The 1-year-old rubber tree clone 93–114 was maintained as described (Cheng et al., 2015). Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as wild-type in this study. Seeds were surface sterilized and sowed in pots (10 cm × 10 cm × 10 cm) containing a mixture of soil and vermiculite (3:1 v/v). After 4 days imbibitions at 4°C, the plates and pots were transferred to a growth chamber at a constant temperature of 20°C under 16 h light (125 μmol m^-2 s^-1)/8 h dark cycles.

To generate the Arabidopsis plants that overexpress HbEBP1 (HbEBP1 OE), the coding region of HbEBP1 was amplified using primers 5′-TCAAAGCTGTAAGCTTATGTCGG-3′ and 5′-CATAAGAATTCCATACAAGGT-3′. The coding sequence fragment was then subcloned between the HindIII and EcoRI sites in pXCS-HAStrep plasmid. Arabidopsis ecotype Col-0 plants were transformed according to the floral dip method (Clough and Bent, 1998) using Agrobacterium tumefaciens strain GV3101(pMP90RK).

Isolation of rubber tree DNA and RNA was carried out as described (Zewei An, 2012). For northern blotting, a HbEBP1 probe was prepared from full-length cDNA with DIG Northern Starter Kit (Roche, USA). Twenty micrograms of total RNA was run on a denature 1.5% agarose gel and the blot was then transferred onto an Amersham Hybond^TM-N⁺ nylon membrane (GE Healthcare, USA). Hybridization and detection were carried out according to the manufacturer’s instructions.

Total RNA was extracted from Arabidopsis seedlings using Qiagen RNeasy Plant Mini Kit (Qiagen, USA). For quantitative RT-PCR (Q-PCR) analysis, the RNA samples were treated with DNase I to remove possible DNA contamination. Two micrograms of total RNA was used for each detection. The first-strand cDNA was synthesized with SuperScript III according to the manufacturer’s instructions (Invitrogen, USA). Q-PCR was carried out using IQ SYBR Green Supermix (Bio-Rad, USA) with gene-specific primers as listed in Supplemental Table S1. PCR was performed on a Bio-Rad Iq5 RT PCR instrument using the following program: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s.

Multiple sequence alignments were performed using ClustalW2 at the EBI ClustalW server¹ using the default parameters (Larkin et al., 2007). The coding sequence was predicted using Bioedit software and confirmed by using BLASTP program at NCBI BLAST server². The protein secondary domains were predicted with Interproscan program (Jones et al., 2014) at EBI server³. MEGA software (version 6.06) was used for phylogenetic analysis (Tamura et al., 2013). A phylogenetic tree was constructed using the neighbor-joining method with bootstrap test. The default parameters were used for the construction.

Southern blotting analysis was carried out as described (Cheng et al., 2015). Briefly, 20 μg Arabidopsis genomic DNA was digested with EcoRI. The DNA was resolved on a 0.8% agarose gel and transferred onto an Amersham Hybond^TM-N⁺ nylon membrane (GE Healthcare, USA); the blot was cross-linked under 0.12 J/cm² UV irradiation. Then the blot was hybridized at 42°C with Digoxigenin-labeled probe for 12 h. After two stringent washes at 68°C, the signal was detected using DIG Nucleic Acid Detection Kit (Roche, USA).

Cold and drought stress and ABA treatment in rubber trees were carried out as described (Cheng et al., 2015). To measure cold resistance in Arabidopsis, an electrolyte leakage analysis was performed as described (Cheng et al., 2013). Briefly, electrolyte leakage was measured using leaves from 2–week-old seedlings that had been frozen to -7°C at a cooling rate of 1°C/h from -1°C with an A28F Thermo Fisher temperature-controlled water bath (Thermo Scientific, USA).

To calculate dehydration rate, leaves were detached from 2-week-old seedlings, weighed and placed on dry filter paper in a drying chamber. The samples were weighed at 30-min intervals and the dehydration rate was calculated as the ratio of the remaining weight to the initial weight at each time point.

The drought tolerance test was carried out by sowing seeds in pots containing vermiculite. Irrigation was performed daily by adding half Hoagland nutrient solution in the tray until all the liquid was absorbed. When the seedlings were 2 weeks old, irrigation was withheld for 1 week. At least three pots of seedlings were observed for each genotype in this test.

