Funneling of gibberellin signaling by the GRAS transcription regulator SCARECROW-LIKE 3 in the Arabidopsis root

SCL3 Acts as a Positive Regulator in the GA Response Pathway.

Because SCL3 expression is actively modulated by bioactive GAs through DELLAs, we initially postulated that SCL3 likely acts as another negative regulator in GA signaling. To test this, we first used a genetic approach by introducing either loss or gain of SCL3 function in backgrounds with defective GA biosynthesis and GA response. Interestingly, the scl3 ga1-3 double mutant exhibited a shorter root phenotype compared with ga1-3 in our root growth assay (Fig. S2 A and C). In contrast, root growth of the SCL3 overexpressors (35S::SCL3) in ga1-3 was indistinguishable from that of ga1-3 (Fig. S2 A and C). In the presence of exogenous GA₃, the phenotype of scl3 ga1-3 was restored, even though both ga1-3 and scl3 ga1-3 roots were slightly shorter than WT roots (Fig. S2 B and C). Furthermore, root growth of scl3 was more sensitive to PAC treatment, whereas 35S::SCL3 seedlings conferred resistance to PAC (Fig. S2 D and E). Additionally, we observed that both rga and scl3 rga were indistinguishable in root growth in the presence of PAC (Fig. S2 D and E), indicating that RGA is epistatic to SCL3. Taken together, our observations indicate that SCL3 is likely a positive regulator in GA signaling. In accordance with our findings, Zhang et al. (26) in this issue of PNAS describe the molecular details on the role of SCL3 as an activator in the GA response pathway.

To maintain GA homeostasis, transcription of GA metabolism genes is subject to feedback regulation in which DELLA repressors play key roles (27, 28). Like DELLAs, SCL3 also belongs to the GRAS transcription regulator family (15, 16, 29). Thus, to investigate the involvement of SCL3 in the maintenance of GA homeostasis, we analyzed expression profiles of the GA metabolism genes with qRT-PCR in the absence or presence of exogenous GA or PAC, as well as in ga1-3 and scl3 ga1-3. Of the GA metabolism genes examined, transcript levels of GA20ox1, GA20ox2, and GA20ox3 were notably up-regulated in scl3 under GA-deficient conditions (in the presence of PAC or ga1-3) (Fig. S2 F and G). Our findings are in accordance with the results by Zhang et al. (26) in this issue of PNAS that the transcription of GA20ox1, GA20ox2, and particularly GA20ox3 is subject to regulation by the SCL3 transcription regulator to maintain GA homeostasis.

Endodermis-Confined GA Signaling by SCL3 Coordinates Root Cell Elongation in the EDZ.

Recent reports demonstrated that GA controls root meristem size by promoting proliferative cell divisions (5, 6) and that GA response in the root endodermis also coordinates cell elongation (3). Endodermis-specific SCL3 expression and root growth inhibition of scl3 in GA-deficiency raised the question as to whether SCL3 plays a role in promoting cell division, cell elongation, or both. To address this question, we analyzed GA-mediated root growth of WT and scl3 seedlings in the absence or presence of ga1-3 or PAC. As described previously (5, 6), we measured the number and length of ground cells from the QC as parameters for root meristem size (Fig. S3A). Under standard conditions, root meristem sizes of both WT and scl3 seedlings were indistinguishable (Fig. S3 B and C). We also monitored the CYCB1::GUS mitotic marker that was shown to correlate with division potential in the root meristem (5, 6). In the presence of PAC, the number of dividing cells was reduced in scl3 to a level similar to that of WT roots (Fig. S3 D–F). In addition, the root meristem size of scl3 ga1-3 double mutants was indistinguishable from that of ga1-3 (Fig. S3 G and H), suggesting that GA-mediated cell proliferation in the root meristem is likely independent of SCL3 function. Thus, we investigated the role of SCL3 in cell elongation, because rapid elongation of cells that exit from the meristem drives postembryonic root growth (30). In the presence of PAC or ga1-3, root cell elongation in scl3 appeared to be reduced when compared with WT, whereas 35S::SCL3 roots were PAC resistant (Fig. S4 A and B). We also found that scl3 rga exhibited a PAC-resistant phenotype in cell elongation indistinguishable from rga (Fig. S4A), further corroborating that SCL3 acts downstream of RGA. Notably, root cell elongation of scl3, 35S::SCL3, rga, and scl3 rga correlated well with the EDZ length of the individuals (Fig. S4 C and D), suggesting that the sum of each cell's elongation contributes primarily to the EDZ length, and consequently to the whole root length.

