Coordination between GROWTH-REGULATING FACTOR1 and GRF-INTERACTING FACTOR1 plays a key role in regulating leaf growth in rice

Abstract

Background: The interactions between Growth-regulating factors (GRFs) and GRF-Interacting Factors (GIFs) have been well demonstrated but it remains unclear whether different combinations of GRF and GIF play distinctive roles in the pathway downstream of the complex.

Results: Here we showed that OsGRF1 and OsGIF1 synergistically regulate leaf growth in rice. The expression of OsGIF1 emerged in all tissues with much higher level while that of OsGRF1 appeared preferentially only in the stem tips containing shoot apical meristem (SAM) and younger leaves containing leaf primordium. Overexpression of an OsmiR396-resistant version of mOsGRF1 resulted in expanded leaves due to increased cell proliferation while knockdown of OsGRF1 displayed an opposite phenotype. Overexpression of OsGIF1 did not exhibit new phenotype while knockdown lines displayed pleiotropic growth defects including shrunken leaves. The crossed lines of mOsGRF1 overexpression and OsGIF1 knockdown still exhibited shrunk leaves, indicating that OsGIF1 is indispensable in leaf growth regulated by OsGRF1. The expression of OsGRF1 could be upregulated by gibberellins (GAs) and downregulated by various stresses while that of OsGIF1 could not.

Conclusion: Our results suggest that OsGIF1 is in an excessive expression in various tissues and play roles in various aspects of growth while OsGRF1 may specifically involve in leaf growth through titrating OsGIF1. Both internal and external conditions impacting leaf growth are likely via way of regulating the expression of OsGRF1.

Keywords: Leaf growth; OsGIF1; OsGRF1; Stress response; miR396.

Fig. 1

The expression patterns of OsGRF1 and OsGIF1 in rice. a The expression levels of OsGRF1 and OsGIF1 in flowers (adult plants) and different tissues of the 4-week-old seedlings in non-transformed rice (NT). Expression was analyzed by qRT-PCR. *, Significant difference at P < 0.05, **, Significant difference at P < 0.01 compared with expression in Older Stems by Student’s t-test (n = 3; means ± SDs). OS: Older Stem, basal internodes in stem of 4-week-old-seedlings; YS: Younger Stem, 5-mm-long shoot tips containing SAM (Shoot Apical Meristem) of 4-week-old-seedlings; OL: Older Leaf, the leaves in basal shoot of 4-week-old seedlings; YL: Younger Leaf, leaf tips and leaf primordium of 4-week-old-seedlings; F: Flowers in adult stage; OR: Older Root, the basal region in roots of 4-week-old seedlings; YR: Younger Root, 5-mm-long root tips of 4-week-old seedlings. b The RNA levels of OsGRF1 and OsGIF1 analyzed by northern blot in different tissues of the non-transformed rice (NT). Total RNA from 1-week-old-seedlings (lane SE) and different tissues as described in (A), including older stem (lane OS), Younger stem containing SAM (Shoot Apical Meristem) (lane YS), older leaf (lane OL), younger leaf (lane YL), the flowers in adult stage (lane F), older root (lane OR), and younger root (lane YR) was loaded and electrophoresed. Then the electrophoretic products were transferred and probed by labeled anti-sense sequences. The rRNA bands were visualized by ethidium bromide staining and served as loading control. c The protein levels of OsGIF1 and OsGRF1 analyzed by western blot. Total protein extracted from 2-week-old seedlings of the non-transformed plants (NT) was immunoblotted by anti-OsGIF1 (the left) and anti-OsGRF1 (the right) respectively. Actin immunoblotted by anti-Actin was served as control

Fig. 2

Response of OsGRF1 and OsGIF1 to GA, ABA, and stresses. Time course analysis of expressions of OsGRF1 and OsGIF1 in response to gibberellins (GA), salt, drought, UV-light, pathogen, and ABA. 2-week-old seedlings were incubated into N6 solution containing 50 μM GA₃ (a) or 200 mM NaCl (b) or 1 μM ABA (F) for designed time. 2-week-old seedlings were transplanted into 25% PEG (polyethylene glycol) (c), or exposed to 100 μmol m^− 2 s^− 1 ultraviolet (d), or sprayed with 3 × 10⁵ spore ml^− 1Magnaporthe grisea (e) for designated time respectively. Expression was analyzed by qRT-PCR. *, Significant difference at P < 0.05, **, Significant difference at P < 0.01 compared with No treatment by Student’s t-test (n = 3; means ± SDs)