For cold treatment in rubber tree, the seedlings were transferred to a 4°C cold culture room. Drought was carried out by detaching the leaf from the seedlings and keeping on the filter paper at room temperature. For ABA treatment, 100 μmol/L ABA solution was sprayed onto the seedlings until the liquid dropped from the leaf. Then the seedlings were covered with a plastic bag. The leaf sample was collected at indicated time points and stored in liquid nitrogen immediately.

The leaf development phases were determined by observing abaxial trichome production as described (Willmann and Poethig, 2011). The emergence of rosette leaves was recorded every day from day 7 after planting based on the criteria that they could be recognized with the naked eyes. Flowering time was recorded as the day on which flower primordia were visible without the aid of a microscope. Leaf length and blade length and width were measured as described (Willmann and Poethig, 2011). For leaf area measurements, the leaves were carefully detached from the seedlings and pasted on a sheet of A4 paper and then were scanned. The images were then analyzed with Image J software (version 1.45s) to calculate leaf area (Schneider et al., 2012).

For root length measurement, seeds were surface sterilized and sown onto the surface of a square plate containing half MS agar medium, and were then cultured vertically. Photos were taken daily, and root length on each day was measured with a ruler. At least 20 individual seedlings were analyzed from each line in these studies.

For measurement of the leaf epidermal cells, ten Arabidopsis plants were selected randomly. The area of the fully expanded seventh leaf was measured at about 25 days after germination in each transgenic line and Col-0 plants. The detail measurement was performed according to the method reported (Wang et al., 2016). Total cell number per leaf was calculated by dividing the leaf area by the average cell size.

All results are presented as the mean ± standard error of the mean (SEM) from five biological replicates for the stress experiments or from the number of replicates indicated. Statistical analysis was performed by using the Student’s t-test, and p < 0.05 was recognized as significant.

We previously identified a cDNA clone that putatively encodes a proliferation-associated 2G4 family member from the cold induced full-length cDNA library (Cheng et al., 2008). This cDNA fragment is 1462 bp in length, and contains an 1188 bp coding region (Figure 1A). The predicted peptide is highly similar to EBP1 proteins in other plant species and was therefore designated as HbEBP1. The deduced HbEBP1 amino acid sequence contains a proliferation-associated PA2G4-like domain, an aminopeptidase APP_MetAP domain and a methionine aminopeptidase (MAP) domain (Supplementary Figure S1). The HbEBP1 gene sequence was then deposited into the GenBank (Accession No: KX661028). A phenogram generated by MEGA analysis demonstrated that EBP1 proteins are highly conserved in eukaryotic species and that EBP1s from plant and animal species are clustered respectively. Rubber tree EBP1 is highly similar to EBP1 from Ricinus communis (accession no. XP_002530504.1) and Jatropha curcas (accession no. XP_012089820.1), which also belong to the Euphorbiaceae family (Figure 1B).

The gene expression profiles under abiotic stress and ABA treatment were analyzed using northern blotting. HbEBP1 expression was moderately expressed under ambient cultural conditions and this expression increased when rubber tree plants were subjected to cold, drought or ABA exposure. HbEBP1 transcripts increased notably after 4 h of treatment with cold stress and reached their highest level at 8 h, with some decrease after 24 h of treatment (Figure 2). A similar expression pattern was found when the seedlings were subjected to drought stress, i.e., induction occurred after 4 h of treatment and reached its highest level after 8 h (Figure 2). During ABA treatment, HbEBP1 expression showed some decrease during the first 4 h, followed by an increase after 8 h (Figure 2). EBP1 genes accumulated in response to auxin in Arabidopsis and tomato, and their expression correlated with genes involved in ribosome biogenesis and function (Zhang et al., 2005; Horváth et al., 2006). The responses toward abiotic stress and ABA suggest that HbEBP1 may be involved in the regulation of abiotic stress resistance in rubber trees.

