Characterization of temperature-sensitive mutants reveals a role for receptor-like kinase SCRAMBLED/STRUBBELIG in coordinating cell proliferation and differentiation during Arabidopsis leaf development
National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
The balance between cell proliferation and cell differentiation is essential for leaf patterning. However, identification of the factors coordinating leaf patterning and cell growth behavior is challenging. Here, we characterized a temperature-sensitive Arabidopsis mutant with leaf blade and venation defects. We mapped the mutation to the sub-2 allele of the SCRAMBLED/STRUBBELIG (SCM/SUB) receptor-like kinase gene whose functions in leaf development have not been demonstrated. The sub-2 mutant displayed impaired blade development, asymmetric leaf shape and altered venation patterning under high ambient temperature (30°C), but these defects were less pronounced at normal growth temperature (22°C). Loss of SCM/SUB function results in reduced cell proliferation and abnormal cell expansion, as well as altered auxin patterning. SCM/SUB is initially expressed throughout leaf primordia and becomes restricted to the vascular cells, coinciding with its roles in early leaf patterning and venation formation. Furthermore, constitutive expression of the SCM/SUB gene also restricts organ growth by inhibiting the transition from cell proliferation to expansion. We propose the existence of a SCM/SUB-mediated developmental stage-specific signal for leaf patterning, and highlight the importance of the balance between cell proliferation and differentiation for leaf morphogenesis.
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Plants rely on temporal and spatial coordination of cell proliferation and differentiation to generate new tissues and organs from meristematic cells at the post-embryonic stage. Leaves are lateral organs derived from the peripheral zone of the shoot apical meristem. In Arabidopsis, leaf development can be divided into three phases: initiation, primary morphogenesis, and secondary morphogenesis. After the leaf initiates, the primordial cells proliferate robustly for a period of time to form the basic leaf shape during primary morphogenesis (Poethig, 1997; Donnelly et al., 1999; Scarpella et al., 2010). At this phase, several patterning processes are established, such as the axial polarities (the adaxial–abaxial, proximo-distal and medio-lateral axes) and the major venation pattern. At the time of the transition from cell proliferation to cell expansion/differentiation, which coincides with the onset of endo-reduplication, vacuole-associated cell expansion contributes to the increase of organ size during secondary morphogenesis (Poethig, 1997; Donnelly et al., 1999; Beemster et al., 2005; Scarpella et al., 2010).
Extensive genetic studies have shown that transcriptional networks and auxin-related pathways play important roles in leaf patterning (Barkoulas et al., 2007; Scarpella et al., 2010). However, the upstream components that integrate developmental and environmental cues to control leaf morphogenesis remain largely unknown. In this study, we identified a temperature-sensitive leaf developmental mutant and found it corresponding to the sub-2 mutant allele of the SCRAMBLED/STRUBBELIG (SCM/SUB) gene encoding a receptor-like kinase (RLK). Previous studies showed that SCM/SUB is required for root epidermal cell specification and cell morphogenesis during the development of floral organs and stems, as well as the orientation of cell divisions in the L1 layer of the outer integument and the L2 layer of the floral meristem (Chevalier et al., 2005; Kwak et al., 2005). Here we report previously unknown functions of SCM/SUB in blade development and venation formation. We found that SCM/SUB is required to maintain the balance between cell proliferation and cell differentiation in both epidermal and internal tissues during leaf development. Our expression pattern analyses and temperature-shift experiments suggest that SCM/SUB probably mediates a developmental-stage specific signal for early leaf patterning.
In order to understand the mechanisms of leaf patterning and identify the hidden component(s), a recessive temperature-sensitive leaf developmental mutant was isolated from an EMS-mutagenized Arabidopsis mutant pool in the Col-0 background (Yan et al., 2006). Map-based cloning showed that a G→A mutation occurred in the coding region of the At1g11130 gene encoding the leucine-rich repeat receptor-like kinase (LRR-RLK) SCM/SUB and corresponded to the sub-2 allele in the Ler background (Figure S1a,b; Chevalier et al., 2005; Kwak et al., 2005). For confirmation that mutation in the SCM/SUB gene contributes to mutant phenotypes, a construct containing the full-length SCM/SUB gene was introduced into mutant plants and successfully complemented the mutant phenotypes (Figure S1c). For consistency, we use SUB as the gene name hereafter. The sub-2 mutant in the Col-0 background was backcrossed three times to wild-type Col-0, and the resultant mutant line was used for subsequent analyses.