Recently, the endodermis-specific disruption of a functional GA response by expressing the nondegradable gai under the SCR promoter (pSCR::gai-GR-YFP) was shown to affect root cell elongation and cell morphology in the EDZ (3). Thus, we introgressed the pSCR::gai-GR-YFP construct into WT, scl3, and 35S::SCL3 plants, respectively. In the absence of dexamethasone (−DEX), we found no difference in root cell elongation in WT, scl3, and 35S::SCL3 seedlings (Fig. 2 A, C, E, and G). When induced with DEX (+DEX), root growth of pSCR::gai-GR-YFP seedlings in the WT background was reduced (Fig. 2B), and cells in the EDZ showed inhibition of elongation (Fig. 2 D and F). Notably, cell elongation of pSCR::gai-GR-YFP seedlings in scl3 was more severely inhibited (Fig. 2 D and F). Under prolonged DEX treatment (+DEX for 3 d), the surface of pSCR::gai-GR-YFP roots in scl3 appeared bulged because the direction of cortex cell elongation was shifted perpendicularly compared with the untreated roots (Fig. S5 A and B). As a result, the average length of individual cells in scl3 was more significantly reduced than in the WT background (Fig. 2H and Fig. S5 B and C). We also observed similar results in WT and scl3 roots harboring the pSCR::rga-GR-YFP construct (Fig. S5 D–F). Interestingly, 35S::SCL3 seedlings with pSCR::gai-GR-YFP suppressed the inhibition of cell elongation when induced with DEX (Fig. 2 B, D, F, and H). These observations indicate that SCL3 attenuates the effects of nondegradable gai and rga in the root endodermis. The results from ref. 26 in this issue of PNAS lend support for the notion that SCL3 acts as an attenuator of DELLAs. Thus, the maintenance of GA signaling by SCL3–DELLA interaction in the endodermis controls coordination of cell elongation for root growth.

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Fig. 2.

Regulation of GA-mediated root cell elongation by SCL3–DELLA interaction in the EDZ. The pSCR::gai-GR-YFP seedlings in Col-0, scl3, and 35S::SCL3 were grown in MS agar plates for 4 d, transferred to MS agar plates supplemented with or without 10 μM of DEX, and incubated for another 3 d (A and B) or for 12 h (C–H). In the absence of DEX (−DEX), neither inhibition of root growth nor cell elongation was observed (A, C, E, and G). In the presence of 10 μM of DEX (+DEX) (B, D, F, and H), pSCR::gai-GR-YFP seedlings in Col-0 showed inhibition of root growth and cell elongation. Root growth and cell elongation of pSCR::gai-GR-YFP seedlings in scl3 were more severely inhibited. Notably, overexpression of SCL3 (pSCR::gai-GR-YFP in 35::SCL3) suppressed the inhibition of root growth and cell elongation. As a result, the average cell length is reduced significantly in scl3, whereas increased in 35S::SCL3 (n > 20) (H). (Scale bars, 5 mm in A and B; 30 μm in C and D; and 10 μm in E and F, respectively.) Statistical significance of differences was determined by Student's t test (error bars: SE, **P < 0.01).

SCL3 and Bioactive GA Levels Modulate the Timing and Extent of the Formative Division for Ground Tissue Maturation.

The asymmetric cell division that generates cortex and endodermis is established during embryogenesis and perpetuated in postembryonic development (31). At later stages, Arabidopsis seedlings undergo the additional formative division giving rise to endodermis and another cortex (MC) for ground tissue maturation (21). Recent work demonstrated that the GA pathway, in conjunction with the SHR/SCR module, controls the timing and extent of MC formation (2, 4). Our results that SCL3 serves as an integrator of the GA/DELLA and SHR/SCR pathways in the endodermis raised the possibility that SCL3 plays a role in the regulation of MC formation. To test this possibility, we focused on the timing and extent of the formative division in WT, scl3, and 35S::SCL3 roots. In WT roots, occurrence of the formative division was first observed at 6 d postgermination (dpg), and half of the seedlings in a given population showed MC formation at 12 dpg under our experimental conditions (Fig. S6A). As roots became mature, scl3 seedlings had already undergone the periclinal division (Fig. 3 A and B). For instance, only 12% of WT roots exhibited the formative ground tissue division, whereas 42% of scl3 seedlings had already formed MC at 7 dpg (Fig. 3E and Fig. S6A). By contrast, 35S::SCL3 roots at 7 dpg exhibited a delay in occurrence of the formative division compared with WT roots (2 vs. 12%) (Fig. 3E and Fig. S6A). Interestingly, a decreased frequency of MC formation conferred by 35S::SCL3 had diminished from 9 dpg onward, implying that SCL3 is likely subject to its own regulation, as demonstrated by Zhang et al. (26) in this issue of PNAS. Our data indicate that SCL3 modulates the timing and frequency of the formative ground tissue division for MC formation.

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Fig. 3.