The EBP1 genes regulate organ size by stimulating both cell proliferation and expansion via the regulation of RBR1 levels (Horváth et al., 2006). To validate these functions, HbEBP1 was overexpressed in Arabidopsis using the floral dip method. PCR amplification was used to confirm gene transfer. Among tens of HbEBP1 OE lines, we selected OE5, OE18 and OE28, each of which contained a single-copy T-DNA insertion and showed high and stable expression, for further study (Figure 3A). The T3 generation seedlings that harbored homozygous T-DNA insertions were used for further analysis. Northern blotting was used to detect HbEBP1 expression in the transgenic lines. As shown in Figure 3B, the OE5, OE18 and OE28 lines displayed comparable high expression. As expected, OE of HbEBP1 led to markedly enlarged organ size in Arabidopsis seedlings. Leaves from all three OE lines were notably larger as compared with leaves from the Col-0 seedlings (Figure 3C).

The area of the first adult rosette leaf (normally the seventh leaf) was measured. As demonstrated in Figures 4A,B, the area of the seventh leaf was significantly larger in OE5, OE18 and OE28 lines, as compared with that in the Col-0 plants (p < 0.01). The wild-type plants had an average leaf area of 1.43 ± 0.25 cm², whereas the average leaf area from OE5, OE18 and OE28 plant was 2.72 ± 0.36, 4.04 ± 0.44 and 3.12 ± 0.49 cm² respectively. In addition, the HbEBP1 OE lines developed more rosette leaves than did the wild type Col-0 seedlings (Figure 4B). We further measured the cell size and calculated total cell numbers. The results showed that the OE leaves have more cell numbers than the Col-0, while the cell size is comparable (Supplementary Figure S4). These results demonstrated HbEBP1 OE promotes cell proliferation in transgenic lines.

Similar results were observed in the roots. Primary root length was measured daily, and roots were found to grow faster in HbEBP1 transgenic seedlings. The OE5, OE18 and OE28 lines all displayed significantly faster root growth than did the Col-0 seedlings from 3 days after germination (p < 0.01) (Figure 4C). By 8 days after germination, the HbEBP1 OE lines had developed obviously longer primary roots than did the wild-type Col-0 seedlings (Figure 4D). At 2 weeks of age, the root systems were more complex in the HbEBP1 OE lines as compared with those in the Col-0 plants (Supplementary Figure S2). Thus HbEBP1 OE facilitated root development in Arabidopsis. The obviously larger leaves and longer primary roots in HbEBP1 OE plants suggested that HbEBP1 has a conserved function related to the regulation of organ size in H. brasiliensis.

The HbEBP1 OE lines displayed a late flowering phenotype. Under long-day conditions, the flower meristem was visible at 22.2 ± 0.9 days after planting for the Col-0 plants. In contrast, for OE5, OE18 and OE28 lines, the flower meristem was visible at 25.8 ± 1.0, 24.8 ± 0.7 and 23.6 ± 1.1 days respectively. The flower meristems in the HbEBP1 OE lines were induced significantly later when compared with those from the wild-type plants (p < 0.05) (Figure 5A).

Late flowering prolonged the vegetative growth period in HbEBP1 OE seedlings, which allowed the plants to grow more rosette leaves. An average of 15 or 16 leaves developed before floral induction for HbEBP1 OE lines (25 days), whereas the number for the Col-0 plants was 12 (22 days) (Figure 5B). We carefully counted rosette leaf number daily, and found that HbEBP1 OE plants developed rosette leaves faster than did the Col-0 plants. As shown in Figure 5B, all the HbEBP1 OE lines grew significantly more leaves than Col-0 from 15 days after germination (p < 0.01). Thus HbEBP1 OE promoted leaf growth in Arabidopsis.

Late flowering is a result of prolonged vegetative growth, in which the juvenile-to-adult and vegetative-to-reproductive phase transitions are delayed (Martínez-Zapater et al., 1995; Poethig, 2003). To examine if the phase transitions were delayed in HbEBP1 OE lines, leaf morphology was investigated at different phases. The leaf developmental stages were determined by observing the onset and the distribution of abaxial trichomes, leaf length and leaf shape (blade length/width ratio) (Telfer et al., 1997). The OE5, OE18 and OE28 lines had a comparable number of juvenile and juvenile-to-adult transition leaves relative to the Col-0 plants (Figure 5C). Both the HbEBP1 OE and wild-type plants developed about four juvenile and two juvenile-to-adult transition leaves. However, the number of leaves that developed during the adult phase was significantly higher for OE5, OE18 and OE28 plants (8.35 ± 1.47, 9.09 ± 1.09, and 9.85 ± 0.97, respectively; p < 0.01), whereas adult leaf number for the Col-0 plants was 4.26 ± 0.73 (Figure 5C). Presumably the increase in the number of adult leaves and their enlarged size (Figures 4A,B) prolonged vegetative growth in the HbEBP1 OE lines.

