Conserved Functions of Arabidopsis and Rice CC-Type Glutaredoxins in Flower Development and Pathogen Response  Zhen Wang, Shuping Xing, Rainer P. Birkenbihl, Sabine Zachgo  Molecular Plant  Volume 2, Issue 2, Pages 323-335 (March 2009) DOI: 10.1093/mp/ssn078 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Phylogenetic Tree of the CC-Type GRXs from Arabidopsis and Rice and Comparison of Amino Acid Sequences of ROXY1 and Its Homologs. (A) An unrooted neighbor-joining tree of 38 CC-type GRXs from Arabidopsis and rice (Oryza sativa), divided into four clades (I –IV). Gene ID numbers starting with ‘At’ and ‘Os’ indicate genes from Arabidopsis thaliana and rice (Oryza sativa), respectively. Active site motifs of the GRXs are given after gene ID numbers. ROXY1, ROXY2, and their closely related homologs from rice, named OsROXY1 (Os04g32300) and OsROXY2 (Os02g30850), are indicated in bold. (B) Amino acid alignment of ROXY1 and its closely related homologs. Conserved amino acids among the four proteins are shadowed in dark blue. Less well conserved amino acids, shared by only three proteins, are marked in red. CCMC active site motifs are underlined and an asterisk indicates the conserved glycine in the putative GSH binding site. Molecular Plant 2009 2, 323-335DOI: (10.1093/mp/ssn078) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Comparison of Rice and Arabidopsis Flower Structures and Expression Pattern of OsROXY1 and OsROXY2. (A) Expression of OsROXY1 and OsROXY2 in different organs including the root, stem, leaf, and spikelet was analyzed by RT–PCR. Sample harvest, total RNA extraction, and PCR were performed as described in Methods. A rice 18S rRNA was used as control (Kim et al., 2003). (B) Schematic comparison of the floret structure from rice (left) and flower structure from Arabidopsis (right). A rice floret consists of two rudimentary glumes and two empty glumes, surrounding four whorls of floral organs: one lemma and one palea are formed in the first whorl, two lodicules emerge in the second whorl and six stamens and one carpel develop the third and fourth whorl, respectively (Figure 2B, left). The Arabidopsis flower consists of four whorls comprising four sepals, four petals, six stamens, and two carpels (Figure 2B, right). ca, carpel; eg, empty glume; le, lemma; lo, lodicule; pa, palea; pe, petal; rg, rudimentary glume; se, sepal; st, stamen. (C–R) RNA in-situ hybridization of OsROXY1 and OsROXY2 during rice spikelet development. Sections shown in (C–F) and (K–N) were hybridized with OsROXY1 antisense probe and, for (G–J) and (O–R), OsROXY2 antisense probe was used. (C, G) Longitudinal sections through the shoot apical meristem that has just converted into a rachis (inflorescence) meristem (IM). The flag leaf primordia, the last leaf to be formed before the spikelet formation, starts to initiate at one flank of the IM. OsROXY1 and OsROXY2 are expressed in young leaf primordia, flag leaf primordia and the IM, a weaker signal being detectable for OsROXY1 in the IM. (D, H) Longitudinal sections of the rachis during the stage of primary branch formation. OsROXY1 is strongly expressed at the tips of primary branch primordia. OsROXY2 RNA is weakly detectable throughout primary branch primordia but it is highly expressed in the rachis, where a vascular bundle will be formed (arrow). (E, I) Longitudinal sections through the spikelet at a stage when lemma and palea primordia are initiated. OsROXY1 is expressed in the lemma primordium and in the central region of the floral meristem; a weaker signal is detectable in empty glume primordia (E). At this stage, OsROXY2 expression is more restricted and only observed in lemma and palea primordia (I). (F, J) Longitudinal sections through the spikelet when second and third whorl organs are initiated. When stamen primordia are just formed in the third whorl, lodicule primordia start to emerge in the second whorl. OsROXY1 and OsROXY2 transcripts are detectable in stamen and lodicule primordia. At this stage, expression of both GRXs ceases in the lemma primorida that started to differentiate and only a weak signal for OsROXY2 is still observable in palea primordia. (K, O) Longitudinal sections through the floret during a later organ differentiation stage. Signals of OsROXY1 and OsROXY2 mRNAs are detectable in anthers. OsROXY1 is also expressed in differentiating lodicules and lemma as well as in carpel primordia. (L, P) Cross-sections through anthers at a pre-meiosis stage. Expression of OsROXY1 and OsROXY2 is mainly restricted to the tapetum. (M, Q) Cross-sections through anthers at a tetrad stage; meiosis is accomplished but microspores are not yet separated. OsROXY1 and OsROXY2 transcripts are detectable in the tetrads. (N, R) Cross-sections through a carpel in the same stage as sections shown in (M) and (Q). OsROXY1 and OsROXY2 are expressed at the tips of ovule primordia and a signal for OsROXY2 is also visible in vascular bundles of the carpel. an, anther; eg, empty glume; fl, flag leaf; fm, floral meristem; im, inflorescence meristem; le, lemma; lf, young leaf; lo: lodicule; o, ovule; pa, palea; pb, primary