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First published online February 18, 2004
doi: 10.1242/10.1242/dev.00992

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Gibberellin regulates Arabidopsis floral development via suppression of DELLA protein function

Hui Cheng^1,2,*, Lianju Qin^3,*, Sorcheng Lee^1,*, Xiangdong Fu⁴, Donald E. Richards⁴, Dongni Cao¹, Da Luo^3,, Nicholas P. Harberd^2, and Jinrong Peng^1,2,

¹ Institute of Molecular & Cell Biology, 30 Medical Drive, Singapore 117609
² Department of Biological Sciences, National University of Singapore, Singapore 117546
³ Institute of Plant Physiology & Ecology, 300 Fenglin Road, Shanghai 200032, China
⁴ John Innes Centre, Colney Lane, Norwich NR4 7UH, UK

View larger version (91K): [in a new window]   Fig. 1. GA regulates stamen filament length via control of cell elongation. (A) SEMs of wild-type flowers at floral stages 10 and 13 are shown. Petals and stamens from the most advanced flower (middle bottom) in ga1-3 resembled the wild-type flower at stage 10 (top left). Stamen filament elongation is dramatically increased in ga1-3 plants lacking RGL1, RGL2 and RGA (Q2) and in ga1-3 plants lacking RGL1, RGL2, GAI and RGA (penta) compared with the wild type. Both Q2 and penta lines produced visible pollen grains. (B) Comparison of stamen and pistil lengths among different genotypes. Filament and pistil lengths were measured from SEM pictures (n=20). (C) SEM of stamen filament epidermal cells. ga1-3 stamen filaments have relatively short epidermal cells compared with those of wild type; cell length was restored in stamen filaments of ga1-3 plants lacking RGL1, RGL2, GAI and RGA (penta). Stamen filament segments shown were all from the middle part of the filament. (D) Average number of epidermal cells per stamen filament in wild type, ga1-3 and ga1-3 lacking RGL1, RGL2, GAI and RGA (penta).

View larger version (71K): [in a new window]   Fig. 2. ga1-3 plants fail to produce tricellular pollen grains. (A) Pollen grains from various genotypes examined by SEM. Pollen grains from ga1-3 plants lacking RGL1, RGL2 and RGA (Q2), or RGL2 and RGA (not shown) were almost identical to wild-type pollen grains, being oval shaped with long indented lines on the surface. In ga1-3 plants, the cells contained in a locule appeared to remain as microspores, being round and, in some cases, with short indented lines on the wall surface. Pollen grains from ga1-3 plants lacking RGL1, RGL2, GAI and RGA (penta) were similar to wild-type pollen grains but were slightly more wrinkled in appearance. (B) DAPI staining showed that pollen grains from ga1-3 plants lacking RGL1, RGL2 and RGA (Q2), or RGL1, RGL2, GAI and RGA (penta) are tricellular (as in the wild-type control). By contrast, the majority of the cells in the anther locule of ga1-3 had either no detectable nucleus or only a single condensed nucleus whereas only 6% had two or more than two nuclei. (C) Frequencies of tricellular pollen grains in anther locules of various genotypes as revealed by DAPI staining as shown in B.

View larger version (103K): [in a new window]   Fig. 3. Histological analysis of microsporogenesis. Transverse sections of anthers are displayed in developmental sequence, showing the progress in microsporogenesis in various genotypes. ga1-3 anthers developed normally up to the tetrad formation stage (stage 7) but after this, they diverted from the normal (compared with stages 9-12 in wild type; ga1-3 stages highlighted with question marks). Eventually, all ga1-3 pollen sacs aborted (last two photos). ga1-3 plants lacking RGL1, RGL2 and RGA (Q2), or RGL1, RGL2, GAI and RGA (penta) successfully completed microsporogenesis and released mature viable pollen grains. Scale bar: 50 µm. Microsporogenesis stages are indicated in bottom right-hand corner of the pictures.

View larger version (67K): [in a new window]   Fig. 4. Pollen development is arrested in ga1-3. (A) The anther-specific markers SDS and ATA7 were used in in situ hybridization analysis to compare microsporogenesis in ga1-3 and wild type. SDS patterns appeared the same in ga1-3 and wild type whereas the ATA7 signal pattern in ga1-3 locules was significantly different to that of the wild-type control. (B) Chromosome spread experiments confirmed that pollen meiosis is successfully completed in ga1-3, resulting in tetrad formation. (C) Aniline Blue staining showed that the tetrads in ga1-3 tend to be clustered and are not found in the form of free microspores as is seen in the wild type. Scale bars: 1 µm in B; 50 µm in C.

View larger version (59K): [in a new window]   Fig. 5. RGA and RGL2 are key GA-response regulators of floral development. (A,B) Comparison of growth of ga1-3 plants lacking single (A) or pairs of (B) DELLA proteins at 48 days. Presence of wild-type gene (and functional protein) is indicated by +, presence of loss-of-function mutation (and lack of functional protein) is indicated by -. (C) ga1-3 plants lacking RGA and RGL2 initially produced sterile non-opening flowers (bottom right; SEM far right), then began to produce fertile open flowers (top right; SEM far right) when they were 50 days old. By contrast, ga1-3 plants lacking RGA alone (middle) never produced any fertile flowers.

View larger version (75K): [in a new window]   Fig. 7. RGL1, RGL2 and RGA repress flower opening, petal and stamen development in ga1-3 plants. (A) Comparison of the flowers of 30-day-old plants; genotypes as indicated. (B) Comparison of the seed set of various genotypes. (Left panel) Segment of main shoots bearing siliques. (Right panel) Seed production in a typical silique from genotypes as shown. Although ga1-3 plants lacking RGL1, GAI and RGA, or RGL2, GAI and RGA both have short siliques, the former bears no seeds whereas the latter is partially fertile. Siliques of ga1-3 plants lacking RGL1, RGL2 and RGA or lacking all four DELLA proteins are similar not only in length but seed production as well as in wild type. Scale bar: 1.0 mm.

View larger version (45K): [in a new window]   Fig. 6. Absence of RGL1, RGL2, GAI and RGA leads to GA-independent plant growth. (A,B) Wild-type plants were compared with ga1-3 plants containing loss-of-function mutations causing lack of various combinations of RGL1, RGL2, GAI or RGA at 22 (A) and 48 (B) days. Note that ga1-3 plants lacking RGL1, GAI and RGA or RGL2, GAI and RGA or all four DELLA proteins bolted earlier than the wild-type control at 22 days. Genotypes are as indicated. (C) Flowering time of various genotypes, measured as number of leaves at time of flowering. In this case, ga1-3 plants under SD did not produce any visible flower buds even though they had more than 50 leaves (marked with *). Under LD condition, ga1-3 plants lacking RGL1, GAI and RGA or RGL2, GAI and RGA or all four DELLA proteins all flowered with one or two leaves less than did the wild-type control, although this small magnitude of difference is hard to discern from the histogram shown here. (D) Plant height of various genotypes at 28 days old under LD growth condition. Results are presented as mean±standard error (n=30).