Subcellular identification of HbSUT5
The H. brasiliensis SUT (HbSUT) gene, HbSUT5, was cloned previously [19, 21], but has not been further characterized. HbSUT5 contained a 1497-bp-long ORF that predicted a protein of 498 amino acids with theoretical molecular weight of 54.1 kDa and pI of 9.39. Phylogenetic analysis (Fig. 1A) revealed that HbSUT5, together with another Hevea tree SUT, HbSUT4, belongs to SUT4 clade SUTs. As predicted by the method of TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) , HbSUT5 consists of 12 transmembrane spans, short cytoplasmic N- and C-terminals, and a short central cytoplasmic loop between transmembrane spans 6 and 7, the characters of which are typical of the SUT4 clade SUT members (Fig. S1) . To further verify the subcellular locations of HbSUT5, the HbSUT5 protein fused with GFP (green fluorescent protein) was transiently expressed in rice protoplasts together the known rice tonoplast intrinsic protein OsTIP1;1 fused with RFP (red fluorescent protein) . The GFP signals overlapped completely with the RFP signals (Figs. 1B-E), indicating that HbSUT5 shares a similar subcellular location with the well-characterized tonoplast protein OsTIP1;1. High-stringency DNA gel blot analysis showed that there are one to three hybridization bands under different restriction analyses, indicating HbSUT5 to be a single or low copy gene in the Hevea genome (Fig. 1F), the results of which has been further verified by our high-quality draft genome of the rubber tree .
Functional analysis of HbSUT5 in baker’s yeast
To test whether HbSUT5 has Suc transport activity, assays with baker’s yeast cells expressing the HbSUT5 cDNA were performed using radiolabeled 14C-Suc. Suc transport into yeast cells expressing the HbSUT5 was found to be nearly linear within the assayed time course (2 to 10 min), reaching approximately 13-fold higher than that of the control yeast harboring the empty vector pDR196 after 10 min (Fig. 2A). These results demonstrated that HbSUT5 encodes a functional Suc transporter.
Suc transport kinetics analysis displayed that the transport activity of HbSUT5 was obviously dependent on the pH of the buffer solution, showing the highest at pH around 5.0 but decreasing progressively with the elevation of buffer pH (Fig. 2B). The Suc affinities of HbSUT5 were measured at pH 5.5 under a Suc substrate range of 0.5-8 mM, and the data were used to perform the Michaelis-Menten analysis (Fig. 2C). The Eadie-Hofstee double-reciprocal plots of the data (Fig. 2D) revealed that the estimated Km was 2.03 mM with a transport rate Vmax of 0.522 Suc min-1 (108 cells)-1, suggesting HbSUT5 belongs to a high-affinity/low-capacity (HALC) Suc transporter .
The substrate specificity of HbSUT5 was detected with several unlabeled sugars as 14C-Suc competitors, including galactose, fructose, inositol, lactose, Suc, maltose and raffinose. The results showed that only Suc and maltose could significantly compete at similar extents with the 14C-Suc uptake (Table 1), indicating the relative substrate specificity of HbSUT5 in transporting sugars. In addition, three sugars, i.e., fructose, inositol and lactose revealed to stimulate Suc uptake somewhat. Further, Suc transport by HbSUT5 was strongly inhibited by the protonophores of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP) (Table 1). This result together with the character of pH-dependent Suc transport ability (Fig. 2B) clearly indicated that HbSUT5 was a H+-Suc symporter.
In planta expression patterns of HbSUT5
As reported previously, among the six HbSUT genes, HbSUT5 is abundantly expressed in the latex, only inferior to HbSUT3, and shows much higher expression than that of the other SUT4-clade SUT gene, HbSUT4 . Besides being an abundant SUT isoform in the latex, HbSUT5 was the predominant isoform in the bark (Fig. 3A), predicting the multifunctional roles of this gene. The transcript abundance of HbSUT5 was then compared by QPCR (quantitative polymerase chain reaction) in five Hevea tissues, including leaf, latex, seed, flower and bark. As shown in Fig. 3B, HbSUT5 was most highly expressed in the bark and seed, followed by the latex and leaf, and the lowest in the flower.