branch; st, stamen; ta, tapetum; te, tetrad; vb, vascular bundle (bars = 50 μm). Molecular Plant 2009 2, 323-335DOI: (10.1093/mp/ssn078) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 Complementation of roxy1-2 Mutants by OsROXY1 and OsROXY2. For complementation studies, OsROXY1 and OsROXY2 were expressed downstream of a 3.6-kb ROXY1 promoter and upstream of a 445-bp ROXY1 3′-end fragment confering an expression identical to the endogenous ROXY1 expression (Xing et al., 2005). (A) RT–PCR analysis of the expression levels of ROXY1, OsROXY1, and OsROXY2 in Arabidopsis wild-type (1), roxy1-2 mutant (2), ROXY1:OsROXY1 (3), and ROXY1:OsROXY2 (4) T1 transgenic plants. ROXY1 transcripts are only detected in Arabidopsis wild-type plants (1) and absent in the roxy1-2 mutant (2). In ROXY1:OsROXY1 (3) and ROXY1:OsROXY2 (4) transgenic plants, OsROXY1 and OsROXY2 transcripts are strongly expressed. (B) Heterologous expression of OsROXY1 or OsROXY2 rescues the petal phenotype of the roxy1-2 mutant. (1) Arabidopsis wild-type flowers form four equally shaped petals. (2) A typical roxy1-2 mutant flower produces less and abnormally formed petals (arrow). (3) A representative flower from a T1 Arabidopsis transgenic plant harboring the ROXY1:OsROXY1 construct in the roxy1-2 background produces four normal petals. (4) A typical flower of a T1 Arabidopsis transgenic plant harboring the ROXY1:OsROXY2 construct in the roxy1-2 mutant background, exhibiting four wild-type-like petals (bars = 500 μm). Molecular Plant 2009 2, 323-335DOI: (10.1093/mp/ssn078) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Overexpression of ROXY1, OsROXY1, and OsROXY2 in Wild-Type Arabidopsis Plants. (A–C) Correlation of the severity of morphological changes observed for 35S:OsROXY1, 35S:OsROXY2, and 35S:ROXY1 T1 Arabidopsis transgenic plants and the respective transgene expression levels. For preparation of total RNA, rosette leaves were harvested from 26-day-old wild-type plants and from T1 plants with weak, intermediate and severe phenotypes, respectively. Twenty-four cycles were used for PCR. In wild-type Arabidopsis plants, no bands for OsROXY1 or OsROXY2 were observed. Severity of transgenic plant phenotypes coincides with stronger expression of the investigated GRXs. (D) Leaves from 26-day-old wild-type and 35S:OsROXY1 transgenic Arabidopsis plants. From left to right, leaf from a wild-type plant and from transgenic plants with weak, intermediate, and severe phenotypes, respectively. (E) Comparison of 6-week-old wild-type and 35S:OsROXY1 transgenic Arabidopsis plants with weak, intermediate, and strong phenotypes shows the impact of the transgene expression on plant growth performance. Molecular Plant 2009 2, 323-335DOI: (10.1093/mp/ssn078) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Overexpression of ROXY1 and OsROXY1 Enhances Susceptibility to Botrytis cinerea and Leads to H2O2 Accumulation. Four-week-old wild-type plants, as well as 35S:ROXY1 and 35S:OsROXY1 T2 plants revealing an intermediate phenotype, were inoculated with Botrytis cinerea. (A) Representative plants are shown before, and 3 and 7 d after inoculation (dpi) with B. cinerea. Wild-type and roxy1-2 mutant plants are resistant to B. cinerea infection, while ROXY1 and OsROXY1 overexpression plants are susceptible to B. cinerea infection and were completely covered with B. cinerea spores 7 d after inoculation. (B) Histochemical detection of H2O2 in leaves from different transgenic plants by DAB staining. The top layer shows whole leaves after DAB staining. The colour precipitate indicative for H2O2 accumulation is stronger in ROXY1 and OsROXY1 overexpression plants than in wild-type and roxy1-2 mutants. Thus, H2O2 production and accumulation are much higher in the leaves of the overexpression plants. Pictures at the bottom layer are taken at a higher magnification to show leaf mesophyll cells. In wild-type and roxy1-2 mutant leaves, H2O2 accumulation was only detected in a few mesophyll cells. However, in leaves of ROXY1 and OsROXY1 overexpression plants, the number of mesophyll cells producing H2O2 was much higher (bars = 50 μm). Molecular Plant 2009 2, 323-335DOI: (10.1093/mp/ssn078) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 Comparison of ROXY1/2 and OsROXY1/2 Functions. Overlapping expression patterns of ROXY1/2 and OsROXY1/2 during flower development, successful complementation of the Arabidopsis roxy1-2 mutant and overexpression studies causing similar phenotypes in the transgenic plants altogether support a conserved activity of these GRXs. The eudicot and monocot GRXs likely exert in both species rather a second whorl-specific than organ-specific function in floral organ initiation and differentiation and might also conduct conserved, redundant activities during anther development. Ectopic expression studies unravel, besides a function in developmental processes, also a role in pathogen interaction, as demonstrated by an enhanced susceptibility to B. cinerea. Molecular Plant 2009 2, 323-335DOI: (10.1093/mp/ssn078) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions