Arabidopsis RETINOBLASTOMA-RELATED is required for stem cell maintenance, cell differentiation, and lateral organ production

Abstract

Several genes involved in the regulation of postembryonic organ initiation and growth have been identified. However, it remains largely unclear how developmental cues connect to the cell cycle. RETINOBLASTOMA RELATED (RBR) is a plant homolog of the tumor suppressor Retinoblastoma (pRb), which is a key regulator of the cell cycle. Using inducible RNA interference (RNAi) against Arabidopsis thaliana RBR (RBRi), we reduced RBR expression levels at different stages of plant development. Conditional reduction or loss of RBR function disrupted cell division patterns, promoted context-dependent cell proliferation, and negatively influenced establishment of cell differentiation. Several lineages of toti- and pluripotent cells, including shoot apical meristem stem cells, meristemoid mother cells, and procambial cells, failed to produce appropriately differentiated cells. Meristem activity was altered, leading to a disruption of the CLAVATA-WUSCHEL feedback loop and inhibition of lateral organ formation. Release of RBR from RNAi downregulation restored meristem activity. Gene profiling analyses soon after RBRi induction revealed that a change in RBR homeostasis is perceived as a stress, even before genes regulated by RBR-E2F become deregulated. The results establish RBR as a key cell cycle regulator required for coordination of cell division, differentiation, and cell homeostasis.

Figure 1.

Quantification of RBR Downregulation and Phenotypic Alterations in β-Estradiol–Induced RBRi Seedlings. (A) RBR mRNA quickly decreases after RBRi induction. Twelve hours after spraying 10-d-old RBRi seedlings with β-estradiol, RBR mRNA in the first two young leaves was reduced to <40% of its initial level (gray columns). Black columns: untreated RBRi seedlings (n = 50 for each time point/genotype treatment). The error bars represent the se from three biological replicates. (B) RBR protein levels in young leaves of 10-d-old seedlings were detected using an antibody against the N-terminal 374 amino acids of RBR. Thirty-six hours after β-estradiol spraying, RBR is barely visible on protein gels. (C) Five days after germination on β-estradiol–containing plates (RBRi+E), RBR is not longer detectable compared with wild-type (WT) or uninduced RBRi seedlings (RBRi-E). The stained gel is shown as a quantitative control. (D) Five-day-old wild-type seedlings germinated on β-estradiol–containing MS plates. (E) Five-day-old RBRi seedlings germinated on β-estradiol–free MS plates. (F) Five-day-old RBRi seedlings germinated on β-estradiol–containing MS plates. (G) Wild-type and RBRi seedlings were germinated on MS medium plus β-estradiol and moved to β-estradiol–free MS plates 5 d after germination. One week after recovery, development of RBRi-induced seedlings remained delayed (left three plants) compared with wild-type seedlings of the same age (right three plants). Organ production in both shoot and root apices is strongly retarded in RBRi seedlings. Bars = 0.5 cm.

Figure 2.

RBRi Leaf Mutant Phenotypes. (A) Both 19-d-old wild-type (left) and RBRi (right) plants were sprayed with β-estradiol for five consecutive days beginning at day 14. The RBRi plant is smaller and leaf production is arrested. (B) Top row: cotyledon and leaf morphogenesis of the 19-d-old wild-type plant. Bottom row: RBRi cotyledons and leaves. Leaf production was arrested in RBRi-induced plants, and leaves produced during the β-estradiol treatment were strongly delayed in development. Bar = 0.5 cm. (C) A wild-type leaf number 6 on the left, compared with RBRi leaves number 5 and 6 from 19-d-old plants. RBRi leaves are small with a strong downward curl.

Figure 3.

SAM Morphological and Genetic Alterations in β-Estradiol–Treated RBRi Seedlings. (A), (C), (E), and (G) Resin-embedded, toluidine blue–stained sections of wild-type SAMs from 3-, 4-, 5-, and 7-d-old seedlings, respectively. The apex acquires the dome shape morphology typical of the Arabidopsis SAM. Asterisks indicate the L1 layer. (B), (D), (F), and (H) Resin-embedded, toluidine blue–stained sections of SAMs from 3-, 4-, 5-, and 7-d-old RBRi plants, respectively, germinated on β-estradiol. Beginning at day 4, morphological changes became visible in mutant SAMs. In the inset in (D), magnification of the boxed area, white arrowheads point to the beginning detachment of L2 from L1. On day 5 (F), the L2 layer became detached from the L1 layer, and disorganized cell divisions were observed in L2 (between square brackets, compare [E] and [F]). On day 7 (H), the L1 layer was still clearly visible, but cell division in L2 and L3 had become disorganized, resulting in the loss of the typical L2 anatomy. Mutant SAMs became flattened by day 5 (F) and afterwards expanded laterally and vertically, possibly because of cell overproliferation in L2 and L3. (I), (K), (M), (O), and (Q) ProCLV3:GUS staining in untreated 3-, 4-, 5-, 8-, and 15-d-old RBRi; ProCLV3:GUS seedlings. The stronger GUS staining (3 h at 37°C) was necessary to compare the β-estradiol untreated and weakly staining treated RBRi seedlings. (J), (L), (N), and (P) ProCLV3:GUS staining in 3-, 4-, 5-, and 8-d-old RBRi; ProCLV3:GUS seedlings germinated on β-estradiol. (R) Thirty percent (n = 60) of the 15-d-old recovered seedlings germinated on β-estradiol developed twin ProCLV3:GUS sectors 1 week after transfer to β-estradiol–free soil. (S) In rare cases, the ProCLV3:GUS signal remained confined to the L1 layer and no organ production was observed even 1 week after recovery from a 5-d-long β-estradiol treatment. (T) Most of the seedlings recovered and showed a wild-type-like, although weaker, ProCLV3:GUS staining pattern. (U), (V), and (V') Thirty percent (n = 60) of the recovered RBRi seedlings produced twin inflorescence stems originating at the first node ([V], arrow points at the bifurcation), or from the basal rosette ([V'], arrow pointing in between the two emerging young inflorescences), while the majority of the recovered RBRi plants had a single inflorescence stem (U). (W) CLV3 and WUS expression in Arabidopsis RBRi shoot apices quantified by real-time PCR. In 5-d-old seedlings germinated on β-estradiol (n = 50 for each time point/treatment/analyzed gene expression), CLV3 is strongly downregulated, as confirmed by ProCLV3:GUS staining. The CLV3-WUS loop appears to be intact 5 d after RBRi induction because WUS became temporarily upregulated. Ten days after continuous induction, CLV3 expression levels was still reduced, while WUS mRNA levels had returned to nearly wild-type levels. Seedlings treated with β-estradiol for 5 d were arrested in organ production (see Figure 4). The error bars represent the se from two biological replicas and two technical replicas. (X) Expression of meristem marker genes in Arabidopsis RBRi shoot apices quantified by real-time PCR. STM and KNAT1 RNA levels fluctuated only slightly at 12 and 24 h after RBR downregulation but remained at constant levels 5 d after germination on β-estradiol (n = 50 for each time point/treatment/analyzed gene expression). Consistent with the decreased organ production we observed in RBRi seedlings after β-estradiol treatment (Figure 4), ANT expression levels were downregulated 5 d after RBRi induction. Five days after germination on β-estradiol, CYCB1;1 was upregulated in the SAM, as we observed in leaves (see Figure 5). BARD1 (reported to limit WUS expression; Han et al., 2008), is strongly upregulated 5 d after induction. The error bars represent the se from two biological replicas and two technical replicas. Bars = 10 μm in (A) to (H), 2.5 μm in the (D) inset, 20 μm in (I) to (V), and 2.5 mm in (V').

Figure 4.

Arrest of Organ Primordia Initiation in RBRi Seedlings Germinated on β-Estradiol. (A) to (C) Five-day-old RBRi seedlings grown on β-estradiol–free medium. (B) and (C) are contiguous sections of (A). Four organ primordia were identified. (D) to (F) Five-day-old RBRi seedlings grown on β-estradiol–containing medium. (E) and (F) are contiguous sections of (D). One organ primordium was identified. p1 to p4, organ primordia; s, stipules. Bars = 20 μm.

Figure 5.

Epidermal Pattern Variations in RBRi Leaves. (A) to (D) Comparison of β-estradiol–treated CYCB1;1:GUS patterns in wild-type and RBRi leaves 2 weeks after germination. The GUS signal is visible on the proximal halves of the 2nd pair of both wild-type (C) and RBRi (D) leaves. Notice the absence of GUS signal in the distal half of wild-type leaves (C') but not in RBRi leaves (D'). (E) and (G) Scanning electron microscopy images of adaxial (E) and abaxial (G) sides of wild-type leaves from 15-d-old plants. (F) and (H) Scanning electron microscopy pictures of adaxial (F) and abaxial (H) sides of RBRi leaves from 15-d-old plants sprayed with β-estradiol for five consecutive days. Bars = 1 mm in (A) and (B), 20 μm in (C) to (D'), 100 μm in (E) to (H), and 20 μm in insets in (E) to (H).

Figure 6.

