RCD1 and SRO1 are necessary to maintain meristematic fate in Arabidopsis thaliana

Abstract

The radical-induced cell death1 and similar to RCD ONE1 genes of Arabidopsis thaliana encode members of the poly(ADP-ribose) polymerase (PARP) superfamily and have pleiotropic functions in development and abiotic stress response. In order to begin to understand the developmental and molecular bases of the defects seen in rcd1-3; sro1-1 plants, this study used the root as a model. Double mutant roots are short and display abnormally organized root apical meristems. However, acquisition of most cell fates within the root is not significantly disrupted. The identity of the quiescent centre is compromised, the zone of cell division is smaller than in wild-type roots and abnormal divisions are common, suggesting that RCD1 and SRO1 are necessary to maintain cells in a division-competent state and to regulate division plane placement. In addition, differentiation of several cell types is disrupted in rcd1-3; sro1-1 roots and shoots, demonstrating that RCD1 and SRO1 are also necessary for proper cell differentiation. Based on the data shown in this article and previous work, we hypothesize that RCD1 and SRO1 are involved in redox control and, in their absence, an altered redox balance leads to abnormal development.

Fig. 1.

RCD1 and SRO1 are expressed throughout the embryo and in the root meristem. Staining for GUS activity in embryos and roots of RCD1::GUS (A–D, I, K) and SRO1::GUS (E–H, J, L) are shown. Arrows indicate end of division zone in (I, J). Scale bars represent 100 μm.

Fig. 2.

Root length and meristem size are reduced in rcd1-3; sro1-1 plants. (A) Root growth curves. (B) Root meristem length. (C) Root meristem cell number. Stars indicate values significantly different from the wild type at P < 0.01. Error bars indicate standard deviation. Col-0, Columbia; DPG, days post-germination.

Fig. 3.

RCD1 and SRO1 control proliferation in the root tip. (A–D) DAPI-stained roots: Col-0 (A, B), rcd1-3; sro1-1 (C, D). (E–H) Staining for GUS activity of CYCB1; 1::GUS for 4 h in the root tip: WT-like (E, F), rcd1-3; sro1-1 (G, H). (I–L) Staining for GUS activity of CYCD4;1::GUS in the root tip: (I, K) 2 h staining and (J, L) 20 h staining; WT-like (I, J) and rcd1-3; sro1-1 (K, L). (M–P) Staining for GUS activity of DEL1::GUS: (M-O), 4 h staining and (P), 20 h staining; WT-like (M, N), rcd1-3; sro1-1 (O, P). All scale bars represent 50 μm. Scale bar in (A) applies to (A–D) and that in (E) applies to (E–P). The green lines indicate the extent of the division zone in A–D. Col-0, Columbia.

Fig. 4.

rcd1-3; sro1-1 roots have abnormal radial patterning. Transverse sections of roots within the maturation zone of wild type (A–C) and rcd1-3; sro1-1 (D–F). (A) and (D) are hand-sections while (B, C, E, F) are thin sections. Small arrows indicate abnormal planes of cell division. All scale bars indicate 50 μm. C, cortex; En, endodermis; Ep, epidermis; Mx, metaxylem; Pc, pericycle; Px, protoxylem.

Fig. 5.

rcd1-3; sro1-1 roots have abnormal division patterns and disorganized root caps. (A–F) Root tip structure, visualized by confocal laser scanning microscopy after cell wall staining with propidium iodide: Col-0 (A), rcd1-3 (B), sro1-1 (C) and rcd1-3; sro1-1 (D–F). Small arrows indicate QC, large arrows indicate columella stem cells, and arrowheads indicate abnormal planes of cell division. (G–K) Columella structure in 5-day-old seedlings: Col-0 (G, H) and rcd1-3; sro1-1 (I–K). Scale bars in (A–F) indicate 50 μm. Scale bar in (G) represent 100 μm and applies to (G–K).

Fig. 6.

Cell fate in the root tip of rcd1-3; sro1-1 plants resemble that of wild type. Wild type-like roots (A, D, G, J, M); rcd1-3; sro1-1 roots (B, C, E, F, H, I, K, L, N, O). PLT1p::GFP (A-C), PLT2p::PLT2-GFP (D-F), SCRp::GFP (G-I), SCRp::SCR-GFP (J-L), SHRp::SHR-GFP (M-O). Scale bars represent 100 μm and scale bar in each row applies to all images in that row.

Fig. 7.

Auxin transport proteins accumulate normally in rcd1-3; sro1-1 embryos and roots. Wild type (A, C, E, G, I); wild type-like (L, O, R); rcd1-3; sro1-1 (B, D, F, H, J, K, M, N, P, Q, S, T). DR5rev::GFP (A-K), PIN1p::PIN1-GFP (L-N), PIN7p::PIN7-GFP (O-Q), PIN7p::GUS (R-T). The arrowheads in (A-H) indicate auxin maxima through DR5rev::GFP expression while the stars in (P, Q) mark gaps in PIN7 protein accumulation. All scale bars represent 100 μm and bars in (I, L, O, R) apply to the corresponding double mutant images for the respective marker line.

Fig. 8.

RCD1 and SRO1 control cell identity within the QC. Staining for GUS activity in the QC marker lines QC184 (A–E) and QC25 (F–J): 2 h of staining (A, B, F, G), 20 h of staining (C, D, E, H, I, J), WT-like (A, C, F, H), rcd1-3; sro1-1 (B, D, E, G, I, J). (K-N) Expression of SCL3p::GFP in the root visualized by confocal laser scanning microscopy after cell wall staining with propidium iodide: Wild type-like (K), rcd1-3; sro1-1 (L-N). Large arrow indicates the endodermis while small arrows indicate other cell layers. All scale bars represent 100μm. Scale bar in (A) applies to (A-J).

Fig. 9.

Cell division, patterning and differentiation are defective in the aerial organs of rcd1-3; sro1-1 plants. Col-0 (A, B, E, F, I, J) and rcd1-3; sro1-1 (C, D, G, H, K, L). (A–D) Transverse sections of leaves stained with toluidine blue: arrowheads indicate misshapen cells and arrows indicate large intercellular gaps. (E–H) Transverse sections of stems stained with toluidine blue, through the base of the inflorescence stem. Small arrows indicate gaps in the vascular cambium while stars indicate interruptions in the continuity of secondary growth. (I–L) Transverse sections of stems stained with toluidine blue, through the apical region. Scale bars in (B) and (D) represent 50 μm, all other scale bars represent 100 μm. Col-0, Columbia; C, cortex; Cm, cambium; Cu, cuticle; Icm, interfascicular cambium; Mx, metaxylem; P, palisade cells; Ph, phloem; Pi, pith; Px, protoxylem; Sm, spongy mesophyll; St, stomata; Xf, xylem fibres.