All of the Arabidopsis lines used in this study were in the Col-0 background. The ER family mutants and overexpression plants have been described previously (Torii et al., 1996; Shpak et al., 2004; Shen et al., 2015). The seeds for er-105, er-/- erl1+/- erl2-/- mutant were kindly provided by Prof. Masao Tasaka (Nara Institute of Science and Technology). The seeds for 35S::ERECTA were kindly provided by Prof. Zuhua He (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). The seeds for DR5::GFP-NLS in wild type were kindly provided by Prof. Yuling Jiao (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). Other Arabidopsis seeds were obtained from self-preserved, genetic hybridization or agrobacterium-mediated transformation. All seeds were sterilized with 70% ethanol and 0.5% Tween for 10 min, followed by washing two times with 95% ethanol and air drying. After sterilization, seeds were sown onto 1/2 Murashige and Skoog (MS) medium containing 1% sucrose and 0.8% agar, then incubated at 4°C in darkness for 2 days, finally grown vertically at 21°C under long-day condition (16 h of light and 8 h of darkness).

For construction of pER::3 × VENUS-NLS, a 1.4-kb upstream sequence of ERECTA before ATG was used as a promoter. For the pERL1::3 × VENUS-NLS, a 4.1-kb upstream sequence of ERL1 before ATG was used as a promoter. For the pERL2::3 × VENUS-NLS, a 3.6-kb upstream sequence of ERL2 before ATG was used as a promoter. For construction of pER::ER-GFP, pERL1::ERL1-GFP and pERL2::ERL2-GFP, the genomic sequence of these three genes were amplified and used the same promoter we described above. The pER::ER-GFP construct was then transformed into the er-105 mutant to rescue its defects, while other constructs were transformed into the Col-0. For pER::iaaM, the 1.4-kb promoter of ERECTA was used to drive iaaM sequences that were obtained from Prof. Yunde Zhao (UC San Diego). This construct was then transformed into the er-/- erl1+/- erl2-/- mutant. The primer sequences used in the plasmid construction are listed in Supplementary Table S1.

Whole seedlings were dissected and immediately transferred to liquid nitrogen. The Tripure Isolation Reagent (Roche) was used to isolate total RNA from the plant samples. The PrimeScriptTM RT Reagent Kit (TaKaRa) was used for cDNA synthesis. Quantitative PCR was performed with the Thermo PIKO REAL96 Real-Time PCR system using the GoTaq^® qPCR Master Mix (Promega) with the following PCR conditions: 95°C for 5 min and 40 cycles of 95°C for 10 s, 57°C for 30 s and 72°C for 30 s, followed by 72°C for 10 min and 20°C for 10 s. TUBULIN was used to normalize the mRNA levels. Primers used for qRT-PCR are listed in Supplementary Table S1.

Propidium Iodide (PI) powder (Sigma) was dissolved with PBS solution to 5 mg/ml as a stock solution, which was stored in dark. The working solution was diluted to 5 μg/ml. The whole seedling was put into the solution for staining 10 min, then washed with water three times and then observed under the confocal microscope (OLYMPUS LSM1200).

For the length of hypocotyls in the seedling stages, pictures were taken under the microscope; The Image J software was used for the measurement and statistical analysis. The cells in longitudinal direction from the top to the base of the hypocotyls epidermis were used for the measurement of each cell length, and 25 hypocotyls were used for biological repetitions, and the Image J software was used for the statistical analysis. The total cells number in the hypocotyls was counted directly under the microscope.

IAA and yucasin powder (Sigma) were dissolved in the DMSO and diluted to 50 μM for IAA and 5 mM for yucasin as the stock solutions. The stock solutions were diluted 1000x and added to the corresponding 1/2MS media. The control group was added with the same amount of DMSO in the media. The plants were grown vertically at 21°C under long-day condition (16 h of light and 8 h of darkness).

Where appropriate, statistical analyses were performed with analysis of variance (ANOVA) test. Otherwise, comparisons between two groups were conducted using Student’s t-test. The p-value level was set to 5%.

