA with a post hoc Tukeys test, and columns with the same letter were not signicantly different (P 0.05).

spikelets were much lower, while Rubisco was undetectable in roots irrespective of plant growth stage (Fig. 1A). Such declines in Rubisco contents were greater than those in total N content (Fig. 1B), resulting in decreased N allocation to Rubisco (Fig. 1C). Rubisco contents at the vegetative stage

were greater than at any other stage. This was associated with greater total leaf N. Expanding leaf blades were used for RNA analyses since the RBCS and RBCL mRNA levels are maximal at this stage (Suzuki et al. 2001). Spikelets just after panicle emergence (94 DAS) were used from the same reason (data not shown). The leaf sheath and root samples used were the same as in Fig. 1AC. Total RNA levels differed depending on tissues and plant growth stages, and showed a similar trend to total N contents (Fig. 1D). The mRNA levels of the RBCS multigene family members, the relative ratio among them and the RBCL mRNA levels were then determined (Fig. 2). In the leaf blades, mRNAs of OsRBCS2, 3, 4 and 5 accumulated to high levels, while OsRBCS1 mRNA levels were very limited. In the sheaths, mRNA levels of the four major RBCS genes were very low, although those of OsRBCS1 were still detectable. The accumulation of RBCS mRNA was well coordinated with RBCL accumulation, and showed a similar trend to that of Rubisco contents (Fig. 1A). In the leaf blades and leaf sheaths, total RBCS mRNA levels were highest in the vegetative stage, as was total RNA, Rubisco and total N contents (Fig. 1). Despite the differences in the absolute mRNA levels, the relative ratio of each member of RBCS mRNA in the leaf blades was similar irrespective of the plant growth stage. For example, OsRBCS3 mRNA levels were highest and accounted for 3045% of total RBCS mRNA, whereas the mRNA levels of OsRBCS2, 4 and 5 accounted for 1030% each. Additionally, a similar trend was also observed in leaf sheaths, although the relative mRNA levels of OsRBCS1 slightly increased, suggesting that the relative expression of each member of RBCS gene family was basically unaffected by plant growth stage in the green tissues of rice. On the other hand, in roots and spikelets, which are non-green tissues, some differences were found in the ratios among the mRNA levels. In roots, the ratios changed greatly depending on plant growth stage, with OsRBCS1 mRNA levels greatly increasing with plant age. In spikelets, the mRNA levels of OsRBCS3 and OsRBCS4 accounted for about 90% of total RBCS mRNA. Although such observations suggest the existence of tissue-specic regulation of expression of RBCS genes, the biological signicance of this seems to be limited because roots and spikelets are non-photosynthetic organs. In all tissues except roots, OsRBCS3 mRNA levels tended to be relatively high. Expression of OsRBCS1 was greatly different from that of the other RBCS genes. Amino acid sequence analyses using the Surveyed conserved motif ALignment diagram and the Associating Dendrogram (SALAD) database (http://salad.dna.affrc. go.jp/salad/en/) indicate that OsRBCS1 protein lacks an amino acid motif located in the transit peptide region common to the products of the other RBCS genes, while other motifs are almost conserved. This implies that OsRBCS1 protein may not be transported into chloroplasts or plastids, though we have not examined this yet.

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Expression of the RBCS multigene family in rice

Fig. 2 Transcript abundances of the RBCS multigene family (A), relative ratio among each RBCS transcript level (B) and transcript abundances of RBCL (C) in leaf blades, leaf sheaths, roots and spikelets of rice at different plant growth stages. For A and C, the insets show enlargements of the data from leaf sheaths, roots and spikelets. Data are presented as means SE (n = 3). Statistical analysis was carried out by ANOVA with a post hoc Tukeys test, and columns with the same letter were not signicantly different (P 0.05).

Fig. 3 Relationships between total RBCS mRNA levels and Rubisco content (A) and RBCL mRNA levels (B). Data are taken from Figs. 1 and 2. Symbols are as follows: diamonds, leaf blade; square, leaf sheath; triangle, root; circle, spikelet. Orange, green, blue and red symbols represent samplings on the 21, 44, 81 and 94/97 DAS, respectively. White and black diamonds represent data obtained with the leaf blades of the wild-type and RBCS-antisense rice in Suzuki et al. (2009), respectively. Insets show enlargements of the data from leaf sheaths, roots and spikelets. For regression lines between total RBCS mRNA and Rubisco and RBCL mRNA, Y = 333 X + 2, r2 = 0.998 and Y = 45.2 X 1.0, r2 = 0.968, respectively.

