Brassinosteroids Control the Proliferation of Leaf Cells of Arabidopsis thaliana

Plant Cell Physiol. 43(2): 239 244 (2002) JSPP 2002 Brassinosteroids Control the Proliferation of Leaf Cells of Arabidopsis thaliana Masaki Nakaya 1, 5, Hirokazu Tsukaya 2, 3, 6, Noriaki Murakami 4 and Masahiro Kato 1 1 Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo, 113-0033 Japan 2 National Institute for Basic Biology/Center for Integrative Bioscience, Myodaiji-cho, Okazaki, 444-8585 Japan 3 Additional affiliations: Form and Function, PRESTO, Japan Science and Technology Corporation, Japan, and School of Advanced Sciences, the Graduate University for Advanced Studies, Shonan Village, Hayama, Kanagawa, 240-0193 Japan 4 Laboratory of Plant Taxonomy and Evolution, Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-8502 Japan The growth of leaves in the model plant, Arabidopsis thaliana (L.) Heynh., is determined by the extent of expansion of individual cells and by cell proliferation. Mutants of A. thaliana with known defects in the biosynthesis or perception of brassinosteroids develop small leaves. When the leaves of brassinosteroid-related mutants, det2 (deetiolated2 = cro1) and dwf1 (dwarf1 = cro2) were compared to wild-type plants, an earlier cessation of leaf expansion was observed; a detailed anatomical analysis further revealed that the mutants had fewer cells per leaf blade. Treatment of the det2 mutants with the brassinosteroid, brassinolide, reversed the mutation and restored the potential for growth to that of the wild type. Restoration of leaf size could not be explained solely on the basis of an increase in individual cell volume, thus suggesting that brassinosteroids play a dual role in regulating cell expansion and proliferation. Key words: Arabidopsis thaliana Brassinosteroid Cell proliferation cro mutants det2 mutant Leaf morphogenesis. Abbreviations: BL, brassinolide; BRs, brassinosteroids. Brassinosteroids (BRs) play an important role in plant cell elongation (Altmann 1998). Arabidopsis thaliana (L.) Heynh. mutants with defects related to the actions of BRs exhibit dwarfism, with stunted shoots and small leaves. For example, the brassinolide-insensitive 1 (bri1) mutant is defective in the perception of brassinolide (BL), the active molecular form of BRs (Li and Chory 1997). The cabbage1 (cbb1 = dwf1-6; dim), cbb2, cbb3, constitutive photomorphogenesis and dwarfism (cpd), de-etiolated 2 (det2), dwf4, and dwf5 mutants have defects in the biosynthesis of BRs (Feldmann et al. 1989, Chory et al. 1991, Kauschmann et al. 1996, Szekeres et al. 1996, Fujioka et al. 1997, Azpiroz et al. 1998, Choe et al. ; 2000). The cells of the stems and leaves of all these mutants are much smaller than those of the wild-type plant. The KORRIGAN gene (Nicol et al. 1998, Sato et al. 2001) appears to be essential for initiation of cell expansion in A. thaliana; expression of the KORRIGAN gene is controlled by the DET2 gene (Nicol et al. 1998). Thus, the stunted morphology of mutants with defects in the biosynthesis or perception of BRs appears to be attributable, at least in part, to a decrease in the elongation of each cell (Azpiroz et al. 1998). Mutants with small, round leaves have been categorized as compact rosette (cro) mutants (Serrano-Cartagena et al. 1999). The cro mutants include det2 (de-etiolated2) and dwf1 (dwarf1), which are characterized by mutations of key genes of the BR biosynthetic pathway (Fujioka et al. 1997, Noguchi et al. 1999, Choe et al. 1999). The leaves of these BR-related mutants are very small, with a base described as truncate to cordate; however, histological analysis of the leaf lamina has not been reported, and the decrease in the size (and number) of leaf cells has not been quantified. This communication reports the findings of a detailed histological examination of the mutants det2 and dwf1. Ecotype Columbia was used as the wild-type strain of A. thaliana in this study. The Nottingham Arabidopsis Stock Center (Nottingham, U.K.) kindly supplied mutants N352, N356, and N416. These mutant strains were genetically identified as alleles of known mutations (Serrano-Cartagena et al. 1999, Choe et al. 1999), so the following nomenclature was adopted: N356 was designated dwf1-5 (= cro2-3); and N352 and N416 were designated