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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
Four out of ﬁve members of the RBCS multigene family (OsRBCS2–OsRBCS5) 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 184.108.40.206) is a key enzyme in photosynthesis and the most abundant leaf protein. Rubisco catalyzes two competing reactions, the ﬁxation 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 15–30% 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 2–22 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 ﬁve or six expressed genes accounted for 60–75% 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 ﬁve 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 OsRBCS2–OsRBCS5 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
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Plant Cell Physiol. 50(10): 1851–1855 (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: email@example.com
Plant Cell Physiol. 50(10): 1851–1855 (2009) doi:10.1093/pcp/pcp120 © The Author 2009.
1 Rubisco (A) and total N (B) contents. 1D). and columns with the same letter were not signiﬁcantly different (P ≤ 0. 1C). whereas the mRNA levels of OsRBCS2. the relative ratio of each member of RBCS mRNA in the leaf blades was similar irrespective of the plant growth stage.Y. while other motifs are almost conserved. In all tissues except roots. and showed a similar trend to total N contents (Fig. total RBCS mRNA levels were highest in the vegetative stage. as was total RNA. resulting in decreased N allocation to Rubisco (Fig. with OsRBCS1 mRNA levels greatly increasing with plant age. although the relative mRNA levels of OsRBCS1 slightly increased. spikelets were much lower. Suzuki et al. Such declines in Rubisco contents were greater than those in total N content (Fig. The leaf sheath and root samples used were the same as in Fig. 1852 Plant Cell Physiol. and showed a similar trend to that of Rubisco contents (Fig. Spikelets just after panicle emergence (94 DAS) were used from the same reason (data not shown). Statistical analysis was carried out by ANOVA with a post hoc Tukey’s test. 2). In the sheaths. In the leaf blades. Total RNA levels differed depending on tissues and plant growth stages. Despite the differences in the absolute mRNA levels. the mRNA levels of OsRBCS3 and OsRBCS4 accounted for about 90% of total RBCS mRNA. This implies that OsRBCS1 protein may not be transported into chloroplasts or plastids. On the other hand. go. OsRBCS3 mRNA levels were highest and accounted for 30–45% of total RBCS mRNA. a similar trend was also observed in leaf sheaths. the biological signiﬁcance of this seems to be limited because roots and spikelets are non-photosynthetic organs. 1). although those of OsRBCS1 were still detectable. In spikelets. mRNAs of OsRBCS2. 4 and 5 accumulated to high levels. For example. 1B). Expression of OsRBCS1 was greatly different from that of the other RBCS genes. 3. Fig. Although such observations suggest the existence of tissue-speciﬁc regulation of expression of RBCS genes. 4 and 5 accounted for 10–30% each. the ratios changed greatly depending on plant growth stage. Additionally.1093/pcp/pcp120 © The Author 2009.affrc. 1A). leaf sheaths. Amino acid sequence analyses using the Surveyed conserved motif ALignment diagram and the Associating Dendrogram (SALAD) database (http://salad. some differences were found in the ratios among the mRNA levels. 50(10): 1851–1855 (2009) doi:10. though we have not examined this yet. . Data are presented as means ± SE (n = 3). the relative ratio among them and the RBCL mRNA levels were then determined (Fig.05). while Rubisco was undetectable in roots irrespective of plant growth stage (Fig. while OsRBCS1 mRNA levels were very limited.dna. which are non-green tissues. The accumulation of RBCS mRNA was well coordinated with RBCL accumulation. 1A–C. In the leaf blades and leaf sheaths. ratios of Rubisco-N to total N (C) and total RNA content (D) in leaf blades. mRNA levels of the four major RBCS genes were very low. roots and spikelets of rice at different plant growth stages. Expanding leaf blades were used for RNA analyses since the RBCS and RBCL mRNA levels are maximal at this stage (Suzuki et al. 2001). Rubisco contents at the vegetative stage were greater than at any other stage. in roots and spikelets. OsRBCS3 mRNA levels tended to be relatively high. The mRNA levels of the RBCS multigene family members. Rubisco and total N contents (Fig.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. 1A). This was associated with greater total leaf N. 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. In roots.
