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First published online 23 February 2005
doi: 10.1242/dev.01725

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ROXY1, a member of the plant glutaredoxin family, is required for petal development in Arabidopsis thaliana

Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany

View larger version (140K): [in a new window]   Fig. 1. The roxy1 phenotype. (A) Wild-type flower. (B-D) Phenotypes of roxy1-3 flowers. Petal number is reduced and the size often smaller, forming an abnormal blade and base. Arrow in D marks a filament-like stalk of a petal with a reduced blade. (E-L) SEM photographs of wild-type (E) and roxy1-3 mutant flowers (F-L) after onset of organ differentiation (stages 8-10) (Smyth et al., 1990). Sepals were partially or completely removed to reveal inner organs. Arrows indicate wild-type and abnormal petals in the second whorl between the medial (ms) and lateral (ls) stamens. Petal primordia often fail to be initiated, indicated by arrowheads (G,H). (I) roxy1-3 petal showing abnormal folding at the tip. (J) Conical cells, similar to wild-type cells, are formed in the inner petal epidermis at the tip of the folded petals. (K,L) Occasionally, roxy1-3 second whorl organs form fused (K) or filamentous structures that carry cells resembling stigmatic papillae at their tips (L). Scale bar: 50 µm.

View larger version (27K): [in a new window]   Fig. 2. Isolation of the ROXY1 gene. (A) T-DNA insertion sites are shown for each allele. The T-DNA insert in roxy1-1 is located in the promoter, 175 bp upstream of the start codon ATG. roxy1-2 and roxy1-3 carry insertions in the single coding exon, 91 and 291 bp downstream of the ATG, respectively. Numbering of coordinates is based on the Arabidopsis genomic clone BACF1C9 that contains the Col-0 ROXY1 gene. Left and right (L and R) T-DNA borders are only indicated where they could be identified. (B) ROXY1 cDNA and predicted amino acid sequence encoding a 136 amino acid protein (GenBank Accession Number AY910752). The dithiol CCMC and the monothiol CSGS motif are underlined. Locus name is At3g02000. (C) Comparison of ROXY1 expression in wild-type and roxy1-2 and roxy1-3 mutants by RT-PCR. RNA was isolated from inflorescences and primers were used allowing specific cDNA amplification. As a control, reactions with 18S rRNA were carried out.

View larger version (75K): [in a new window]   Fig. 3. Contribution of conserved cysteines to the ROXY1 function in petal development. (A) Alignment of open reading frames from GRXs of different species. Identical amino acids are in dark shading, similar ones in light shading. Two cysteines in the active site CXXC, conserved among all GRXs, are indicated by arrows (C49, C52). Another cysteine at position 90 (C90) is part of a putative monothiol CXXS motif specific to ROXY1. (B-E) Complementation analysis of mutagenized ROXY1 cysteines. roxy1-3 mutants were transformed with the wild-type (B) and mutagenized ROXY1 genes, where the three cysteines C49, C50 and C90 were exchanged into serines (C-E) and expressed under the control of the 35S promoter. (B) Wild-type protein complements 41/64 transgenic T1 roxy1-3 plants. (C) Mutagenesis of the N-terminal cysteine in the dithiol motif (C49S) disables the complementation capacity of the protein. C49S plants (88/92 T1 plants) resemble the roxy1-3 mutant. (D,E) C52S and C90S proteins were able to restore over 50% of the T1 roxy1-3 mutants (35/68 and 37/54, respectively), forming flowers with almost wild-type like petals. Photographs show representative inflorescences from transgenic T1 plants. Accession Numbers: E. coli, NP_415370; S. cerevisiae, AP_009895; Homo sapiens, AAH05304 Oryza sativa, CAA54397