Increased Suc loading to the laticifers is one of the most important mechanisms responsible for ethylene-stimulated latex production [19, 41, 42]. To further investigate the functions of HbSUT5, its expression patterns were analyzed in the latex and bark tissues by QPCR after ethephon (2-chloroethylphosponic acid, an ethylene releaser) bark treatment. HbSUT5 expression was significantly down-regulated both in the latex and bark tissues, attaining at 24h post treatment about 40% of its initial level in the latex and 15% in the bark (Fig. 4A). To further probe the roles of decreased HbSUT5 expression under the ethephon treatment, the Suc contents were examined in latex cytosol (C-serum) and lutoids (B-serum). The results showed that the C-serum Suc was decreased significantly by the treatment, but the B-serum Suc was much less affected, suggesting a function of the lowered HbSUT5 expression in reducing Suc export from lutoids (Fig. 4B). The tapping of virgin (never tapped before) Hevea trees is an ideal model to identify and characterize latex regeneration-related genes due to the conspicuous stimulating effect of tapping on latex production for the first few tappings [19, 43]. As shown in Fig. 3C, the expressions of HbSUT5 in the latex markedly depressed with the first four consecutive tappings, reaching less than 4% of its initial level, and maintaining low levels thereafter, the pattern of which correlates negatively with the conspicuous increase in latex yield with the tappings . Bark wounding that stimulates latex production  was also found to decrease HbSUT5 expression (Fig. 3D), reaching the lowest level at 24 h after treatment. The decreased HbSUT5 expression in the latex by tapping was hypothesized to function in a similar mechanism as it did under the ethephon treatment.
Characterization of the HbSUT5 promoter
To explore the transcriptional characters of HbSUT5, its putative promoter sequence of about 1.5-kb-long genomic sequence upstream from its start codon (GenBank: KU529197)  was characterized by in-silico analysis and heterogeneous transient expression. As revealed by the NNPP (neural network promoter prediction) promoter predictor (http://www.fruitfly.org/seq_tools/promoter.html), this sequence contained a high score (0.99) transcription promoter with a putative transcriptional start site (marked as C+1) located 342 bp upstream of the start codon (ATG) (Fig. 5A). The PLACE (plant cis-acting regulatory DNA elements) software (http://www.dna.affrc.go.jp/PLACE/) was then used to analyze the putative cis-acting elements harbored by the promoter. A total of 54 distinct cis-regulatory elements were predicted, many of which are implicated in multiple biological processes, including stress and hormone responses, signaling pathways, and tissue-, organ- or metabolism-specific expressions (Fig. 5A; Table S1). The harboring of multiple minimal elements such as four TATA box and 21 CAAT box motifs suggested its capability to initiate accurate basal transcription in most plant species. Experimentally, its promoter activity was confirmed by the transient promoter expression analysis, which revealed a strong GFP fluorescence in tobacco leaf protoplasts transfected with the GFP expression cassette driven by the putative HbSUT5 promoter (pSUT5-GFP) (Fig. 5B).
It is noteworthy that three classical ethylene-responsive elements (EREs) were predicted in the HbSUT5 promoter, with one (-785) on its sense orientation, and the other two (-767 and -327) on its antisense orientation. Besides that, there were 9 cis-acting elements with more than ten locations in the HbSUT5 promoter, among which were those involved in dehydration response (MYB (myeloblastosis) and MYC (myelocytomatosis) recognition sites) [45, 46], tissue- or organ-specific expression (E-Box "CANNTG", POLLEN1 element “AGAAA” and tetranucleotide "YACT") [46-48], overall expression activation (CT-rich motif "TCTCTCTCT", GATA-Box and ARR1 recognition site "NGATT") [50-52], and sugar metabolism (DOF core recognition site " AAAG").