One of the most striking defects in the ERECTA family mutants (erf) mutants is the dwarf phenotype (Shpak et al., 2004). The er-105 single mutant showed reduced plant height comparing to that of wild type, whereas the plant height was increased in the ERECTA overexpressing plants (Supplementary Figure S1A) (Shen et al., 2015). In the er erl1 erl2 triple mutant, we observed even severe phenotypes in the reduction of the plant height (Supplementary Figure S1A), suggesting that ERECTA, ERL1 and ERL2 were functionally redundant in control of the plant height. To further support these observations, we measured the plant height of the 35S::ERECTA, er-105 and er erl1 erl2 plants. We observed a slight increase of the plant height in the 35S::ERECTA plants, however, a significant decrease in the er-105 and er erl1 erl2 mutants (Supplementary Figure S1B).

Consistent with the observation of plant height, in the 6-day-old seedling, we observed that the length of hypocotyl in the 35S::ERECTA plants was significantly increased comparing to that of wild type seedlings (Figures 1A,B). Conversely, the hypocotyl lengths in the er-105 and er er1 erl2 mutants were dramatically reduced (Figures 1A,B). We conclude that the ERECTA gene family are involved in the regulation of hypocotyl length in Arabidopsis.

Given the fact that disturbing the functions of ERECTA gene family causes the severe defects in the hypocotyl length, we assumed that ERECTA gene family are expressed in the hypocotyl cells. To test whether the ERECTA family genes are transcribed in the hypocotyl cells, we first performed the promoter activation analysis. To this end, we constructed pER::3 × VENUS-NLS, pERL1::3 × VENUS-NLS, pERL2::3 × VENUS-NLS transgenic plants. We observed evenly distributed fluorescence signals in the whole hypocotyl of these three transgenic plants (Figures 2A–C), suggesting that ERECTA, ERL1 and ERL2 genes are expressed in the whole hypocotyl of Arabidopsis.

Because ERECTA family genes encode leucine-rich repeat receptor-like kinases, we then test whether the ERECTA, ERL1 and ERL2 proteins are localized in the hypocotyl cells. We transformed the pER::ER-GFP construct into the er-105 mutant and observed a fully rescue of the mutant defects. By the confocal microscopy, we observed that ERECTA proteins were strongly accumulated in the hypocotyls (Figure 2D).

To check the protein localization of two homologous genes of the ERECTA, the pERL1::ERL1-GFP, pERL2::ERL2-GFP transgenic plants were also analyzed under the confocal microscope. Consistent with the observation of ERECTA proteins, we found that the both of proteins, ERL1 and ERL2, were also localized in hypocotyls (Figures 2E,F).

The organ size and shape of multicellular organism is directly related to the cells division and the size of cells. Therefore, the individual cell length and the total number of cells could determine the hypocotyl length in Arabidopsis. Previous studies have shown that ERECTA family genes may regulate cell number in some organs, including stomata, ovule and stamen (Shpak et al., 2004; Woodward et al., 2005; Pillitteri et al., 2007; Hord et al., 2008). However, whether the ERECTA family genes control the cells division and cell elongation in the hypocotyl is still unclear. To test this, we performed Propidium Iodide (PI) staining on the hypocotyls of the wild-type plants, 35S::ERECTA plants, er-105 single mutant plants, and er erl1 erl2 triple mutant plants (Figures 3A–D). By the observation under the confocal microscope, we found that the individual cell size of hypocotyl in the 35S::ERECTA overexpression plants was large than that of wild type plants (Figure 3B). On the contrary, we observed size largely reduced cells in the hypocotyls of the er-105 single mutant and er erl1 erl2 triple mutant plants comparing to that of wild type plants (Figures 3C,D). By the quantitative analysis of the average cells length in the middle column of the hypocotyl epidermis, we found that the average cells length in the 35S::ERECTA overexpression plants was significantly increased, whereas it was remarkably decreased in the er-105 and er erl1 erl2 mutants (Figure 3E).