View larger version (90K): [in a new window]   Fig. 4. ROXY1 expression. (A) RT-PCR analysis of ROXY1 in wild-type organs. For first-strand cDNA synthesis, RNA was isolated from roots (R), stems (S), leaves (L), inflorescences (I), mature flowers (F) and siliques (Si). 18S rRNA was used as a control. (B-F) In situ analysis of ROXY1 expression in wild-type flowers. (B,C) Longitudinal (B) and transverse (C) sections through the tip of an inflorescence. Onset of ROXY1 expression is visible in the inflorescence apex where a future primordium will be initiated (pre-stage 1). Then, signal is detectable when a flower primordium emerges (stage 1), in a flower primordium (stage 2) and in the area where the sepal primordia are formed (stage 3). (D,E) ROXY1 expression in longitudinal sections of a wild-type flower at stage 4 (D) and stage 7 (E). Expression is detected in petal (arrowhead) and stamen primordia (arrow) that are just initiated (D). ROXY1 mRNA is still expressed throughout young petals but confined to the vasculature in stamens (E). (F) Cross-section through a bud at stage 8 shows that the signal becomes restricted at a later stage to the central vasculature of both older petals and stamens. (G) Top view of a transgenic inflorescence meristem revealing expression of the ROXY1-GFP fusion protein comparable to the in situ staining shown in C. (H) Arrowheads indicate ROXY1-GFP expression in petal primordia in a flower bud at stage 8. se, sepal; pe, petal; st, stamen; ca, carpel. 1, 2, 3 indicate developmental stages (Smyth et al., 1990). Asterisks in C and G indicate the position of the inflorescence meristem (im). Scale bar: 50 µm.

View larger version (61K): [in a new window]   Fig. 5. Double mutant analysis with class B and C genes affecting organ identity. (A) ap3-3 mutant flowers produce sepals instead of petals in the second whorl and stamens are replaced by carpeloid, filamentous organs. (B) In the roxy1-3 ap3-3 double mutant, fewer sepals of a reduced size are formed in the second whorl. They are often bent (arrow) and resemble filamentous structures. (C) ag-1 mutant flowers display a transformation of stamens into petals and a new mutant flower is initiated in the fourth whorl. (D-F) roxy1-3 ag-1 double mutant flowers produce fewer petals in the second whorl that are often reduced in size (arrowheads in E). However, third whorl petals are not affected and occasionally formation of a new flower (arrow) can be observed in the second whorl (F).

View larger version (55K): [in a new window]   Fig. 6. Changes in AG expression in roxy1-3 ap1-10 double mutants. (A) ap1-10 mutants develop leaf-like first whorl organs; petals are absent in the second whorl, but an additional flower is formed in flowers arising on the inflorescence at later stages. (B) Strong feminization of the first whorl organs in the roxy1-3 ap1-10 double mutant (arrow). (C) In situ analysis of AG expression in young ap1-10 mutant inflorescences. Onset of AG expression is detectable at stage 3 and is confined to the two inner floral whorls. (D) AG is prematurely expressed in the roxy1-3 ap1-10 double mutant and expression is already detectable in the inflorescence meristem (im). Furthermore, AG is ectopically expressed in sepal primordia at stage 3. (E,G) AG-I::GUS expression in ap1-10 flowers resembles wild-type AG expression, being confined to the third and fourth whorl organs. (F,H) AG-I::GUS analysis in inflorescences and mature flowers of roxy1-3 ap1-10 mutants shows continued ectopic AG expression in the feminized first whorl organs (arrow).

View larger version (66K): [in a new window]   Fig. 7. Phenotypes of double mutants between roxy1-3 and ap2-5, lug-1, ufo-2 and rbe-2. (A-D) Enhancement of first whorl carpeloidy of roxy1 in combination with negative regulators of AG. (A) ap2-5 mutant flower showing transformation of sepals into carpeloid organs. (B) In roxy1-3 ap2-5 double mutants, first whorl carpeloidy is strongly enhanced and fused organs are topped with stigmatic papillae. (C) The lug-1 single mutant forms feminized first whorl organs. (D) In the roxy1-3 lug-1 double mutant carpeloidy of the first whorl organs is enhanced. (E) Mutants of the strong ufo-2 allele display defects in all floral whorls, affecting most severely second and third whorl organs resembling sepaloid and stamenoid structures, respectively. (F) roxy1-3 ufo-2 double mutant flowers form a reduced number of carpeloid second whorl organs. One first whorl sepal was removed to reveal inner organs. (G) rbe-2 single mutant, where sepals were removed. Arrows indicate filamentous structures formed instead of petals in the second whorl. (H) roxy1-3 rbe-2 double mutant flower, sepals were removed. Extra stamens develop in the second whorl (arrowheads), instead of petals or filamentous structures. ls, lateral stamen.