Confocal Laser Scanning Microscopy of Leaf Surfaces of Wild-Type and RBRi Plants. Green channel, ProTMM:TMM-GFP; red channel, propidium iodide. (A) to (C) In 13-d-old wild-type leaves, ProTMM:TMM-GFP expression was confined to stomates and one or two nearby pavement cells. (D) to (F) In 13-d-old RBRi leaves from plants sprayed with β-estradiol for three consecutive days, all small proliferating cells expressed ProTMM:TMM-GFP. (G) to (I) Wild-type leaf surface 2 weeks after β-estradiol treatment was discontinued. (J) to (L) Two weeks after β-estradiol treatment was discontinued in RBRi plants, the small proliferating cells had not differentiated into stomata. The cells remained smaller and appeared to be less differentiated compared with the typical puzzle-shaped epidermal cells of wild-type or uninduced plants. (M) to (O) In the wild type (M), asymmetric cell divisions of MMCs gave rise to epidermal spacer cells and founders of stomata (asterisks). In RBRi plants treated with β-estradiol ([N] and [O]), cell division was symmetric and disorganized. (P) to (S) Differential interference contrast microscope ([P] and [R]) and 4',6-diamidino-2-phenylindole (DAPI) fluorescence ([Q] and [S]) images of RBRi leaves from 15-d-old plants treated with β-estradiol for five consecutive days. Clusters ([P] and [Q], white arrow) or single ([R] and [S], white arrowheads) four-celled stomates were observed in addition to wild-type-like two-celled stomates (black arrows). Bars = 30 μm in (A) to (L), 20 μm in (M) to (O), and 20 μm in (P) to (S).

Figure 7.

Context-Dependent Overproliferation or Arrest of Meristematic Cells in RBRi Plants. (A) to (H), (M), and (N) Transversal sections of resin-embedded tissues stained with Toluidine blue. (A) and (B) Vascular bundles from the 4th leaf of 3-week-old RBRi plants treated with β-estradiol for 5 d (B) appear more convex than those from the wild type (A). (C) and (D) Comparison of wild-type (C) and RBRi (D) vasculature shows that the procambium in RBRi plants treated with β-estradiol developed five to six cell layers (asterisks) instead of the two to three in wild-type plants. (E) and (F) Stem lateral sections of 5-week-old wild-type (E) and RBRi plants treated with β-estradiol for nine consecutive days (F). (G) and (H') A higher magnification of the vasculature meristem showed that the fascicular procambium (fc and highlighted in orange in [G'] and [H']) between the fascicular phloem (fp) and the fascicular xylem (fx) is thicker in RBRi plants treated with β-estradiol ([H] and [H']) compared with the wild type ([G] and [G']). (I) and (J) RBRi induction impairs inflorescence elongation and affects cauline leaves. Three-week-old plants were sprayed with β-estradiol for five consecutive days. Wild-type plants (left) are taller and cauline laves are not bent downwards when compared with RBRi plants (right). (K) Inflorescence meristem activity was arrested when 3-week-old flowering plants were subjected to 2 weeks of consecutive β-estradiol treatment. Wild-type plants (left) still produced flowers and lateral branches, whereas RBRi plants did not (right). (L) and (M) Wild-type apex (asterisk) with two emerging lateral buds compared with arrested RBRi inflorescences from 5-week-old plants, treated with β-estradiol for fifteen consecutive days when 3 weeks old. Meristem organization was lost, and organ production was arrested. (N) and (O) Top view of wild-type (left) and RBRi (right) inflorescence apices treated as in (F). The last organ produced by the RBRi inflorescence may result in a pin-like structure. (P) and (Q) Thirty-day-old wild-type and RBRi plants sprayed with β-estradiol for two consecutive weeks. RBRi induction in flowering plants induces sterility. (R) and (T) Five-week-old wild-type Arabidopsis inflorescences and flower, respectively. (S) and (U) Five-week-old RBRi inflorescence, 10 d after recovery from a 5-d treatment with β-estradiol. Phyllotaxis is disturbed (asterisks), as multiple siliques may emerge from the same node, and internode length is altered. In 5% of the cases, flowers with increased numbers of petals and stamens (U) were observed. Bars = 100 μm in (A) and (B), 20 μm in (C) and (D), 0.5 mm in (E) and (F), 100 μm (G) to (H'), and 100 μm in (L) and (M).

Figure 8.

Nuclear DNA Ploidy Analysis. Leaf numbers 3 and 4 (old) and 5 to 8 (young) were collected from 16- and 24-d-old plants, respectively. The DNA content of purified nuclei was analyzed by flow cytometry. Asterisk indicates DNA content of nuclei from RBRi plants treated with β-estradiol. No asterisk indicates the DNA content from uninduced RBRi plants. dag, days after germination.