Fig. 3

The Phenotypes of the non-transformed rice, OsGRF1OE, and mOsGRF1OE.a The regions complementary to OsmiR396 in OsGRF1 and mOsGRF1 (mutated OsGRF1) mRNA and the corresponding amino acid sequence. The mutated sites (shown in blue) in mOsGRF1 were artificially brought into to reduce the degree of the complementarity to OsmiR396 without alteration of amino acid sequence. b The RNA levels of OsmiR396 and OsGRF1 in the seedlings of the non-transformed plants (NT), OsGRF1OE, and mOsGRF1OE. OsmiR396 was detected by northern blot and U6 was served as loading control. OsGRF1 was analyzed by qRT-PCR, **, Significant difference at P < 0.01 compared with expression in the non-transformed plants (NT) by Student’s t-test (n = 3; means ± SDs). c The length of different leaves in 4-week-old seedlings of the non-transformed plants (NT), OsGRF1OE, and mOsGRF1OE. *, Significant difference at P < 0.05 compared with the non-transformed plants (NT) by Student’s t-test (n = 5; means ± SDs). d The morphology of the incomplete leaf in 4-week-old seedlings of the non-transformed plants (NT), OsGRF1OE, and mOsGRF1OE

Fig. 4

The phenotypes of transgenic lines with ectopic expression of OsGRF1. a The expression levels of OsGRF1 in 2-week-old seedlings of the non-transformed plants (NT) and OsGRF1RNAi. Expression was analyzed by qRT-PCR. **, Significant difference at P < 0.01 compared with expression in the non-transformed plants (NT) by Student’s t-test (n = 3; means ± SDs). b The phenotype of the complete leaf from second to fourth position in the 3-week-old seedlings of the three backgrounds of OsGRF1 (WT, mOsGRF1OE, OsGRF1RNAi). Bar = 10 cm. c The growth curve of the biomass of the suspension-cultured cells in the three backgrounds of OsGIF1. The suspension-cultured cells were harvested, dried, and weighed at given time. DW: dry weights. The data were the means of three biological repetition ± SE. d The morphology and size of the cells in suspension-cultured system originated from leaf calli in the three backgrounds of OsGRF1. Bar = 50 μm. e The expression levels of cycOs1 and cycOs2 in the leaves of three backgrounds of OsGRF1. Expression was measured by qRT-PCR. **, Significant difference at P < 0.01 compared with expression in the non-transformed plants (NT) by Student’s t-test (n = 3; means ± SDs)

Fig. 5

The phenotypes of the transgenic lines with ectopic expression of OsGIF1. a The phenotypes of the three genetic backgrounds of OsGIF1 (NT, OsGIF1OE, OsGIF1RNAi). b The expression level of OsGIF1 measured by qRT-PCR in the three genetic backgrounds of OsGIF1. **, Significant difference at P < 0.01 compared with expression in the non-transformed plants (NT) by Student’s t-test (n = 3; means ± SDs). c The phenotypes of the complete leaf from second to fourth position in the 3-week-old seedlings of the three backgrounds of OsGIF1 as well as crossed line: OsGIF1RNAi × mOsGRF1OE. d The morphologic features of roots of the three backgrounds of OsGIF1. e The morphologic features of seeds and spikes of the three backgrounds of OsGIF1. f The expression level of OsFRF1 and OsGIF1 measured by qRT-PCR in 2-week-old seedlings of the non-transformed plants and crossed lines of mOsGIF1RNAi × mOsGRF1OE. **, Significant difference at P < 0.01 compared with expression in the non-transformed plants (NT) by Student’s t-test (n = 3; means ± SDs). g The expression level of cycOs1 and cycOs2 measured by qRT-PCR in leaves of the three backgrounds of OsGIF1 as well as crossed line, mOsGIF1RNAi × mOsGRF1OE. **, Significant difference at P < 0.01 compared with expression in the non-transformed plants (NT) by Student’s t-test (n = 3; means ± SDs)

Fig. 6

Model for the functions of OsGRF1 and OsGIF1 in regulating growth. ABA, OsmiR396, and various stresses such as pathogens, ultraviolet (UV), drought, salt, etc., can downregulate the expression of OsGRF1, which is usually in a lower level, while gibberellin (GA) upregulates it. The interaction between OsGRF1 and OsGIF1 is necessary in promoting leaf growth via promoting the expression of cell-cycle-related genes. OsGIF1, which expresses in a higher level, may also work with other factor(s) to regulate other aspects of growth