suggesting that maximal Rubisco contents are primarily determined by the total RBCS mRNA level irrespective of tissue and plant growth stage in rice. Data are taken from Figs. 81 and 94/97 DAS.968. For example. square. respectively.2 X – 1. In leaf blades. For A and C. (2009). A highly positive correlation was observed. 2007). 44. Orange. 3 Relationships between total RBCS mRNA levels and Rubisco content (A) and RBCL mRNA levels (B). Statistical analysis was carried out by ANOVA with a post hoc Tukey’s test.0. Y = 333 X + 2. Fig. the insets show enlargements of the data from leaf sheaths. antisense suppression of RBCS reduces the Rubisco content in several plant species including tobacco Plant Cell Physiol.000 except in the sheath at the seedling stage. both tissues where accumulation of Rubisco protein was low. Symbols are as follows: diamonds. 2 Transcript abundances of the RBCS multigene family (A).Expression of the RBCS multigene family in rice Fig.05). four functional RBCS genes appear to be used in the leaf blades to produce the large amount of Rubisco required for high photosynthetic activity. 1853 . the RBCS mRNA level is considered to be one of the major determinants of maximal Rubisco contents. 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. 50(10): 1851–1855 (2009) doi:10. spikelet. changes in the amounts of Rubisco synthesis are positively correlated with those in the mRNA levels of RBCS during leaf expansion. roots and spikelets. circle. root. and columns with the same letter were not signiﬁcantly different (P ≤ 0. leaf sheath. triangle. For regression lines between total RBCS mRNA and Rubisco and RBCL mRNA. Thus. White and black diamonds represent data obtained with the leaf blades of the wild-type and RBCS-antisense rice in Suzuki et al. Additionally. We next analyzed the relationship between total RBCS mRNA levels and Rubisco contents (Fig. and almost all Rubisco protein is synthesized by full leaf expansion (Suzuki et al. leaf blade. r2 = 0. and in the roots. 3A). respectively. whereas a low demand for Rubisco protein in leaf sheath and spikelets correlates with low levels of RBCS expression. 2001). Insets show enlargements of the data from leaf sheaths. roots and spikelets. Furthermore. 1 and 2. the molecular ratio of mRNA and the active site of the Rubisco protein was fairly constant and its average was estimated to be 1 : 358.998 and Y = 45. roots and spikelets of rice at different plant growth stages. Data are presented as means ± SE (n = 3). r2 = 0. relative ratio among each RBCS transcript level (B) and transcript abundances of RBCL (C) in leaf blades. blue and red symbols represent samplings on the 21. green.1093/pcp/pcp120 © The Author 2009. respectively. Additionally.000 ± 20. leaf sheaths.
. the Japan Society for the Promotion of Science (Grants-inAid for Scientiﬁc Research No. Suzuki et al. Similar factors might be involved in the coordination of mRNA levels between RBCS and RBCL in rice. 2009. All collected tissues were weighed and immediately frozen in liquid N2. Plant Mol. 2007. Samples for RNA analyses were collected between 11:00 h and 13:00 h. Rev. 2009). J. Tamaki. (1989) Photosynthesis and nitrogen relationships in leaves of C3 plants. (2004) with slight modiﬁcations (Suzuki et al. Suzuki et al. Hudson et al.. (1988). T. Materials and Methods Rice (O. .. RBCS and RBCL mRNA levels simultaneously declined in RBCS-antisense rice (Makino et al. while the levels of these mRNAs were low in other tissues. (1986) The relationship between CO2-limited photosynthetic rate and ribulose-1. Total RNA was extracted as described in Suzuki et al. Suzuki et al. 4: 3055–3061. Suzuki et al. Suzuki et al. Oecologia 78: 9–19. suggesting that under antisense conditions. Evans. It has been suggested that transcriptional regulation of plastome-encoded genes is controlled by nuclear geneencoded sigma factors (for a review. RBCL mRNA levels declined in sig6 mutant leaves of Arabidopsis (Loschelder et al.1093/pcp/pcp120 © The Author 2009.M. Forestry and Fisheries of Japan (Genomics for Agricultural Innovation GPN 0007 to A. 3A.M. Biol. 1988. Evans. References Dean. However. sativa L. In RBCSantisense tobacco. 