as variants of det2, with N352 corresponding to det2-13 (= cro1-3), and N416 to det2-18 (= cro1-8). All mutants were backcrossed three times to the Columbia wild type before characterization. Plants were grown on rockwool or vermiculite moistened with MGRL medium (Tsukaya et al. 1991) at 22 C under continuous white fluorescent light, as previously described (Tsuge et al. 1996). The matured leaves of mutants were much smaller than those of wild-type plants (Table 1). A kinetic analysis of leaf growth was performed to determine the nature of the defect in 5 Present address: Research Laboratory for Packaging, Technology Development Department, Production Division, Kirin Brewery Co., Ltd. 1-17- 1 Namamugi Tsurumi-ku, Yokohama, 230-8628 Japan 6 Corresponding author: E-mail, tsukaya@nibb.ac.jp; Fax, +81-564-55-7512. 239

240 Fig. 1 Kinetics of leaf growth. Primordia of the fifth foliage leaves of Columbia wild-type (WT), dwf1-5, det2-13, and det2-18 plants were examined. For each strain, more than five primordia were continuously observed. Each dot indicates data from a leaf primordium at a particular stage. Data were plotted as length of leaf blade vs. width of leaf blade (A) and length of leaf blade vs. time (B). The correlation coefficients for regression lines in panel (A) are 0.99 (WT), 0.98 (dwf1-5), 0.86 (det2-13), and 0.93 (det2-18), respectively. the leaf development of the BR-related mutants (Fig. 1). Plots of leaf length vs. leaf width showed that the two-dimensional growth of leaf blades was altered in all of the mutants. The rate of growth of each leaf, in terms of the relationship between the length and width of the leaf blade, was lower in all mutants than in wild-type plants (Fig. 1A). All strains demonstrated a similar pattern of leaf elongation during the early stages of the process (i.e. until the leaf blade was ca. 3 mm in length; Fig. 1B). These data indicate that mutant leaves stopped growing in the longitudinal direction earlier than wild-type leaves. While all A. thaliana mutants with defects in the biosynthesis or perception of BRs have characteristically stunted round leaves (Feldmann et al. 1989, Kauschmann et al. 1996, Szekeres et al. 1996, Azpiroz et al. 1998), these data suggest that BRs regulate longitudinal growth of leaf blades more strongly than they regulate growth of leaf width. This observation is especially interesting, considering that polarized longitudinal growth of A. thaliana leaves is controlled by the ROTUNDIFOLIA3 gene, which has high similarity to genes involved in biosynthesis of BRs (Kim et al. 1998). The fifth foliage leaves of mutant and wild-type plants were collected 25 35 d after sowing (close to bolting), when these leaves were fully expanded. Samples were embedded in Technovit 7100 resin (Kulzer & Co. GmbH, Wehrheim, Germany) and sectioned as previously described (Tsukaya et al. 1993, Tsuge et al. 1996). The numbers of parenchymatous mesophyll cells in a longitudinal section parallel to the midrib, and in a transverse section from midrib to leaf border taken at the exact center of the leaf blade were counted. Cell counts were used to estimate the decline in number of parenchymatous cells in each mutant leaf blade. The number and dimensions of the uppermost palisade cells in similar sections were counted and measured and used to calculate the decrease in expansion of mutant leaf cells. The leaf-length and leaf-width parameters used in the analysis of leaf blades have been defined elsewhere (Tsuge et al. 1996). The leaves of mutant plants had smaller intercellular spaces than those of wild-type plants (Fig. 2), which might explain the darker green color of the mutant leaves. The ratio of palisade cells to mesophyll cells was not altered in the mutants (ca. three; Table 2), indicating that the number of cell layers in the leaf blade was unaffected by the mutations. Distri- Table 1 Strain The lengths and widths a of the fifth foliage leaves of wild-type and mutant plants Length of leaf blade Width of leaf blade Number of leaves examined (n) Wild type 17.2 1.7 9.6 0.8 11 dwf1-5 4.3 0.2 5.1 0.3 8 det2-13 7.2 1.1 7.6 0.9 10 det2-18 5.7 0.8 6.1 0.7 10 a Mean values standard deviations. All data from mutant strains are significantly different (P <0.01; t-test) from those of wild-type plants.