Compared with the wild-type, the sub-2 leaves displayed varying degrees of defects at 30°C, including reduced leaf outgrowth, bilateral asymmetry, incomplete lamina and twisted petiole (Figure 1a,b). In severe cases, the deformed leaf blade had >180° rotation of petiole (Figure 1c). The shape and size of sub-2 cotyledons appeared to be similar to Col-0, indicating a role for SUB in post-embryonic leaf development. When grown at 22°C, the mutant phenotypes were weakened, except for the reduced leaf size and slightly rough leaf surface (Figure 1d,e). The reduced leaf area in sub-2 was mainly due to the reduced leaf width and incomplete development of the leaf blade, leading to a significant increase in the leaf index (radio of leaf length to leaf width; Figure 1f–i). In addition, sub-2 exhibited temperature-sensitive twisting of the stem and reduced plant height (Figure 1j–l). Furthermore, a null T-DNA insertion mutant scm-2 in the Col-0 background (Kwak et al., 2005), as well as the sub-2 and sub-3 mutants in the Ler background, also showed temperature-sensitive leaf defects (Figures S1d and S2). Therefore, we conclude that the mutation in the SUB gene is responsible for the mutant phenotypes.
Altered cell proliferation and expansion during early leaf patterning in sub-2
To monitor the timing of SUB action during leaf development, we first examined the early leaf morphology at 30°C using scanning electron microscopy (SEM). At leaf stage 1 (Carland and McHale, 1996), the leaf shape and size of sub-2 appeared similar to that of Col-0 (Figure 2a,b). However, an irregular leaf margin appeared at leaf stage 2 in sub-2 plants (Figure 2c,d), and morphological abnormalities progressed at leaf stage 3, including reduced leaf outgrowth, bilateral asymmetry, and an irregular leaf margin with ectopic hydathodes (Figure 2e–h). The elongated epidermal cells along the leaf margin and midrib appeared to be distorted in sub-2 plants (Figure 2i–m). Study of the growth dynamics of leaf primordia also confirmed that early leaf outgrowth was largely inhibited in the medio-lateral axis of sub-2, leading to the reduced leaf size (Figure S3). At the late developmental stage, non-fully expanded epidermal cells were visible in both the adaxial and abaxial leaf surfaces of the sub-2 blade (Figure 2n–q). The uneven expansion of these cells may lead to the rough and crinkled leaf surfaces as shown in Figure 2(u). In addition, the abaxial epidermal cells along the midvein were obviously twisted (Figure 2r,s), and the shape and arrangement of mesophyll cells were irregular in the sub-2 leaves (Figure 2t,u). These results indicate that the SUB gene is required for cell shape/expansion in both epidermal and internal tissues.
Transverse sections through the apex of 5-day-old seedlings (approximately leaf stage 3) grown at 30°C showed that the number of proliferating cells was reduced, but more vacuolated cells and intercellular gaps appeared in young leaves of sub-2, suggesting an early transition from cell proliferation to expansion (Figure 3a,b). Statistical analyses showed that the number of exterior epidermal cells at this stage and the number of palisade cells in the 25-day-old first leaf of sub-2 plants were reduced, indicating that cell proliferation was impaired during sub-2 leaf development (Figure 3c,d).
During leaf development, basipetal exit of cell division can be visualized by accumulation of the cell-cycle marker CYCB1;1–GUS (Colon-Carmona et al., 1999; Donnelly et al., 1999). The CYCB1;1–GUS activity decreased earlier in sub-2 plants than in Col-0 at both temperatures (Figure 3e,f and S4), consistent with the reduced cell number as shown in Figure 3(d). As transition from cell proliferation to cell expansion is coupled with the onset of endo-reduplication (Beemster et al., 2005), an alternative cycle in which cells undergo rounds of DNA replication without division, we measured the DNA content using flow cytometry to examine cell growth in the developing first leaves. As shown in Figure 3(g), the proportions of 4C and 8C cells in sub-2 leaves were prematurely increased, while that of 2C cells was reduced. These results indicate that the mutation in SUB results in reduced cell proliferation and earlier onset of endo-reduplication during leaf development.
