A novel gibberellin-induced gene from rice and its potential regulatory role in stem growth

Abstract

Os-GRF1 (Oryza sativa-GROWTH-REGULATING FACTOR1) was identified in a search for genes that are differentially expressed in the intercalary meristem of deepwater rice (Oryza sativa L.) internodes in response to gibberellin (GA). Os-GRF1 displays general features of transcription factors, contains a functional nuclear localization signal, and has three regions with similarities to sequences in the database. One of these regions is similar to a protein interaction domain of SWI2/SNF2, which is a subunit of a chromatin-remodeling complex in yeast. The two other domains are novel and found only in plant proteins of unknown function. To study its role in plant growth, Os-GRF1 was expressed in Arabidopsis. Stem elongation of transformed plants was severely inhibited, and normal growth could not be recovered by the application of GA. Our results indicate that Os-GRF1 belongs to a novel class of plant proteins and may play a regulatory role in GA-induced stem elongation.

Figure 1

Sequence analysis of Os-GRF1. A, Amino acid sequence of Os-GRF1. The QLQ domain is boxed in blue, the WRC domain in gray, and the TQL domain in green. The His-rich region is underlined with dashes, and the acidic region with a solid line. In the WRC domain, amino acids that may be involved in nuclear localization are indicated by circles above the respective letters. B, Alignment of the QLQ domain with that of other organisms (multiple alignment by Clustal W; Thompson et al., 1994; http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.html). O.s., Oryza sativa (accession no. for Os-GRF1 is AF201895); A.t., Arabidopsis; H.s., Homo sapiens (accession nos. for BRG1 and hSNF2α are P51532 and S45251, respectively); G.g., Gallus gallus (accession nos. for BRG1 and BRM are X91637 and X91638, respectively); D.m., Drosophila melanogaster (accession no. for brahma is P25439); S.c., Saccharomyces cerevisiae (accession no. for SNF2 is P22082). The dotted line denotes a region of a predicted α-helix. C, Alignment of the WRC domain by Clustal W. The letters on yellow background comprise the potential metal-binding motif CX₉CX₁₀CX₂H. The solid line denotes the region rich in basic amino acids.

Figure 2

Nuclear localization of Os-GRF1. A, Localization of the Os-GRF1::GUS fusion protein in the nucleus of an onion epidermal cell. The staining reaction was performed with the GUS substrate 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-gluc). B, Staining of the nuclei shown in A with the DNA-specific dye DAPI. C, Localization of Os-GRF1::GUS and of GUS (control) in the nucleus (N), nucleus and cytoplasm (N/C), and cytoplasm (C). Seventy-nine and 240 cells were scored to determine the distribution of Os-GRF1::GUS and GUS, respectively.

Figure 3

Expression of the Os-GRF1 gene in rice. A, Rice stem sections were incubated in 50 μm GA₃ for the times indicated above the lanes. Expression of Os-GRF1 and D40170, a rice EST that contains both the QLQ and WRC motifs, was determined in the intercalary meristem by RNA gel-blot analysis (20 μg of total RNA per lane). E37 served as loading control. B, Quantitative analysis of Os-GRF1 expression shown in A using a PhosphorImager. The values were normalized to the E37 loading control. C, Expression of Os-GRF1 in submerged plants. Whole plants were submerged for the times indicated above the lanes. Expression of the Os-GRF1, histone H3, and cycOs1 genes in the intercalary meristem was determined by RNA gel-blot analysis (20 μg of total RNA per lane). E37 served as loading control. D, Tissue-specific expression of Os-GRF1. N2, Second highest node; N1, highest node containing the shoot apex; L2b, basal 2 cm of second youngest leaf blade; L2s, basal 2 cm of second youngest leaf sheath; L1, youngest leaf; Co, coleoptile 3 d after germination; Ro, root 3 d after germination; 0–3, internodal region 0 to 3 mm above N2 containing the intercalary meristem; 3–8, internodal region 3 to 8 mm above N2 containing the elongation zone; 8–18, internodal region 8 to18 mm above N2 containing the upper part of the elongation zone and the differentiation zone; old, oldest part of the internode. E37 served as loading control. E, Schematic representation of a rice stem section containing the uppermost internode.

Figure 4

DNA gel-blot analysis of Os-GRF1. Rice genomic DNA was digested with BamHI (B), EcoRI (E), HindIII (H), or PstI (P). The blot was probed with a random-prime-labeled insert derived from the 3′ 737-bp region of Os-GRF1. Lane M, Molecular size markers (kb). The washing conditions were 0.1× SSC, 0.1% (w/v) SDS at 65°C.

Figure 5

Copy number and expression of Os-GRF1 in Arabidopsis. A, DNA gel-blot analysis of BamHI-digested DNA from the individual T₁ lines denoted above the lanes. The blot was hybridized with a random-prime-labeled probe derived from the full-length Os-GRF1 cDNA. B, Os-GRF1 transcript levels in individual T₂ lines. The RNA gel blot was hybridized with a random-prime-labeled insert derived from the 3′ 737-bp region of Os-GRF1. The arrow indicates the position of the full-length Os-GRF1 mRNA. C, Control plants transformed with the vector alone.

Figure 6

Growth of Arabidopsis plants expressing Os-GRF1 (Line 13). A, Five-week-old plants that flowered without concomitant bolting and showed curly leaves. B, GA₃ (1 μg) was applied in a 25-μL drop of a 0.01% (v/v) Tween 20 solution to the center of rosettes of 3-week-old plants transformed with Os-GRF1 or with the vector alone (control) before bolting of the control plants had started. This treatment was repeated three times. For the minus GA treatment, GA₃ was omitted from the Tween 20 solution. The photograph was taken when the plants were 7.5 weeks old. C, Plants transformed with Os-GRF1 were sprayed with 0.02% (v/v) Tween 20 solution with or without 50 μm GA₃ beginning 21 d after germination and then twice a week for 22 d. The plants were grown for an additional month without further hormone treatment. The primary stem had died off when the photograph was taken, but secondary shoots had continued to grow. D, Growth curve of the primary stem of plants (n = 7) treated with GA₃ as described in C. The arrow denotes the start of hormone treatment, which was repeated twice a week for 17 and 22 d for the control plants and for plants expressing Os-GRF1, respectively. The growth of control plants was measured until d 38 after germination; that of the plants transformed with Os-GRF1 until d 43. The inset is a magnification of the growth curve for the primary stem of Os-GRF1-expressing plants treated with and without GA.