Control of the formative ground tissue division in the MZ by the interaction of SCL3 and GA/DELLA pathways. MC formation in Col-0 (A) and scl3 roots (B) at 10 dpg. The scl3 root exhibited the additional formative periclinal division giving rise to endodermis (en) and middle cortex (mc). Arrowheads indicate endodermis (en), middle cortex (mc), and cortex (co), respectively. The nascent MC layer loses the endodermis identity marked by pSCR::GFP-SCR (Insets in B and C). Occurrence of the formative division in scl3 in the presence of 1 μM of PAC (C) and 10 μM of GA₃ (D). The treatment with PAC increased, whereas exogenous GA₃ decreased the MC formation. (E) Quantitative evaluation of MC formation confirms that GA deficiency causes an increased frequency of MC formation in Col-0, scl3, and 35::SCL3. In addition, loss and gain of SCL3 function also have opposing effects on the MC formation: increase in scl3 and decrease in 35::SCL3. (F–I) Occurrence of MC formation in ga1-3, scl3 ga1-3, and 35::SCL3 in ga1-3. Similarly, the frequency of the formative division was also modulated by loss and gain of SCL3 function. (J) Quantitative evaluation of MC formation confirms that exogenous bioactive GA, conversely, decreases the frequency in Col-0, scl3, and 35::SCL3. (K–M) MC formation in gai rga and scl3 gai rga in the absence or presence of 1 μM of PAC. As predicted, gai rga roots were extremely resistant to PAC, whereas scl3 gai rga triple mutants showed far more increased occurrence of the formative division in the presence of PAC. (N) In the absence or presence of DEX, the pSCR::gai-GR-YFP seedlings in Col-0 and scl3 were grown in MS agar plates for 4 d, transferred to MS agar plates supplemented with or without 10 μM of DEX, and incubated for 12 h. In the presence of DEX (+DEX), pSCR::gai-GR-YFP seedlings in scl3 showed a dramatically increased frequency of MC formation compared with pSCR::gai-GR-YFP seedlings in Col-0. (Scale bar, 30 μm.)

Previously, it was shown that GA deficiency (either by PAC or ga1-3) caused premature MC formation, and conversely exogenous bioactive GA application delayed MC formation, indicating that bioactive GA levels regulate ground tissue maturation (2, 4). Thus, we next investigated the mutual relation between SCL3 and bioactive GA levels in the control of the timing and extent of the formative division. In the presence of PAC, WT roots showed precocious MC formation compared with the untreated WT roots at 7 dpg (24 vs. 12%) (Fig. 3E and Fig. S6 B and C). In the same GA-deficient condition, scl3 roots also showed an increase in frequency of MC formation compared with the untreated scl3 roots (64 vs. 42%) (Fig. 3 B, C, and E). Similarly, we observed an increased frequency of MC formation in scl3 ga1-3 than in ga1-3 roots (85 vs. 29% at 6 dpg) (Fig. 3 F, G, and I). These observations indicate that scl3 and GA deficiency additively or synergistically cause an increased frequency of precocious MC formation. In contrast, 35S::SCL3 roots in GA-deficient conditions (by PAC or ga1-3) showed a resistant phenotype in the MC formation compared with WT and scl3 (Fig. 3 H and I). In the presence of exogenous GA₃, only 18% of WT roots exhibited MC formation at 12 dpg compared with the untreated WT (50%) (Fig. 3J and Fig. S6 D and E). Similarly, when applied with exogenous GA₃, scl3 roots showed a decreased frequency of MC formation at 12 dpg compared with the untreated scl3 (30 vs. 62%) (Fig. 3 D and J). In addition, application of exogenous GA₃ further reduced the frequency of MC formation in 35S::SCL3 roots compared with the untreated 35S::SCL3 (18 vs. 34%) (Fig. 3J). Taken together, our results indicate that SCL3 and bioactive GA levels additively or synergistically regulate the timing and extent of the formative division for ground tissue maturation.

Maintenance of a Functional GA Signaling by SCL3–DELLA Interaction Regulates the MC Formation.

Next, we interrogated genetically the mutual relation between SCL3 and GA response in MC formation. To this end, we generated gai rga double mutants (loss-of-function mutations in two major DELLAs) and scl3 gai rga triple mutants. Under standard conditions, gai rga roots showed a decreased frequency of MC formation compared with scl3 gai rga roots (6 vs. 16%) (Fig. 3M). In the presence of PAC, gai rga roots, as predicted, were extremely PAC resistant. Surprisingly, the difference in the frequency of MC formation between gai rga and scl3 gai rga became clearly evident at 7 dpg (6 vs. 42%) in the presence of PAC (Fig. 3 K–M). Our data indicate that a PAC-sensitive phenotype of scl3 gai rga in MC formation is likely conferred by loss of SCL3 function. The results from ref. 26 in this issue of PNAS also support the notion that SCL3 regulates its own expression to maintain a functional GA signaling. Next, we examined the MC formation in roots with defects in GA response by targeting the nondegradable rga in the endodermis under the SCR promoter (pSCR::rga-GR-YFP). In the presence of DEX, pSCR::rga-GR-YFP in WT showed an ∼10-fold increase in the occurrence of the MC formation compared with the untreated seedlings (Fig. 3N). Notably, pSCR::rga-GR-YFP in scl3 seedlings dramatically increased the frequency of the MC formation in the given population (Fig. 3N), further corroborating that SCL3 attenuates the endodermis-specific disruption of GA signaling conferred by the GA-insensitive rga function.

