Functional categories with reference to tapping-stimulated latex regeneration
Tapping can stimulate the regeneration of latex, especially in virgin Hevea trees [12, 20]. A number of early studies have shown that the first few tappings greatly stimulate the metabolism of laticifers, accompanied by the enhanced expression of several specific genes involved in latex regeneration [18, 21, 29-30]. The latex flows out of laticifers after tapping, and in order to compensate for the loss of cytoplasm (latex) and maintain the balance of intracellular metabolism, the laticifers require large amounts of RNA and proteins to be synthesized before the next tapping. Of the 366 DE-TDFs identified with known function (Table 2), 26.2% were classified into the functional category of transcription and protein synthesis (Fig. 2), representing the largest category, 42.7% of which were up-regulated by the tapping treatment. These results indicated that tapping significantly affects the ways of laticifers to synthesize RNA and proteins, thus laying a foundation for their subsequent physiological response to the tapping treatment . Laticifers are believed to be a defense system for Hevea trees to cope with biological and abiotic stresses, and the latex exuded after bark wounding has been found to play roles in resisting pathogen infection, insect feeding and abiotic stress . The tapping itself is a kind of abiotic stress upon Hevea trees. Therefore, it is reasonable that “stress and defense” also accounted for a large portion of the functional DE-TDFs identified responsive to tapping (Table 2; Fig. 2), ranking the second place in functional categories. The harvesting stress response has been suggested to be one of the key factors affecting latex regeneration and rubber productivity in Hevea trees .
The category of transporters and intracellular was the third largest among the 11 functional categories, accounting for 12.3% of the total functional DE-TDFs (Fig. 2). This corresponds well to the sink effect caused by the large loss of latex after tapping. The process of regenerating the expelled latex involves the synthesis, transport, loading and subcellular localization of a large number of organelles, proteins, nucleic acids, sugars, etc., all of which require the active involvement of transporters and intracellular transport-related proteins [3, 33]. DE-TDFs involved in signal transduction were also highly represented, accounting for a proportion of 11.5% for the total DE-TDFs (Fig. 2). A variety of signaling pathways within Hevea laticifers, including ethylene, jasmonic acid and wound signaling, have been reported to be extensively participate in latex regeneration and regulation [4, 17, 29, 32, 34-35]. The proportions for the two categories, protein degradation and storage and primary metabolism were also high, covering, respectively, 9.3% and 8.2% of the total functional DE-TDFs. Their high representation suggested that with the progress of tapping, in order to meet the balance of supply and demand of all substances in latex regeneration, protein turnover rate becomes faster and primary metabolism gets active. In a word, these results indicated that the latex regeneration regulated by tapping involves a complex multi-gene regulatory network, as well as a physiological and biochemical response process.
Sugar metabolism and rubber biosynthesis in tapping-stimulated latex production
In regularly tapped Hevea trees, the main metabolic activity of the laticifers is latex regeneration, which centers on the biosynthesis of rubber hydrocarbon that accounts for about 90% of the dry weight of fresh latex . Sucrose has been identified as the precursor material for rubber biosynthesis in laticifers, providing the carbon skeleton and energy required for latex regeneration [11, 36]. In Hevea trees normally tapped at intervals of 2-4 days, each tree produces dozens to hundreds of milliliters of fresh latex, and the removed latex could be effectively recovered before the next tapping to ensure the sustained productivity of the tree [3, 12]. Therefore, the laticifers are an active carbon sink, and the effective supply of sucrose is a key factor to determine the yield of latex [10, 37]. In this study, the genes of a sucrose transporter and a sugar transporter were among the DE-TDFs identified, both of which were significantly up-regulated with the increase of tappings (Table S1). Interestingly, the former is just the sucrose transporter HbSUT3 we previously identified to be critical in sucrose uptake into laticifers and rubber production in exploited Hevea trees . The up-regulation of these two transporters indicated an active involvement of sucrose and sugar transport in tapping-stimulated latex regeneration. Sucrose catabolism and the following pathways of glycolysis, tricarboxylic acid cycle and pentose phosphate provide essential components, i.e. the carbon skeleton (acetyl CoA), the reducing power (NADPH) and the energy (ATP) for the final rubber biosynthesis pathway [3, 11]. Therefore, sugar metabolism becomes one of the core metabolic pathways contributing to latex regeneration and rubber biosynthesis in Hevea [3, 11, 38, 40-41]. This study identified multiple DE-TDFs involved in sucrose cleavage and the three above mentioned sugar metabolism pathways (Table 4; Tables S1 to 3), including those encoding neutral/alkaline invertase, fructokinase, phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase, pyruvate dehydrogenase, and glucose-6-phosphate dehydrogenase, etc. Most of them were up-regulated in the latex for the first few tappings (Table S1; Fig. 3). It is worth noting that the up-regulated DE-TDF (M16-A7-1) as identified by both cDNA-AFLP (Table S1) and qRT-PCR (Fig. 3) turned out to be HbNIN2, the neutral/alkaline invertase that is responsible for sucrose catabolism in Hevea laticifers .
There are 20 gene families directly involved in the rubber hydrocarbon biosynthesis and termed as rubber biosynthesis genes [17, 31]. The DE-TDFs identified in this study involved six of these families, including cis-prenyltransferase, hydroxymethylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, farnesyl diphosphate synthase, rubber elongation factor and small rubber particle protein (Table 4). Among the nine DE-TDFs, eight were demonstrated by qRT-PCR to be up-regulated with the tappings (Fig. 3). In addition, a DE-TDF (M8-A5-6) annotated as inorganic pyrophosphatase was also bolstered by the tapping treatment (Fig. 3). A vacuolar type of inorganic pyrophosphatase has been found to locate on rubber particles and essential for the incorporation of IPP monomers into elongating rubber molecules .
Strength and weakness of the cDNA -AFLP technique
The cDNA-AFLP technique has been widely applied in various eukaryotes including the Hevea tree for transcript profiling due to its advantages of stringency, reproducibility, cost-effectiveness, genome-wide coverage and the ability to distinguish among highly homologous genes [24, 39, 41-43]. In this study, as determined by sqRT-PCR and qRT-PCR, about 84% and 90%, respectively, of the selected DE-TDFs were verified for their cDNA-AFLP profiles (Tables 3 & 4; Fig. 3), reflecting a high reliability of this technique in screening tapping-responsive DE-TDFs in Hevea latex. According to a previous in-silico estimation , about 84% of the genes expressed in Hevea latex could be visualized using the silver-staining cDNA-AFLP technique with the restriction enzyme pair of Apo I and Mse I exploited here. The sucrose transporter HbSUT3  and the neutral/alkaline invertase HbNIN2  that have been reported to be up-regulated in the latex of virgin Hevea trees by the tapping treatment were among the DE-TDFs identified in this study (Table 4; Fig. 3; Table S1), demonstrating a high transcript coverage of this technique. However, compared with the currently popularly used next generation RNA-sequencing technique that relies on expensive DNA sequencers and specialized bioinformatics , the cDNA-AFLP is labor-intensive. Nevertheless, the cDNA-AFLP technique still have its niche among the various transcript profiling techniques, and can be readily established in a mediocrely equipped and stringently funded lab to fulfill its customized transcript profiling task.