Altered venation and auxin patterning in sub-2
During leaf development, the formation of leaf shape and venation pattern are temporally coordinated (Dengler and Kang, 2001; Scarpella et al., 2010). We found that the complexity and continuity of sub-2 venation was significantly reduced at 30°C, with fewer branching points and areoles but visible vascular islands (Figure 4a and Table 1). Similar to leaf shape, these defects were also alleviated at 22°C, although the venation complexity was still reduced because of the decreased number of higher-order veins in sub-2 leaves (Figure 4b and Table 1). To examine the early vascular development, we introduced ATHB8–GUS, a marker for procambial cells (Baima et al., 1995), into sub-2 plants. Although expression of ATHB8–GUS showed no obvious difference between sub-2 and Col-0 at leaf stage 1 (Figure 4c), the subsequent formation of continuous loops was disturbed in the sub-2 leaves (Figure 4d,e). It is known that polar auxin transport plays important roles in venation patterning (Mattsson et al., 1999; Sieburth, 1999; Mattsson et al., 2003; Scarpella et al., 2006). Expression of the auxin efflux carrier PIN-FORMED1 (PIN1) precedes expression of ATHB8–GUS in leaf primordia, and thus represents an earlier marker for procambium formation (Scarpella et al., 2006). We used a functional PIN1–GFP reporter (Benkova et al., 2003) to examine PIN1 expression. During formation of continuous veins in wild-type leaf primordia, the PIN1–GFP expression domain was broad at first but was gradually restricted to a narrow site and became connected to the midvein PIN1–GFP expression domain or other pre-existing PIN1–GFP expression domains (Scarpella et al., 2006). In contrast, some PIN1–GFP expression domains were ectopic or even missing, or failed to extend in sub-2 plants (Figure 4f), suggesting that the auxin flow may be interrupted. However, we found that induction of vascular continuity along leaf margin by naphthylphthalamic acid, an inhibitor of polar auxin transport, was decreased in the sub-2 leaves at 30°C (Figure S5). These results indicate that both SUB and polar auxin transport are required for venation patterning.
Table 1. Analysis of venation pattern in the first leaves of 12-day-old sub-2, axr1-3 and sub-2 axr1-3 plants
Number of areoles
Number of branching points
Number of island veins
Values represent means ± SD. Values that are significantly different from Col-0 are indicated in bold; values for double mutants that are significantly different from the single mutant are indicated in italics (Student’s t test, P ≤0.01).
10.65 ± 1.99
26.30 ± 3.70
7.17 ± 1.95
19.50 ± 3.09
6.47 ± 1.68
16.07 ± 3.43
4.15 ± 1.04
12.50 ± 2.59
9.90 ± 1.66
25.60 ± 5.40
2.50 ± 1.08
11.80 ± 2.49
0.50 ± 0.84
4.88 ± 1.25
13.88 ± 2.75
1.90 ± 1.66
9.00 ± 2.16
1.10 ± 1.29
We further examined the auxin response using a synthetic auxin reporter DR5–GUS (Ulmasov et al., 1997). Expression of DR5–GUS in sub-2 leaves was slightly weaker in both tip and vascular zones but increased in the ectopic hydathodes (Figure S6a,b). Exogenous application of indole-3-acetic acid (IAA) induced DR5–GUS expression in both Col-0 and sub-2 seedlings, showing that auxin perception and response were normal in the sub-2 mutant (Figure S6c,d). In addition, AXR1 encodes an ubiquitin-activating enzyme E1 that is required for auxin signaling (Leyser et al., 1993). Compared with the sub-2 or axr1-3 single mutant, the sub-2 axr1-3 double mutant showed reduced complexity of venation patterning at both temperatures (Figure 4g,h and Table 1). Discontinuous veins were significantly increased and more vascular islands appeared in sub-2 axr1-3 leaves, particularly at 30°C (Figure 4h and Table 1), suggesting that SUB acts synergistically with AXR1 in venation patterning.
SUB-mediated early leaf patterning is critical for the final leaf shape
Leaf shape may be affected at various stages during leaf development. To determine the stage(s) that is severely affected by the sub-2 mutation, we performed temperature-shift experiments (Figure 5). Both Col-0 and sub-2 seedlings grown at 22°C were transferred to 30°C between 1 and 14 days after germination (DAG), and vice versa (experiments 22/30 and 30/22, respectively). The length and width of the first rosette leaf was measured at 16 DAG, and the leaf index was used as a parameter to reflect the degree of leaf defects. In both experiments, the leaf index of Col-0 plants was relatively steady and similar to that of seedlings continuously grown at 22 or 30°C. In contrast, the leaf index of sub-2 seedlings shifted at between 1 and 7 DAG in the 22/30 shift experiment was significantly reduced, especially between 4 and 7 DAG (from 1.84 to 1.37; Figure 5a,b). In the 30/22 shift experiment, the sub-2 mutant shifted at between 1 and 7 DAG displayed more severe defects the longer they were held at 30°C (Figure 5c,d). However, the leaf index of mutant seedlings shifted after 7 DAG changed little in either experiment. Based on an analysis of leaf developmental stages at the time of shifting (Figure S7), we conclude that SUB predominantly functions in the early stage of primary morphogenesis during leaf development (leaf stages 1–4).