SCL3 and the SHR/SCR Module Regulate the MC Formation.

At later stages, the radial patterning mutant scr also occasionally undergoes the additional formative division, resulting in the sporadic production of two ground tissue layers: an inner layer with fused characteristics of endodermis and cortex (termed mutant layer; mu) and an outer layer with cortex characteristics (Fig. 4 A and B) (2, 4). Thus, we genetically interrogated the mutual relation between SCL3 and the SHR/SCR module in the control of the formative division. We generated the double mutant combinations of scl3 shr and scl3 scr, respectively, and examined the MC formation. In scl3 shr double mutants, we observed no additional formative division regardless of PAC or GA₃, resulting in the single ground tissue layer (Fig. S7 A–H). These observations are in accordance with the previous results that SHR is required for the formative ground tissue division (2). Interestingly, scl3 scr roots showed much earlier occurrence of the formative division than in scr; at 4 dpg, 55% of scl3 scr had already generated two ground tissue layers (Fig. 4 C–E and Fig. S7I). By contrast, 35S::SCL3 in scr delayed the MC formation compared with scr roots (8 vs. 32% at 5 dpg) (Fig. 4F and Fig. S7I). In addition, we found no effects of 35S::SCL3 on the delay in the MC formation from 6 dpg onward (Fig. S7I), further corroborating that SCL3 regulates its own expression. Our findings indicate that scl3 and scr additively or synergistically cause precocious MC formation.

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Fig. 4.

Control of MC formation by the interplay of the SCL3, SHR/SCR, and GA/DELLA pathways. (A and B) MC formation in scl3 roots marked by pCO2::H₂B-YFP. At 7 dpg, scl3 roots showed the additional formative periclinal division giving rise to two ground tissue layers. Arrowheads indicate mutant layer (mu) and middle cortex (mc), respectively. (C–E) Occurrence of the formative division in scl3 scr. The scl3 scr double mutants exhibited uncoordinated formative divisions, which generate an inner layer with the Casparian stripe (CS) and an additional cortex layer. Black arrows indicate the additional formative division (D), and white arrows indicate the Casparian stripe (CS) (E). (F) 35::SCL3 in scr reduced the occurrence of the formative division (at 5 dpg). Quantitative evaluation of MC formation in scr, scl3 scr, and 35::SCL3 in scr in the absence or presence of PAC (G) or GA₃ (H). Bioactive GA levels additively or synergistically have influence on MC formation with opposing effects: increase by PAC and decrease by GA₃. (I) Occurrence of MC formation in rga scr and scl3 rga scr in the absence or presence of 1 μM of PAC. The scl3 rga scr roots showed a far more increased frequency of the formative division regardless of PAC. (Scale bar, 30 μm.)

Treatment and Root Assay.

Root growth analysis was performed as described previously (6). For detailed description on the analysis of root meristem size and periclinal division, see SI Materials and Methods.

Molecular Cloning and Transgenic Plants.

To generate pSCL3::GUS, pSCL3::SCL3-GFP, and 35S::SCL3, the Gateway recombination cloning technology (Invitrogen) was used as described previously (29, 33). Primers used for plasmid construction are listed in Table S2. For detailed description on plasmid construction and transgenic plant production, see SI Materials and Methods.

Expression Analysis.

Total RNA extraction and reverse transcription-associated quantitative RT-PCR (qRT-PCR) were performed as described previously (29, 33). Primers used for qRT-PCR are listed in Table S3. mRNA in situ hybridization was performed as described previously (19, 20). (See SI Materials and Methods for detailed procedures.)

We thank Philip Benfey, Malcom Bennett, John Celenza, Renze Heidstra, Ben Scheres, Tai-ping Sun, Susana Ubeda-Tomás, the Arabidopsis Biological Resource Center, and the Nottigham Arabidopsis Stock Center for materials. We are grateful to Tai-ping Sun for sharing unpublished results and critical reading of the manuscript. We also thank Philip Benfey for critical reading and suggestions. This work was supported by National Research Foundation Grant 2009-0079304/R0602494 (to M.M.L.) and Grant NRF2009-0077753 (to J.L.) and by the Konkuk University (J.L.).