HbEBP1 transcription was affected by cold and drought stress and ABA treatment, suggesting that this gene may be involved in abiotic resistance in plants. To assess its involvement in drought resistance, the dehydration rates were calculated for detached leaves. The OE18 and OE28 leaves dehydrated significantly slower than did the wild-type leaves from 2 to 8 h. For the OE5 leaves, the dehydration rate was significantly slower from 6 to 8 h (Figure 6A). Thus HbEBP1 OE plants might lose water more slowly than Col-0 plants under drought conditions.

To evaluate drought tolerance in vivo, 2-week-old HbEBP1 OE and Col-0 seedlings were subjected to drought treatment by withdrawing water for 1 week. The HbEBP1 OE5, OE18 and OE28 lines showed a more resistant phenotype as compared with the wild-type plants (Figure 6B). After 7 days of withheld irrigation, all the Col-0 plants were wilted and the rosette leaves had become chlorotic, whereas the HbEBP1 OE lines had leaf blades that green and turgid, although their petioles were purple and displayed some aspects of a drought stressed phenotype (Figure 6B).

Similar results were obtained when HbEBP1 OE plants were subjected to cold stress. Electrolyte leakage analysis was used to evaluate cold resistance. Significantly less leakage was detected in the HbEBP1 OE plants when compared with the Col-0 plants (p < 0.05). In this analysis, the control plants had a leakage rate of 44.5%, whereas the rates were 30.4, 26.9, and 22.6% for OE5, OE18 and OE28 lines, respectively. HbEBP1 OE enhanced resistance to drought and cold stress in Arabidopsis, suggesting that this gene may function as a positive regulator of abiotic stress resistance in rubber trees.

EBP1 regulates organ size by promoting cell cycle transitions during leaf development (Horváth et al., 2006). The expression of the critical cell cycle regulator of the G1 to S and G2 to M transitions, CYCD3;1, was examined by Q-PCR (Dewitte et al., 2007; Blomme et al., 2014; Collins et al., 2015). Two-week-old seedlings were used for the analysis, and gene expression was quantified by comparing the fold changes in expression between the genes of interest and the reference ACTIN7. In the HbEBP1 OE lines, CYCD3;1 expression were significantly increased by 1.7- to 2.1- fold as compared with that in the Col-0 plants (Figure 7A). This effect may explain why HbEBP1 OE led to enlarged organ size in Arabidopsis.

To unravel the mechanism by which HbEBP1 OE enhances drought resistance in Arabidopsis, expression of the drought-responsive genes RD22 and RD29a was examined (Yamaguchi-Shinozaki and Shinozaki, 1993b, 1994; Msanne et al., 2011; Harshavardhan et al., 2014). Leaves were detached from 2-week-old seedlings and subjected to dehydration for 4 h. Then the expression of dehydration-responsive genes was detected. In the seedlings without dehydration, RD22 and RD29a expression was very low in both the Col-0 and HbEBP1 OE lines and showed no significant differences (p > 0.05; Figures 7B,C). After 4 h of dehydration, RD22 and RD29a expression was highly induced in both the wild-type Col-0 and HbEBP1 OE lines, but the HbEBP1 OE lines accumulated higher levels of transcripts than did the wild type plants. For RD22, the expression levels were 3.0-, 3.5- and 3.4- fold for OE5, OE18 and OE28 lines, respectively, when compared with that in the Col-0 seedlings (Figure 7B). For RD29a, expression was increased by 45, 62, and 57% in OE5, OE28 and OE28 lines, respectively, relative to wild type (Figure 7C). The genes CBF1, CBF2 and CBF3, which regulate RD29a (Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997; Jaglo-Ottosen et al., 1998; Liu et al., 1998; Medina et al., 1999), were also tested. Their expression did not differ between the wild-type and OE line seedlings (Supplementary Figure S3). These findings are similar to the reported effects of AmEBP1 (Cao et al., 2008), demonstrating that the HbEBP1 gene regulates drought resistance by other but not CBF pathway.