An F et al. (2015), Regulation of HbPIP2;3, a Latex-Abundant Water ...

XB-ART-50552

PLoS One 2015 Jan 01;104:e0125595. doi: 10.1371/journal.pone.0125595.

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Regulation of HbPIP2;3, a Latex-Abundant Water Transporter, Is Associated with Latex Dilution and Yield in the Rubber Tree (Hevea brasiliensis Muell. Arg.).

An F , Zou Z , Cai X , Wang J , Rookes J , Lin W , Cahill D , Kong L .

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Rubber tree (Hevea brasiliensis) latex, the source of natural rubber, is synthesised in the cytoplasm of laticifers. Efficient water inflow into laticifers is crucial for latex flow and production since it is the determinant of the total solid content of latex and its fluidity after tapping. As the mature laticifer vessel rings are devoid of plasmodesmata, water exchange between laticifers and surrounding cells is believed to be governed by plasma membrane intrinsic proteins (PIPs). To identify the most important PIP aquaporin in the water balance of laticifers, the transcriptional profiles of ten-latex-expressed PIPs were analysed. One of the most abundant transcripts, designated HbPIP2;3, was characterised in this study. When tested in Xenopus laevis oocytes HbPIP2;3 showed a high efficiency in increasing plasmalemma water conductance. Expression analysis indicated that the HbPIP2;3 gene was preferentially expressed in latex, and the transcripts were up-regulated by both wounding and exogenously applied Ethrel (a commonly-used ethylene releaser). Although regular tapping up-regulated the expression of HbPIP2;3 during the first few tappings of the virginal rubber trees, the transcriptional kinetics of HbPIP2;3 to Ethrel stimulation in the regularly tapped tree exhibited a similar pattern to that of the previously reported HbPIP2;1 in the virginal rubber trees. Furthermore, the mRNA level of HbPIP2;3 was associated with clonal yield potential and the Ethrel stimulation response. Together, these results have revealed the central regulatory role of HbPIP2;3 in laticifer water balance and ethylene stimulation of latex production in Hevea.

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Species referenced: Xenopus laevis
Genes referenced: slc12a3 tbx2

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	Fig 2. Multiple sequence alignment of HbPIP2;3 with aquaporins reported in rubber tree.Sequences were aligned using ClustalX2 [47]. Predicted transmembrane helices (TM1-TM6) and the two short helices forming NPAs (HB and HE) are shaded and labeled with black lines above the alignment. The two conserved NPA motifs are shown in bold letters. Residues comprising the ar/R filter are underlined and labeled H2, H5, LE1 and LE2. P1–P5 residues from N- to C-terminus are indicated by italic.
	Fig 3. Phylogenetic analysis of HbPIP2;3 with other aquaporins.Predicted amino acid sequences were aligned using ClustalX2 [47] and the phylogenetic tree was constructed using bootstrap neighbour-joining tree (1000 replicates) method and MEGA4 software [48]. The distance scale denotes the number of amino acid substitutions per site. The similarity of each protein with HbPIP2;3 is shown on the right.
	Fig 4. Water channel activity of HbPIP2;3.(A) Time-dependent cell volume changes and, (B) water permeability (Pf) values of Xenopus oocytes expressing rubber tree aquaporins. The oocytes were injected with 50 ng HbPIP2;3 and HbPIP2;1 (positive control) cRNA solution or RNase-free water (negative controls) were incubated in full strength MBS for 24 h and then transferred into a 5-fold diluted MBS at time 0 for cell volume measurements. The cell volume change was recorded every 20 s for up to 200 s or until the cell burst. The oocytes for the HgCl2 inhibition group were subjected to treatment with MBS containing 0.3 mM HgCl2 10 min before the swelling assay. The Pf data are given as mean ± SD (n = 10) from two independent experiments. Bars with different letters are significantly different (Duncan one-way ANOVA, P<0.05).
	Fig 5. Relative transcript abundance of HbPIP2;1, HbPIP2;3 and HbPIP2;7 aquaporins in rubber tree latex.The relative gene expression is the ratio of each aquaporin transcript to the HbYLS8 house-keeping gene using 2Δt method. The PR107 rubber trees regularly tapped for three years without Ethrel stimulation were used as the plant materials. The Control rubber trees were brushed with only 1g of 1% CMC, while the 24h rubber trees were brushed with 1g of 2.5% (w/w) Ethrel in 1% CMC, at just above the tapping cut 24 hour before sampling. Different letters indicate a significant difference (P<0.05) in gene transcript expression between control and Ethrel-treated trees.
	Fig 6. Expression of HbPIP2;3 in different rubber tree tissues.The relative gene expression was calculated by using HbYLS8 as the house-keeping gene and bark as the normalization sample. The tissues were obtained from tissue cultured CATAS7-33-97 rubber tree saplings (about eight-month-old). All quantitative RT-PCR assays were conducted in triplicate.
	Fig 7. Effect of consecutive tappings on latex yield and HbPIP2;3 expression.(A) Latex yield and total solid content (TSC), (B) HbPIP2;3 transcript variation of virginal CATAS7-33-97 rubber trees subjected to six consecutive tappings. Data are means from three different trees and SD (n = 3). Different letters signify statistical differences between means at P<0.05.
	Fig 8. Comparison of wounding and Ethrel stimulation on latex yield and HbPIP2;3 expression.(A) Change in latex yield and total solid content (TSC) and, (B) HbPIP2;3 transcript expression in the virgin rubber trees (Control) and rubber trees subjected to drawing pin wounding (DP) and Ethrel (ET) treatments. The rubber clone was CATAS7-33-97. Data are given as means ± SD (n = 3) from three trees. Different letters indicate significant differences between means (P<0.05).
	Fig 9. Kinetics of latex yield (A) and TSC (B) subjected to different duration of Ethrel treatment.The regularly tapped rubber clones with different responses to Ethrel stimulation were compared. The yield of PR107 responds well to Ethrel stimulation whereas CATAS8-79 rubber clone displays a relatively poor response to Ethrel stimulation. Data are means ± SD (n = 3) for each including three trees. Different letters represent latex yield or TSCs that are significantly different (P<0.05).
	Fig 10. Variations of HbPIP2;3 transcripts following Ethrel stimulation.The PR107 and CATAS8-79 rubber trees regularly tapped for three years were used as the plant materials. The reference gene was HbYLS8 and the relative expressions of HbPIP2;3 in the latex and bark samples of both PR107 and CATAS8-79 rubber clones were calibrated respectively by using the PR107 latex and bark samples without Ethrel stimulation (i.e. 0 hour after treatment) as the control sample. Data are means ± SD (n = 3). Different letters indicate that the transcript expression of HbPIP2;3 was significantly different (P<0.05) among various treatment durations in the same clone.
	Fig 1. The transcriptional profile of ten PIP genes identified in the rubber tree latex.The expression analysis was based on the Solexa sequencing data of RRIM928 latex transcriptome downloaded from NCBI (SRA accession number SRX278514). RPKM refers to reads per kilobase per million (see details in methods). HbPIP1;1 and HbPIP2;1 were previously reported by Tungngoen et al. [23, 28], whereas HbPIP1;4 and HbPIP2;7 were reported as HbPIP1 and HbPIP2 by Zhuang et al. [31], respectively.

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