The expression pattern of SUB during leaf development
To examine the expression pattern of SUB during leaf development, promoter activity was analyzed using the proSUB:GUS reporter line (Figure 6a–o). The GUS signal was weak in the shoot apical meristem but increased throughout young primordia, with higher activity in provascular tissues and the adaxial–abaxial juxtaposition region. During leaf expansion, GUS expression was basipetally reduced and finally restricted to vascular cells that were still dividing and differentiating.
To further examine the accumulation of SUB protein during leaf development, we generated transgenic plants expressing SUB–YFP under the control of a native SUB promoter (proSUB:SUB-YFP) in the sub-2 background. The SUB–YFP fluorescence signal was very weak in the leaf primordia of most transgenic plants, although the leaf phenotypes of sub-2 were rescued. We thus selected complemented plants with strong YFP signal but reduced leaf size, which resemble the phenotypes of weak SUB-OE plants (see below), for analysis. Consistent with the GUS activity of proSUB:GUS, SUB–YFP accumulation in young primordia was basipetally diminished during leaf development and became restricted to developing vascular cells (Figure 6p–r). In addition, SUB–YFP accumulated in the cell periphery of epidermal tissues (Figure 6s), similar to previous reports in root and sepal epidermal cells (Kwak and Schiefelbein, 2008; Yadav et al., 2008). Interestingly, accumulation of SUB–YFP was predominant at the junctions of two neighboring procambial cells (arrows in Figure 6t), suggesting a potential polar localization of SUB during vascular differentiation. These results indicate that SUB expression is temporally and spatially regulated during leaf development, and the expression pattern coincides with its roles in early leaf development and vascular formation.
Constitutive expression of SUB inhibits leaf development
To learn more about SUB function, we generated transgenic plants expressing SUB under the control of the CaMV 35S promoter in the Col-0 background. Among 86 independent SUB over-expression (SUB-OE) T1 plants, 54 lines showed varying degrees of dwarfing and reduced organ size (Figure 7a,b). Real-time RT-PCR analysis showed that SUB acts in a dose-dependent manner, and the individuals with strong phenotypes accumulated more SUB transcripts (Figure 7c). In severe cases, growth of almost all the organs was inhibited from seedling stage to adult stage (Figure 7a,b and S8).
We then examined the cellular basis of impaired leaf development in the SUB-OE plants with strong phenotypes. Compared with the fully expanded cells in Col-0 (Figure 7d,g,i), a large number of small cells were clustered together in the epidermal and internal tissues of SUB-OE leaves (Figure 7e,h,j). Both cell size and the number of single-layer palisade cells were decreased (Figure 7m–o), and the number of cell layers between adaxial and abaxial epidermis was increased in the SUB-OE leaves (Figure 7h). Some smaller cells were observed in both epidermis and internal tissues, suggesting that progression of cell growth was inhibited and was less synchronous in SUB-OE plants than in Col-0 (arrows in Figure 7f,h,j). In addition, development of vascular tissues was also inhibited in SUB-OE leaves with reduced complexity (Figure 7k,l,p,q). Nuclear ploidy analysis showed that the population of 4C cells was increased, but the populations of 16C and 32C cells were greatly reduced in the leaves of SUB-OE plants compared with Col-0 (Figure 7r–t), indicating that endo-reduplication was inhibited in the leaves of SUB-OE plants. Together with the cellular observations, these results show that constitutive expression of SUB restricts organ growth by inhibiting progression from cell proliferation to cell expansion, in contrast to the loss-of-function mutant.
