55. The first leaf growth promoted by gibberellins in a dwarf mutant of rice B.Matsukura and J. Yamaguchi

Bioscience Center and School of Agricultural Sciences, Nagoya University. Chikusa-ku, Nagoya, 464-01 Japan

 Gibbercllins (GAs) stimulate leaf growth in many species of monocots. Although some physiological aspects of this phenomena have been reported, regulation mechanism(s) of GA-induced growth remains obscure in these plant systems and further analyses are needed. In the present communication, we investigated GA-induced first leaf growth in a dwarf mutant of rice, Tan-ginbozu, carrying d35 mutation.

The effect of GA on shoot growth was quantitatively determined by a modification of the "microdrop method" of Murakami ( 1968). The seedlings were pretreated with a GA-biosynthesis inhibitor, uniconazole, to reduce the endogenous GA level and to enhance the sensitivity to exogenous GAs. By application of exogenous GA3 at 300 pmol/plant to the 4 day-old seedlings, the first leaf were restored to the normal phenotype. Under these conditions, the growth started after a 1-d lag period and ceased 2 d after application of GA3. The final length of the first leaf after GA treatment was 2.5 to 3 fold longer than that without GA treatment (Fig. 2, see increment).

To investigate the effects of GA on elongation and division of cells in dwarf rice seedlings, we anatomically examined the first leaf at 0-48 h after application of GA, to 4 day-old seedlings (Fig. 1 ). Seedlings were fixed with 4% paraformaldehyde and 0.25% glutaraldehyde in 50 mM Na-P buffer, dehydrated through tertial butyl alcohol series, embedded in Paraplast plus tissue embedding medium (Dmsion Sherwood Medical, St. Louis. Mo., USA), sectioned longitudinally at 10-12 m m with a rotary microtome, and mounted on silane-coated slides. The sections were stained to visualize nuclei by Feulgen reaction with a counterstain of Fast Green. Cell size was measured by an ocular micrometer. The percentage of dividing cells was determined for each 0.5 mm section from the base to tip of the first leaf in two parenchyma cell files, which corresponded to second and third cell files from the adaxial epidermis. For evaluating the effect of GA3 on cell elongation, first leaf length was divided into every 0.25 mm section from the base to the tip. The mean length of parenchyma cells was calculated by dividing the length of each section (0.25 mm) by the number of cells contained. At least three seedlings were measured at each sampling time. Three-dimensional histograms shown in Figs. 2A and B were constructed using a graphic software (DeltaGraph Pro ver. 3.5, DeltaPoint Inc. Monterey, CA, USA).

The average length of parenchyma cells in each 0.25 mm long section was determined along the leaf length (Fig. 2A). During the first 6 h after the application of GA,, the parenchyma cells were about 10 to 20 mm in length. Thereafter, the cells rapidly elongated until the 48 h. The final cell length except the basal and apical regions was

154 Rice Genetics Newsletter Vol. 14

Fig. 1 Histochemical analyses of GA-treated first leaf. For details see text. Section 2: view of 0.25 - 0.5 mm from the base of the leaf, Section 6: view of 1.25-1.5 mm from the base of the leaf. Bar = 0.02 mm. about 60 to 80 mm, which was 3 to 4 fold longer than that in the basal region, and 5 to 6 fold longer than that in the leaf without GA3 application. We also calculated the percentage of cells in mitosis (late prophase to telophase) for every 0.5 mm long section of the first leaf (Fig. 2B). At the time of GA3 application, dividing cells were observed in the basal region of the first leaf both in the seedlings treated and not treated with GA3 and the mitotic index in this region was 2-3% during up to the first 6 h. However, the cell division activity disappeared completely in whole leaf at 12 h after GA3 application, followed by the striking growth of the first leaf. The total number of cells in the longitudinal parenchyma cell files were approx. 200 in both leaves treated with or without GA3. These results clearly indicated that the first leaf growth promoted by GA was mainly caused by cell elongation but not by cell division, that is, GA has little effect on cell division in the leaf. Similar results have been reported for the first leaves of the GA-responsive barley dwarf mutant, in which exogenous GA promoted cell elongation but cell division only slightly for the first leaf growth (Zwar and Chandler 1995). On the other hand, GA-

Research Notes

Fig. 2A: Effect of exogenous GA3 on cell elongation in the first leaf 

B: Effect of exogenous GA3 on cell division in the first leaf.

 promoted elongation of internode in submerged deepwater rice is based on an increase in cell production rate in the intercalary meristem as well as in cell extension rate in the elongation zone just above it (Sauter and Kende 1992). In the first leaf of rice, the growth is caused by only cell elongation, which is a different situation from the case of internode growth. We speculate that GA generally promotes cell elongation in most immature cells but cell division in limited cells/tissues.

References 

Murakami, Y., 1968. A new rice seedling test for gibberellins, 'microdrop method', and its use for testing

extracts of rice and morning glory. Bot. Mag. 81: 33-43. Sauter, M. and H. Kende, 1992. Gibberellin-induced growth and regulation of the cell division cycle in

deepwater rice. Planta 188: 362-368.

Zwar, J.A. and P.M. Chandler. 1995. a -Amylase production and leaf protein synthesis in a gibberellin-respon-

sive dwarf mutant of "Himalaya' barley (Hordeum vulgare L.). Planta 197: 39-48.