We next analyzed the relationship between total RBCS mRNA levels and Rubisco contents (Fig. 3A). A highly positive correlation was observed, suggesting that maximal Rubisco contents are primarily determined by the total RBCS mRNA level irrespective of tissue and plant growth stage in rice. Additionally, the molecular ratio of mRNA and the active site of the Rubisco protein was fairly constant and its average was estimated to be 1 : 358,000 20,000 except in the sheath at the seedling stage, and in the roots, both tissues where accumulation of Rubisco protein was low. Thus, four functional RBCS genes appear to be used in the leaf blades to produce the large amount of Rubisco required for high photosynthetic activity, whereas a low demand for

Rubisco protein in leaf sheath and spikelets correlates with low levels of RBCS expression. In leaf blades, the RBCS mRNA level is considered to be one of the major determinants of maximal Rubisco contents. For example, changes in the amounts of Rubisco synthesis are positively correlated with those in the mRNA levels of RBCS during leaf expansion, and almost all Rubisco protein is synthesized by full leaf expansion (Suzuki et al. 2001). Additionally, positive correlations have been noted between total RBCS mRNA levels in expanding leaves and Rubisco contents in fully expanded leaves of rice grown under different N levels (Suzuki et al. 2007). Furthermore, antisense suppression of RBCS reduces the Rubisco content in several plant species including tobacco

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(Rodermel et al. 1988, Hudson et al. 1992), Flaveria (Furbank et al. 1996) and rice (Makino et al. 1997), suggesting that under antisense conditions, at least, mRNA levels are limiting for Rubisco synthesis. In fact, when the RBCS mRNA level in leaf blades of RBCS-antisense rice was plotted against Rubisco content, the data fell on the same regression line as those of the control plants (black diamond in Fig. 3A; Suzuki et al. 2009). Total RBCS mRNA levels were also highly positively correlated with RBCL mRNA levels (Fig. 3B). Coordination between changes in these mRNA levels was observed during greening of etiolated pea seedlings (Sasaki et al. 1987) and during leaf expansion in rice (Suzuki et al. 2001). Our results suggest that these mRNA levels are regulated in a well coordinated manner irrespective of tissue and plant growth stage, implying that such a coordinated mechanism(s) also operates in other rice tissues. This coordination may be important for the regulation of Rubisco synthesis. For instance, an increase in Rubisco content of leaf blades with increasing N supply was accompanied by increases in mRNA levels of both total RBCS and RBCL in rice (Suzuki et al. 2007). Sole overexpression of RBCS did not lead to the overproduction of Rubisco to a similar extent as an increase in total RBCS mRNA levels (Suzuki et al. 2007, Suzuki et al. 2009). However, it remains unclear which factors regulate coordinated gene expression between RBCS and RBCL. In RBCSantisense tobacco, although gene expression of RBCL appears to be modulated at the translational level by the availability of the RBCS protein in chloroplasts, large changes in RBCL mRNA levels were not observed (Rodermel et al. 1996, Wostrikoff and Stern 2007). In contrast, RBCS and RBCL mRNA levels simultaneously declined in RBCS-antisense rice (Makino et al. 2000, Ishizuka et al. 2004, Suzuki et al. 2009; black diamond in Fig. 3B). Possibly, there are some unknown mechanisms that coordinate transcript levels of RBCS and RBCL. It has been suggested that transcriptional regulation of plastome-encoded genes is controlled by nuclear geneencoded sigma factors (for a review, see Lysenko 2007). RBCL mRNA levels declined in sig6 mutant leaves of Arabidopsis (Loschelder et al. 2006). Similar factors might be involved in the coordination of mRNA levels between RBCS and RBCL in rice. In summary, four out of the ve RBCS genes contributed to the high levels of total RBCS mRNA in expanding rice leaf blades, while the levels of these mRNAs were low in other tissues. Among them, OsRBCS3 expression tended to be predominant in all tissues. Although some growth-dependent changes in total RBCS mRNA levels were noted, changes in relative expression of each member of the RBCS gene family were small. Rubisco protein contents were always correlated with total RBCS mRNA levels, and coordinated gene expression between RBCS and RBCL occurred not only in leaf blades but also in other tissues including the sheaths and developing spikelets.

Materials and Methods

Rice (O. sativa L. cv Notohikari) plants were grown hydroponically in a greenhouse using a nutrient solution containing N at a concentration of 2 mM (Makino et al. 1988) with only tap water supplied after panicle emergence. Leaf blades and leaf sheaths on the main culms and roots were collected at the seedling stage (21 DAS), vegetative stage (44 DAS) and reproductive stage (81 DAS). At 2 d before sampling at the vegetative and reproductive stages, a nutrient solution containing 2/7 strength of N (<0.28 mM N) was supplied since a full-strength (2 mM) N supply stimulates a substantial increase in RBCS expression (Imai et al. 2008). Samples for RNA analyses were collected between 11:00 h and 13:00 h. All collected tissues were weighed and immediately frozen in liquid N2, then stored at 80C until analysis. Rubisco and total nitrogen contents were determined as described in Makino et al. (1988). Total RNA was extracted as described in Suzuki et al. (2004) with slight modications (Suzuki et al. 2007, Suzuki et al. 2009). RNA analyses were carried out as described in Suzuki et al. (2009) except for the primer pair used for OsRBCS1 (5-TCGATGATCATCAGTCACAG-3 and 5-CATTGTGCTCATTATACTCGAAG-3).

Funding
The Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation GPN 0007 to A.M.); the Japan Society for the Promotion of Science (Grants-inAid for Scientic Research No. 19780165 to Y.S. and No. 20380041 to A.M.).

Acknowledgments
We thank Drs. T. Yamaya, T. Hayakawa and S. Kojima (Tohoku University) for the use of their real-time PCR apparatus. We also thank Dr. L. J. Irving (Tohoku University) for his critical comments on this study.

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(Received July 7, 2009; Accepted August 20, 2009)

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