The hidden roles of SUB in leaf patterning
Although SUB was previously shown to be required for pattern formation in root epidermis and floral organ development (Chevalier et al., 2005; Kwak et al., 2005), its roles in leaf development have not been reported. We observed that several leaf patterning processes were affected in the sub mutant at high ambient temperature, suggesting that either SUB has specialized functions in distinct developmental processes or SUB is involved in a general aspect of leaf development such as cell patterning. As a developmental trigger, the phytohormone auxin has been recognized to modulate organ growth and patterning formation (Benkova et al., 2003; Zgurski et al., 2005; Scarpella et al., 2006, 2010; Bilsborough et al., 2011). The altered expression pattern of DR5–GUS and PIN1–GFP in sub-2 plants suggests that SUB may coordinate multiple processes together with the auxin-mediated pathway.
An intriguing question is why loss-of-function mutations in the SUB gene cause severe leaf developmental defects under high temperature. One possibility is that SUB-mediated early leaf patterning processes are sensitive to high temperature. The temperature sensitivities of several leaf developmental mutants such as as1 and as2 (Qi et al., 2004), phantastic (Waites and Hudson, 1995; Waites et al., 1998) and varicose (Deyholos et al., 2003) support this possibility. Alternatively, other genes that are functionally redundant with SUB may complement the sub-2 phenotypes at 22°C but not at 30°C. SUB belongs to an LRR-V RLK sub-family with nine members whose expression in rosettes has been detected to varying extents (Eyuboglu et al., 2007). The functions and temperature responses of other members remain to be investigated.
SUB coordinates cell proliferation, differentiation and leaf development
During leaf development, expression of SUB is initially ubiquitous in the young leaf primordia, then basipetally reduced along the lamina and finally restricted to the leaf veins (Figure 6). Our results demonstrated that precise spatio-temporal expression of SUB is important for cell proliferation and differentiation/expansion during leaf development. First, high expression of SUB in young leaf primordia is required for cell proliferation, as the cell number and CYCB1;1–GUS activity were largely reduced in sub-2 leaves. Reduced cell proliferation in sub-2 plants may contribute to incomplete lateral organs and reduced leaf outgrowth, similar to that in jag, trn1 and trn2 mutants (Dinneny et al., 2004; Ohno et al., 2004; Cnops et al., 2006). Second, the early expanded cells in the sub-2 leaves appear not to differentiate well later, given the mis-shapen epidermal and mesophyll cells in mature leaves. This suggests that SUB mediates a differentiation signal for cell expansion in both epidermal and internal tissues. Third, timely arrest of SUB expression in differentiating cells is also important for subsequent organ growth. Constitutive expression of SUB inhibited cell expansion and endo-reduplication. Fourth, in contrast to the AN and ROT genes that modulate cell proliferation or expansion along specific polarity axes (Kim et al., 1998, 2002; Narita et al., 2004), cell number was reduced in the horizontal direction and increased along the adaxial–abaxial axis of SUB-OE leaves, indicating intricate spatiotemporal regulation of cell proliferation during leaf development. Finally, expression of SUB also maintains the synchronous progression of cell proliferation or expansion in bilateral leaf primordia. Mutation of SUB results in asymmetric cell growth (Figure 3b), which may partially contribute to the asymmetric leaf shape (Semiarti et al., 2001; Zgurski et al., 2005; Cnops et al., 2006).
Our temperature-shift experiments indicated that SUB predominantly functions in the early stage of leaf development. As SUB encodes an RLK, we propose that SUB may mediate a developmental stage-specific signal to coordinate cell proliferation and differentiation for leaf patterning. It has been recognized that RLKs modulate cell proliferation and differentiation in a wide range of growth and developmental processes (De Smet et al., 2009). For example, the ERECTA RLK family members control aerial organ shape and size primarily by promoting cell proliferation, and specify stomatal patterning by repressing asymmetric cell divisions during guard-cell differentiation (Shpak et al., 2003, 2004, 2005; Pillitteri et al., 2007). Our results also show that SUB promotes cell proliferation and participates in vascular differentiation during leaf development. These findings suggest a dual function of these RLKs in cell proliferation and patterning of specialized cells during organ growth. The BARELY ANY MERISTEM 1 (BAM1), BAM2 and BAM3 RLKs are required for leaf development (DeYoung et al., 2006). Similar to sub-2, the bam1 bam2 and bam1 bam2 bam3 mutants exhibit reduced leaf size, asymmetric leaf shape and venation patterning (DeYoung et al., 2006), indicating functional redundancy of these RLKs for leaf patterning. Recently, direct interaction of peptide ligands and ERECTA family receptors was demonstrated for specification of stomatal patterning (Lee et al., 2012). In addition, over-expression of a small peptide ROT4 also decreases cell proliferation and alters leaf shape (Narita et al., 2004). These findings indicate the existence of peptide-mediated signaling pathways for leaf development. Further determination of SUB-interacting proteins or ligands will shed light on the molecular mechanism of RLK-mediated leaf development.