241 Fig. 2 Transverse sections of the fifth foliage leaves of Columbia wild-type and mutant plants. (A) Wild type; (B) dwf1-5 mutant; (C) det2-13 mutant; (D) det2-18 mutant. Bar = 100 mm. Table 2 Dimensions and numbers of cells in the uppermost layer of the palisade tissue of the fifth foliage leaves of wild-type and mutant Arabidopsis plants Strain Cell size (number of cells examined) Leaf-length Leaf-width Leaf-thickness Cell numbers in longitudinal or transverse sections a In a longitudinal section In half a transverse section Palisade cells Mesophyll cells Palisade cells Mesophyll cells Wild type dwf1-5 det2-13 det2-18 40.3± 6.1 (66) 40.7± 5.9 (43) 22.4± 6.3 b (128) 26.3± 6.8 b (78) 32.1± 9.3 b (117) 35.0±11.0 b (67) 34.3±10.1b (95) 31.6± 8.5 b (95) 37.5±11.5 (109) 37.5±12.8 (206) 41.4±12.6 b (194) 47.8±16.5 b (190) 332±23.1 218± 8.5 b 197± 7.6 b 160±12.9 b 1,008±10.6 620±18.7 b 605±23.6 b 495±29.0 b 110±11.0 101±13.6 115±18.0 93±11.2 b 339±35.0 378±33.1 360±54.9 301±19.7 Each value is given as a mean ± standard deviation and is based on data from more than three individual plants. Number of palisade cells in the uppermost layer and total mesophyll cells counted in the longitudinal section along the midrib (left two columns), and those counted in half a transverse section at the exact center of the lamina (right two columns) are shown. Four leaves for the wild type and three leaves for each mutant were examined. b Data significantly different from those for the wild type (P <0.05; t-test). a

242 Fig. 3 Effects of BL on the size of leaves and palisade cells in det2-13 (det2; upper lane) mutant and wild-type (wt; lower lane) plants. Sizes of leaves and cells in the leaf-width direction (left) and in the leaf-length direction (right) are shown. Open circles, data from leaves without BL treatment; closed circles, data from leaves supplied with 0.2 M of BL. Data are shown as mean SD (number of cells examined: 10 to 20). See text for details. bution and size of leaf cells were uniform for each section observed (data not shown), permitting estimation of the effects of mutations on numbers of leaf cells from the sample sections. Analysis of cell number and dimension revealed that the difference in leaf shape was a function of significant decreases in both cell size (P <0.05; t-test) and cell number (P <0.05; t- test) (Table 2). Data were consistent for all mutants, suggesting that BRs play a dual role in regulating cell expansion and cell proliferation in the leaf lamina. To confirm the effect of BRs on leaf histology, 10-day-old aseptically cultured plantlets, grown as previously described (Tsukaya et al. 1991), were treated with 5 ml of 0.2 M brassinolide (BL) diluted in MS0 medium. The Petri dishes containing the plantlets were continuously agitated (ca. 50 cycles min 1 ) (NR-30, TAITEC, Tokyo, Japan). The mutant hypocotyl and petiole lengths increased quickly after exposure to BL, followed by an increase in the blade area of stunted leaves (data not shown). Detailed histological analysis was conducted on newly expanded first leaves of the wild type and the BR biosynthetic mutant, det2-13 (Fujioka et al. 1997), with and without BL treatment. These experiments were designed to show the relationship between treatment with BL and the reversal of

243 Table 3 plants The lengths and widths a of the first foliage leaves of wild-type and det2-13 mutant Strain Treatment b Width of leaf blade Length of leaf blade Number of leaves examined Wild type control 5.7 0.5 7.3 1.1 7 +BL 7.2 0.8 c 6.6 0.5 14 det2-13 control 3.6 0.6 c 2.9 0.3 c 16 +BL 5.4 0.9 7.7 1.2 10 a Mean values standard deviations. b Control: treated with MS0 medium, +BL: treated with 0.2 M BL dissolved in MSO medium. c Data significantly different from those of the control wild type (P <0.05; t-test). the defects in size or number of det2 leaf cells (Table 2). Treatment with BL completely restored the stunted leaves of the det2-13 mutant plants to normal size (Fig. 3A and Table 3). The dimensions of the treated det2-13 leaves were not significantly different (P >0.05; t-test) from wild-type leaves (without BL treatment) (Table 3). Anatomical analysis revealed that the size of cells in both the leaf-length and leaf-width directions of the det2-13 mutants increased following treatment with BL (Fig. 3B), and approached the size of cells in wild-type plants (no BL treatment). Cells of the wild-type leaves (n = 7) were 20.1 2.1 m long and 20.4 2.6 m wide compared to 17.9 3.5 m and 17.5 3.1 m, respectively, for the det2 leaves treated with BL (n = 10). BL-treated det2 leaf cells were slightly, but significantly, smaller (P <0.05; t-test) than those of the control wild-type leaves, demonstrating recovery of leaf area could not be explained solely on the basis of an increase in cell volume (Fig. 3B). These data indicate that defects in cell expansion and cell proliferation of det2 leaves were restored by BL-treatment to the level of control wild-type leaves, and support the idea that the defect in the stunted leaves of the BRdeficient det2 mutant is attributable to a decrease in both the number and size of leaf cells. Careful examination of the changes in cell size of the det2 leaves after treatment with BL showed that there was a polarity-dependent effect. Mutant leaves treated with BL were found to increase an average of 1.5 times in width (5.4 mm vs. 3.6 mm; Table 3), while mutant cell size increased an average of 1.3 times in width (17.5 m vs. 13.7 m; Fig. 3B). This correlation suggests that the expansion in the width of det2 leaves as a result of BL treatment is primarily attributable to expansion of individual leaf cells. However, there was no corresponding correlation between the average increase in the length of mutant leaves (2.7-fold: 7.7 mm vs. 2.9 mm; Table 3) and the increase in cell length (1.4-fold: 17.9 m vs. 13.0 m; Fig. 3B), suggesting that the increase in the length of det2 leaves following BL treatment is dependent on both cell expansion and cell proliferation. Recent work showed that BRs are involved in promoting cell division in suspension culture systems (Hu et al. 2000). In A. thaliana, leaf expansion depends not only on the expansion of leaf cells (Tsukaya et al. 1993, Tsukaya et al. 1994, Neff and Van Volkenburgh 1994, Kauschmann et al. 1996, Szekeres et al. 1996, Tsuge et al. 1996, Kim et al. 1998, Kim et al. 1999, Nicol et al. 1998), but also on the proliferation of such cells (Van Volkenburgh 1999, Donnelly et al. 1999). In the current study, the abnormal shape of leaves was associated with a decrease in cell size and number in the dwf1-5, det2-13, and det2-18 mutants. Moreover, exogenously applied BL increased both the number and size of leaf cells in det2 mutants while ultimately restoring the gross morphology of the stunted det2 leaves to that of the wild type. Thus, BR-related pathways appear to be involved not only in the expansion of cells, but also in the proliferation of cells in the lamina, despite previous studies reported that dwarfism in BR-related mutants of A. thaliana is caused by a defect in the expansion or elongation of cells. Further analyses may reveal other overlooked roles of BRs in plant organogenesis. Acknowledgments The authors thank the Nottingham Arabidopsis Stock Center (Nottingham, U.K.) for kindly supplying the mutant lines. Several reports on the morphological adaptations of leaves were brought to the authors attention by Dr. Momoe Ishibashi (University of Tokyo, Japan) and Mr. Jun Yokoyama (Tohoku University, Japan). Ms. Kazuko Kabeya (NIBB, Japan) kindly helped in the analysis of the anatomical data for the mutants. This study was supported in part by a grant from the Sumitomo Foundation to M.K., a grant from the Bio- Design Program, Ministry of Agriculture, Forestry and Fishes of Japan to HT, and by Grants-in-Aid of Scientific Research from the Japanese Ministry of Education, Sports, Science, and Culture (nos. 10182102 and 12440214 to H.T. and nos. 09740638 and 08740663 to N.M.). References Altmann, T. (1998) Recent advances in brassinosteroid molecular genetics. Curr. Opin. 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