1996. 20380041 to A. four out of the ﬁve RBCS genes contributed to the high levels of total RBCS mRNA in expanding rice leaf blades. 2009). changes in relative expression of each member of the RBCS gene family were small. Plant Physiol. C. Ishizuka et al. S. Yamaya. 2007. (2009) except for the primer pair used for OsRBCS1 (5′-TCGATGATCATCAGTCACAG-3′ and 5′-CATTGTGCTCATTATACTCGAAG-3′). At 2 d before sampling at the vegetative and reproductive stages. This coordination may be important for the regulation of Rubisco synthesis. J. Rubisco and total nitrogen contents were determined as described in Makino et al. Our results suggest that these mRNA levels are regulated in a well coordinated manner irrespective of tissue and plant growth stage. E. Coordination between changes in these mRNA levels was observed during greening of etiolated pea seedlings (Sasaki et al. although gene expression of RBCL appears to be modulated at the translational level by the availability of the RBCS protein in chloroplasts. 2008).5-bisphosphate-carboxylase content in two nuclear–cytoplasm substitution lines of wheat and coordination of ribulose-bisphosphate-carboxylation and electron-transport capacities. 19780165 to Y. C. EMBO J. (1985) Differential expression of the eight genes of the petunia ribulose bisphosphate carboxylase small subunit multi-gene family. 2004. Flaveria (Furbank et al. (1989) Structure.Y. We also thank Dr. 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. OsRBCS3 expression tended to be predominant in all tissues.R. Rubisco protein contents were always correlated with total RBCS mRNA levels. Funding The Ministry of Agriculture. 2000. 40: 415–439. 2007). a nutrient solution containing 2/7 strength of N (<0. cv Notohikari) plants were grown hydroponically in a greenhouse using a nutrient solution containing N at a concentration of 2 mM (Makino et al. J. and Bedbrook. Leaf blades and leaf sheaths on the main culms and roots were collected at the seedling stage (21 DAS).S. 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. For instance. Dean. 1854 Plant Cell Physiol. RNA analyses were carried out as described in Suzuki et al.V. when the RBCS mRNA level in leaf blades of RBCS-antisense rice was plotted against Rubisco content. Wostrikoff and Stern 2007).R. J. mRNA levels are limiting for Rubisco synthesis. black diamond in Fig. Elzen. L. Pichersky. Planta 167: 351–358. Total RBCS mRNA levels were also highly positively correlated with RBCL mRNA levels (Fig. Possibly. Hayakawa and S. Dunsmuir. 2001). 1988) with only tap water supplied after panicle emergence. large changes in RBCL mRNA levels were not observed (Rodermel et al. the data fell on the same regression line as those of the control plants (black diamond in Fig. it remains unclear which factors regulate coordinated gene expression between RBCS and RBCL. vegetative stage (44 DAS) and reproductive stage (81 DAS). 50(10): 1851–1855 (2009) doi:10. Although some growth-dependent changes in total RBCS mRNA levels were noted.28 mM N) was supplied since a full-strength (2 mM) N supply stimulates a substantial increase in RBCS expression (Imai et al. 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. at least. see Lysenko 2007). 3B). then stored at −80°C until analysis. and No. there are some unknown mechanisms that coordinate transcript levels of RBCS and RBCL. P. implying that such a coordinated mechanism(s) also operates in other rice tissues. P. Among them. T. Annu. Kojima (Tohoku University) for the use of their real-time PCR apparatus. 1987) and during leaf expansion in rice (Suzuki et al. 1997).).. (Rodermel et al. 3B).). evolution and regulation of rbcS genes in higher plants. and Dunsmuir. In summary. 1992). In fact. 2009). Irving (Tohoku University) for his critical comments on this study. 1996) and rice (Makino et al. Acknowledgments We thank Drs. In contrast. P. 2006).
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