Roles of SUB in vascular development
Our results showed that venation patterning was altered in sub-2 leaves, consistent with strong expression of SUB in leaf veins. The simultaneous alteration of venation patterning and leaf shape in sub-2 indicates a strong connection between the two developmental processes. Auxin has been proposed as an important modulator for both leaf blade development and venation patterning (Scarpella et al., 2010). It has also been shown that auxin maxima at the leaf margin and the PIN1 protein-mediated polar auxin transport in marginal epidermal tissues play important roles in positioning vascular strands (Scarpella et al., 2006). The overlapping functions of auxin and SUB and the irregular leaf margin in sub-2 suggest that SUB may coordinate venation patterning and leaf shape by directly or indirectly modulating auxin patterning at the leaf margin. However, the impaired venation in sub-2 may not be the consequence of altered leaf shape, because the complexity of venation was still reduced in sub-2 leaves with the nearly normal leaf shape at 22°C. SUB strongly accumulates in developing procambial cells (Figure 6t), and the number of vascular cells was reduced in both leaves (Figure 2u) and stems (Chevalier et al., 2005) of sub mutants. These findings suggest a direct role for SUB in vascular differentiation. Thus, SUB may be required for vascular patterning throughout the lamina and within vascular bundles.
During leaf development, expansion/differentiation of mesophyll cells may interfere with extension of procambial domains (Scarpella et al., 2004). Decreased cell proliferation in leaves also reduced the complexity of the venation pattern (Kang et al., 2007). Consistent with these findings, sub-2 exhibited impaired vascular development as well as reduced cell proliferation and premature cell expansion. However, the venation complexity was also reduced in SUB-OE leaves in which cell expansion was inhibited. Similar phenotypes were also observed in our previous analysis with gibberellin-deficient plants (Zhang et al., 2011), suggesting the importance of the balance between cell proliferation and expansion/differentiation for vascular formation. It was shown that a high auxin level in developing leaves is strongly correlated with high cell-division activity, whereas a low auxin level triggers the mitosis to endo-reduplication transition (Ljung et al., 2001; Ishida et al., 2010). As the genetic analysis showed that SUB acts synergistically with AXR1 in venation patterning (Figure 4g,h), auxin and SUB RLK-mediated signaling pathways probably together control the balance between cell proliferation and differentiation for vascular development.
Plant materials and growth conditions
The Arabidopsis sub-2 mutant in the Col-0 background was isolated from an EMS-mutagenized mutant pool (Yan et al., 2006). Seeds of scm-2 (SALK_086357C) were obtained from the Arabidopsis Biological Resource Center. The marker lines ATHB8–GUS (Col-0) and PIN1–GFP (Col-0) were obtained from the Nottingham Arabidopsis Stock Centre ( identification numbers are N296 and N9362, respectively). Seeds of sub-2 and sub-3 in the Landsberg erecta (Ler) background (Chevalier et al., 2005), and the reporter lines DR5–GUS (Col-0) and CYCB1;1–GUS (Col-0) were kindly provided by Kay Schneitz (Plant Developmental Biology, Science Center Weihenstephan, Technical University of Munich), Hai Huang and Jirong Huang (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), respectively. The reporters were introduced into sub-2 (Col-0) by crossing. Seeds were surface-sterilized and sown on half-strength Murashige and Skoog medium with 0.7% agar. After 3–4 days at 4°C in the dark, plates were transferred to a growth chamber at 20–22°C or 28–30°C under light (120 μmol m−2 sec−1) with a 16 h light/8 h dark cycle. At 6 DAG, seedlings were transferred to the soil.
