The paralogous genes RADICAL-INDUCED CELL DEATH1 and SIMILAR TO RCD ONE1 have partially redundant functions during Arabidopsis development

Abstract

RADICAL-INDUCED CELL DEATH1 (RCD1) and SIMILAR TO RCD ONE1 (SRO1) are the only two proteins encoded in the Arabidopsis (Arabidopsis thaliana) genome containing both a putative poly(ADP-ribose) polymerase catalytic domain and a WWE protein-protein interaction domain, although similar proteins have been found in other eukaryotes. Poly(ADP-ribose) polymerases mediate the attachment of ADP-ribose units from donor NAD(+) molecules to target proteins and have been implicated in a number of processes, including DNA repair, apoptosis, transcription, and chromatin remodeling. We have isolated mutants in both RCD1 and SRO1, rcd1-3 and sro1-1, respectively. rcd1-3 plants display phenotypic defects as reported for previously isolated alleles, most notably reduced stature. In addition, rcd1-3 mutants display a number of additional developmental defects in root architecture and maintenance of reproductive development. While single mutant sro1-1 plants are relatively normal, loss of a single dose of SRO1 in the rcd1-3 background increases the severity of several developmental defects, implying that these genes do share some functions. However, rcd1-3 and sro1-1 mutants behave differently in several developmental events and abiotic stress responses, suggesting that they also have distinct functions. Remarkably, rcd1-3; sro1-1 double mutants display severe defects in embryogenesis and postembryonic development. This study shows that RCD1 and SRO1 are at least partially redundant and that they are essential genes for plant development.

Figure 1.

RCD1 and SRO1 are similar proteins. A, Predicted amino acid sequences of the RCD1 and SRO1 proteins. SRO1 is 76% similar to RCD1. Asterisks, colons, and periods indicate identical, similar, and semiconserved amino acid residues, respectively. Hyphens correspond to gaps introduced to improve the alignment. The blue boxes mark the WWE domain, and the red boxes indicate the putative PARP catalytic domain. The insertion sites in rcd1-3 and sro1-1 are indicated by green (inverted) and blue (upright) triangles, respectively. B, SRO1 is expressed in all plant parts tested. RT-PCR was done using primers SRO1-F and SRO1-R to amplify SRO1 and Actin-F and Actin-R to amplify the actin control gene (Supplemental Table S1). IN, Inflorescence; S, 7-d-old seedlings; RL, rosette leaves; CL, cauline leaves; RT, roots. C, The T-DNA insertions in the mutant alleles disrupt gene expression. rcd1-3 and sro1-1 do not accumulate any detectable full-length transcript. RT-PCR was done using primers RCD1-F/RCD1-R and SRO1-F/SRO1-R, respectively. Col-0, Columbia. D, Transcription upstream and downstream of the T-DNA insertion site is seen in sro1-1. RT-PCR upstream (top panel; using primers SRO1-150F and SRO1-1360R) and downstream (middle panel; primers SRO1-1600F and SRO1-R) of the T-DNA insertion produces products.

Figure 2.

RCD1 and SRO1 have opposite roles in control of bolting. rcd1-3 plants bolt early in both long days and short days, as measured by number of rosette leaves at bolting (A and B) and days to bolting (C and D). sro1-1 plants bolt late in both long and short days. Stars indicate values significantly different from the wild type at P < 0.01. Error bars indicate se. Col-0, Columbia.

Figure 3.

Root length and architecture are controlled by RCD1 and SRO1 function. A, The length of the primary root is shortened in rcd1-3 seedlings, while sro1-1 seedlings have longer primary roots. Eleven-day-old seedlings are shown. B, rcd1-3 and sro1-1 seedlings have extra lateral roots. C, In both mutant backgrounds, lateral roots are longer. D, The lengths of the primary roots in both mutants differ from that in the wild type. rcd1-3 primary roots are shorter, while sro1-1 primary roots are longer. Stars indicate values significantly different from the wild type at P < 0.01. Error bars indicate se. Col-0, Columbia. [See online article for color version of this figure.]

Figure 4.

Loss of RCD1 function causes formation of aerial rosettes. A, Under short-day conditions, rcd1-3 plants form many aerial rosettes (arrowhead) before the formation of cauline leaves with axillary branches (arrow). B, sro1-1 plants do not form aerial rosettes and form fertile flowers (stars). C to E, Examples of aerial rosettes formed on rcd1-3 plants. F and G, Examples of extra leaves formed in the axils of cauline leaves of sro1-1 (F) and Columbia (Col-0; G) plants in short-day conditions. H, FLC expression is misregulated in rcd1-3 plants. RT-PCR was done using aerial rosettes (rcd1-3) or cauline leaves and associated structures (sro1-1 and Col-0). The number of amplification cycles is indicated.