To test whether cells division is involved in the ERECTA family genes controlled hypocotyl elongation, we directly counted the total number of cells in the middle column of epidermis, and found that there were no significant difference in all the genotypes we test including the wild type, 35S::ERECTA, er-105 and er erl1 erl2 plants (Figure 3F). We concluded that the ERECTA gene family regulates the hypocotyl length by controlling the cell elongation rather than the cell division.

Given the fact that the cell elongation was significantly reduced in the erf mutants, we then asked by what means does the ERECTA gene family regulate individual cell length in the hypocotyl? Previous studies have shown that the auxin plays an important role in regulation of cell elongation (Rayle and Cleland, 1992; Galinha et al., 2007). The endogenous auxin increased mutants exhibit hypocotyls-elongated phenotypes, whereas auxin deficient mutants have the reduced size of cells (Romano et al., 1991; Zhao et al., 2001; Cheng et al., 2006). To examine whether the auxin is involved in the ERECTA gene family mediated controls of cell elongation, two different auxin sensors, DR5::GFP and DII-VENUS, were applied to detect the endogenous auxin levels in the er erl1 erl2 mutants. The DR5::GFP is a positive auxin sensor by which GFP transcriptions are activated by auxin (Ulmasov et al., 1997; Benkova et al., 2003). Under the confocal microscope, we observed evenly distributed fluorescence signals of DR5::GFP in the entire hypocotyl (Figure 4A), suggesting that the auxin was distributed in all the hypocotyl of Arabidopsis. However, we can barely detect the fluorescence signals in the er erl1 erl2 mutant hypocotyl (Figure 4B), suggesting that the endogenous auxin levels were dramatically reduced in the triple mutant hypocotyl. To confirm this observation, the transgenic plants that harbored the negative auxin sensor DII-VENUS by which fluorescence proteins were degradated by auxin (Brunoud et al., 2012) were further analyzed. Consistent with the observation of DR5::GFP, there were only extremely weak signals can be detected in the wild type hypocotyl (Figure 4C), but stronger DII-VENUS signals in the er erl1 erl2 triple mutants (Figure 4D). At the molecular level, we tested the transcriptional levels of auxin early response gene IAA19, which is commonly used to monitor the endogenous levels of auxin in Arabidopsis. We observed that the expression levels of IAA19 in the 35S::ERECTA transgenic plants were up regulated, whereas significantly down regulated in the er-105 and er erl1 erl2 triple mutants (Figure 4E). These data suggest that the ERECTA gene family is essential to maintain the endogenous auxin levels in the hypocotyl.

To shed light on the molecular mechanism underlying the ability of the ERECTA gene family controlling auxin levels in the hypocotyl, we examined the expression levels of the YUCCA family genes, which encodes the major auxin biosynthesis genes (Cheng et al., 2006). We observed that the transcriptional levels of YUC2, YUC3 and YUC5 were significantly increased in the ERECTA overexpression plants compared with wild type (Figure 4F). Conversely, these three key auxin biosynthesis genes were markedly decreased in the er-105 and er erl1 erl2 triple mutants; especially the expression levels of YUC3 were drastically reduced in the er erl1 erl2 triple mutants (Figure 4F). We draw the conclusion that the ERECTA gene family positively regulates auxin biosynthesis via activating the expressions of YUC2, YUC3 and YUC5 genes.