Plasmid constructions and Arabidopsis transformation
The primers used for cloning are listed in Table S1. For complementation, the 8.4 kb genomic fragment containing the full-length SUB gene plus 3430 bp upstream and 406 bp downstream sequences was released and cloned into the vector pCAMBIA1301 (GenBank accession number AF234297), resulting in p1301-gSUB. The 3056 bp promoter of SUB was amplified using primers pSUB(SalI)-F and pSUB(SmaI)-R, and cloned into vector pBI101.1 (GenBank accession number U12639.1) to generate the proSUB:GUS reporter. For the proSUB:SUB-YFP construct, two fragments including the coding region and introns without the stop codon were amplified from genomic DNA using primers gSUBma(KpnI) F and gSUBa(BamHI)-R, and gSUBb-F and gSUBmb(BamHI)-R, and then fused with YFP. 3′ UTR sequence of SUB amplified using UTR(SacI)-F and UTR(EcoRI) primers, fusion fragment of gSUB-YFP, and the 3430 bp SUB promoter were sequentially cloned into a pCAMBIA1301 vector, generating proSUB:SUB-YFP. For the over-expression construct, two genomic fragments were amplified using primers gSUBa(KpnI)-F and gSUBa(BamHI)-R, and gSUBb-F and gSUBb(BamHI)-R, and then sequentially placed downstream of the 35S promoter in the overexpression vector pCAMBIA1300S (provided by Yinong Yang, Department of Plant Pathology, the Pennsylvania State University). All the constructs were introduced into Arabidopsis plants by the Agrobacterium tumefaciens-mediated transformation procedure (Clough and Bent, 1998).
Microscopy and histology
Leaf morphology and cell number were analyzed as described by Horiguchi et al. (2006). The images were photographed using an Olympus SZX7 stereomicroscope or BX51 compound microscope (http://www.olympus.com/). Leaf length, width and size were measured using ImageJ software (http://www.rsb.info.nih.gov/ij). Scanning electron microscopy, confocal laser-scanning microscopy and preparation of half-thin resin sections were performed as described by Zhang et al. (2011). To examine venation patterning, leaves were fixed in FAA solution (3.7% formaldehyde, 5% acetic acid, and 50% ethanol), and then incubated with clearing solution (80 g chloral hydrate, 30 ml water, 10 ml glycerol) overnight. The cleared leaves were photographed under dark-field illumination. Histochemical detection of GUS activity was performed as described by Scarpella et al. (2004). The samples were incubated in staining solution containing ferricyanide (3 mM for ATHB8–GUS; 1 mM for proSUB:GUS; 0.5 mM for DR5–GUS and CYCB1;1–GUS) at 37°C for 2 h (ATHB8–GUS and proSUB:GUS) or overnight (DR5–GUS and CYCB1;1–GUS) before clearing.
For serial sections, 12-day-old seedlings expressing proSUB:GUS were fixed in 100 mM sodium phosphate buffer (pH 7.0) containing 0.5% formaldehyde and 0.1% Triton X-100 for 45 min, rinsed three times for 5 min each in 100 mM sodium phosphate, and then incubated in staining solution containing 1 mM ferricyanide for 6 h at 37°C. After washing for 5 min with the phosphate buffer, the seedlings were fixed in FAA solution overnight, dehydrated via a series of ethanol gradients followed by substitution with xylene, and then embedded in Paraplast (Sigma-Aldrich, http://www.sigmaaldrich.com/). Serial 7 μm thick sections were cut using a rotary microtome.
Flow cytometric analysis
Ploidy levels were measured using a MoFlo XDP flow cytometer (Beckman Coulter, http://www.beckmancoulter.com/). The nuclei were released using 4′,6-diamidino-2-phenylindole (DAPI)-containing nuclear isolation medium (NIM-DAPI 10, Beckman Coulter) by chopping fresh leaves with a razor blade followed by filtration through a 40 μm cell strainer (BD Falcon, http://www.bdbiosciences.com/). For each sample, at least 10 000 nuclei from approximately 30 leaves were counted. Each assay was repeated at least twice with similar results.
RNA isolation and real-time RT-PCR
Total RNAs were extracted from the shoots of 4-week-old SUB-OE and Col-0 plants using TRIzol reagent (Invitrogen, http://www.invitrogen.com/). cDNAs were synthesized from 3 μg total RNAs using oligo(dT) primer and SuperScript III reverse transcriptase according to the manufacturer’s instructions (Invitrogen). Real-time RT-PCR analysis was performed using SYBR Premix Ex Taq (TaKaRa, http://www.takara.com.cn/) and gene-specific primers (Table S1).
We thank Kay Schneitz, Hai Huang and Jirong Huang for providing seeds, Yinong Yang for plasmid, and Jiqin Li, Yu Kong and Weifang Jiang for technical assistance. This research was supported by a grant from the National Natural Science Foundation of China (91117018).