Figure 5.

rcd1-3 and sro1-1 display pleiotropic developmental defects. A, Double mutant seedlings are small and have malformed, light green leaves. Fifteen-day-old seedlings are shown: rcd1-3; sro1-1 (top), rcd1-3 (bottom left), sro1-1 (bottom center), and Columbia (Col-0; bottom right). B, RCD1 controls plant height. Plants shown are 45 d old. C, Complementation of rcd1-3 mutants by 35S∷RCD1. Four independent transgenic lines are shown. D, sro1-1 is dominant in an rcd1-3 mutant background, causing further reduction in plant height. In each pot, rcd1-3; +/+ plants are at left and rcd1-3; sro1-1/+ plants are at right. E, The rcd1-3; sro1-1 plants are dwarf (mature height averages about two inches; see ruler) and have a bushy habit. The plant shown is 55 d old. F, Complementation of sro1-1 mutants by a 5-kb genomic fragment covering the SRO1 gene. Complementation is shown in the rcd1-3 background, where restoration of SRO1 function confers an rcd1-3 mutant phenotype. Three independent lines are shown.

Figure 6.

Scanning electron microscopy images of epidermal cells of the inflorescence stems of wild-type (A), rcd1-3 (B), and rcd1-3; sro1-1 (C) plants. The double mutant stems have less cell elongation. The middle portion of two representative stems from each genotype is shown. Bar = 100 μm.

Figure 7.

rcd1-3; sro1-1 seedlings respond to exogenous gibberellic acid (GA₃) and brassinosteroid (BR). A, Mock treatment. B, Treatment with 10 μm GA₃. The hypocotyls of the GA₃-deficient mutant (ga1-2; Sun and Kamiya, 1994) and the wild type elongate on application of GA₃, while only some rcd1-3; sro1-1 seedlings respond due to the fact that these seedlings have short or absent hypocotyls. C, Mock treatment. D, Treatment with 0.1 μm BR. The leaves of rcd1-3; sro1-1 seedlings expand in response to BR, similar to the wild type and a BR-deficient mutant (det-2). However, petioles are absent on rcd1-3; sro1-1 leaves. Plants shown in A and B are 1 week old, and those shown in C and D are 3 weeks old.

Figure 8.

RCD1 and SRO1 are necessary for embryogenesis and seed development. A, Wild-type seeds. B, Class I rcd1-3; sro1-1 seeds are oddly shaped but of approximately wild-type size. C, Class II rcd1-3; sro1-1 seeds are both oddly shaped and shrunken. D, Mature wild-type embryos. E and F, Mature rcd1-3; sro1-1 embryos. These embryos are abnormally shaped and sized. The roots and hypocotyls are most affected. Pink arrowheads, Absence of hypocotyls; red arrowheads, short hypocotyls; green arrowheads, abnormal shape and structure of cotyledons; yellow arrowhead, arrest of embryo development at approximately the globular stage. G and H, Siliques of rcd1-3; sro1-1 plants. G, A young rcd1-3; sro1-1 silique in which seeds appear to be of normal size and shape. H, A mature rcd1-3; sro1-1 silique. By this stage in fruit development, the seeds have become small and shrunken.

Figure 9.

RCD1 and SRO1 control ovule morphogenesis and embryogenesis. A and B, Ovules 4 d after emasculation. A, The wild type. B, rcd1-3; sro1-1 mutant ovule is misshapen and small. C to E, Early globular stage embryos. C, The wild type. D and E, rcd1-3; sro1-1 globular embryos are normal. F to H, Heart-stage embryos. F, The wild type. G and H, rcd1-3; sro1-1 heart-stage embryos are broader than the wild type. I to N, Mature embryos. I, The wild type. J to N, rcd1-3; sro1-1 mature embryos display a range of phenotypes and do not fill seed. Bars = 50 μm. Each row has one bar that applies to all images in that row.

Figure 10.

Both the regulatory regions and the coding regions of RCD1 and SRO1 vary in activity. A, Complementation of rcd1-3 mutants by pRCD1∷SRO1g and pSRO1∷RCD1. Three independent transgenic lines are shown for each transgenic construct to illustrate variation in complementation. B, Complementation of rcd1-3; sro1-1 mutants by pRCD1∷SRO1g and pSRO1∷RCD1. Five independent transgenic lines are shown for pRCD1∷SRO1g (center) and two independent lines for pSRO1∷RCD1 (extreme right). The ability of pRCD1∷SRO1g to rescue the double mutant is variable. Plants shown are 45 d old.