Given the fact that the erf mutants contain a very low amount of auxin and the key auxin biosynthesis genes are under the positive control of ERECTA gene family, we hypothesized exogenous increase of auxin might rescue the defects of cell elongation and short hypocotyl phenotypes of the erf mutants. While, previous studies have shown that the high concentrations of auxin might inhibit the elongation of hypocotyls in wild-type Arabidopsis (Collett et al., 2000; Rashotte et al., 2003), which was also found in our experiments (data not shown). To exclude the possibility the high auxin inhibits the cell elongation in our test, we carefully select a very low concentration of IAA (50 nM), and performed our analysis. By applying this low concentration of IAA to the wild type seedlings, we did not observe any significant differences in term of the hypocotyl length comparing with the mock treatments (Figures 5A,B). However, this low concentration of IAA almost fully rescued the hypocotyl-shortened phenotypes of the er-105 single mutants and er erl1 erl2 triple mutants (Figure 5A). By quantifying the hypocotyl length, we observed significant increases of the hypocotyl length in the er-105 and er erl1 erl2 mutants upon the auxin treatments, which showed no differences to that of wild type seedlings (Figure 5B), suggesting that the hypocotyl-shortened defects in the erf mutants are due to the lacking of auxin, and the increase of exogenous auxin completely restores the mutant phenotypes. To further confirm this observation, we examine the cell elongation in the erf mutants after the auxin treatments. In the wild type hypocotyl, the 50 nM auxin treatments did not change the cell length comparing with the mock treatments (Figures 6A,B). However, in the er-105 and er erl1 erl2 mutants, the same amount of auxin effectively promoted the cell length to that of mock treatments (Figure 6A), and the cell length was significantly increased to the levels that comparable with the wild type seedlings (Figure 6B). To test the possibility whether the cell division was also involved in the rescue of erf mutants defects during the low auxin treatments, we directly counted the total number of cells in the middle column of epidermis with or without the low auxin treatments. We observed no significant difference among all the genotypes after the auxin treatments (Supplementary Figure S2). Therefore, we conclude that the hypocotyl-shortened phenotypes in the erf mutants are due to the cell elongation defects, which can be fully rescued by the treatments of exogenous auxin.

Given the fact that the ERECTA gene family positively regulates auxin biosynthesis in the hypocotyl by activating the expressions of several YUCCA family genes, and the overexpression of ERECTA causes the elevated expressions of YUCs (Figure 4F), we then tested this interaction genetically. We treated the 35S::ERECTA seedlings with the chemical yucasin, which have been shown to reduce the exogenous auxin levels by inhibiting the expression of YUCCA genes (Nishimura et al., 2014). We carefully select a low concentration of yucasin (5 μM) to perform the analysis, which showed no effect on the hypocotyl length in the wild type seedlings (Supplementary Figures S3A,B). While in the 35S::ERECTA seedlings, the hypocotyl length was significantly decreased (Supplementary Figures S3A,B). Likely, the hypocotyl cell length of the wild type plants did not respond the low yucasin treatments. However, the cell length of the 35S::ERECTA seedlings with increased YUCCA expressions, reduced dramatically upon the low yucasin treatments (Supplementary Figure S4), demonstrating that the ERECTA gene family controls the cell elongation in hypocotyls by positively regulating the auxin biosynthesis.

The bacterial auxin biosynthesis gene iaaM, which was firstly identified in the T-DNA of agrobacterium, is commonly used in plants to increase the endogenous auxin levels (Comai and Kosuge, 1982). To further investigate the mechanism by which the ERECTA gene family mediated auxin synthesis controls the cell elongation in hypocotyls, we expressed the iaaM gene under the control of the ERECTA promoter, and transformed the pER:iaaM construct into the er-/- erl1+/- erl2-/- mutants. By analyzing the expression levels of the iaaM and the IAA19 genes in eight independent transgenic lines, we observed a correlation between these two genes, suggesting that the ectopically expressed iaaM increased the endogenous auxin levels in the er erl1 erl2 triple mutant background (Supplementary Figure S5A). We measured the hypocotyl length of eight independent transgenic lines and selected three independent T1 transgenic lines with high expression of iaaM for the further analysis. (Supplementary Figure S5B), and observed that hypocotyls and individual cell lengths of pER:iaaM transgenic plants were higher than those of er erl1 erl2 triple mutants (Figures 7A–D). Thus we conclude that the ERECTA gene family mediated endogenous auxin biosynthesis controls the cell elongation in hypocotyls.