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. 2023 Feb;113(3):504-520.
doi: 10.1111/tpj.16063. Epub 2023 Jan 9.

Downregulation of HbFPS1 affects rubber biosynthesis of Hevea brasiliensis suffering from tapping panel dryness

Zhiyi Nie  1   2 , Guijuan Kang  1   2 , Dong Yan  1 , Huaide Qin  1   2 , Lifu Yang  3 , Rizhong Zeng  1   2
Affiliations

Downregulation of HbFPS1 affects rubber biosynthesis of Hevea brasiliensis suffering from tapping panel dryness

Zhiyi Nie et al. Plant J. 2023 Feb.

Abstract

Tapping panel dryness (TPD) is a century-old problem that has plagued the natural rubber production of Hevea brasiliensis. TPD may result from self-protective mechanisms of H. brasiliensis in response to stresses such as excessive hormone stimulation and mechanical wounding (bark tapping). It has been hypothesized that TPD impairs rubber biosynthesis; however, the underlying mechanisms remain poorly understood. In the present study, we firstly verified that TPD-affected rubber trees exhibited lower rubber biosynthesis activity and greater rubber molecular weight compared to healthy rubber trees. We then demonstrated that HbFPS1, a key gene of rubber biosynthesis, and its expression products were downregulated in the latex of TPD-affected rubber trees, as revealed by transcriptome sequencing and iTRAQ-based proteome analysis. We further discovered that the farnesyl diphosphate synthase HbFPS1 could be recruited to small rubber particles by HbSRPP1 through protein-protein interactions to catalyze farnesyl diphosphate (FPP) synthesis and facilitate rubber biosynthesis initiation. FPP content in the latex of TPD-affected rubber trees was significantly decreased with the downregulation of HbFPS1, ultimately resulting in abnormal development of rubber particles, decreased rubber biosynthesis activity, and increased rubber molecular weight. Upstream regulator assays indicated that a novel regulator, MYB2-like, may be an important regulator of downregulation of HbFPS1 in the latex of TPD-affected rubber trees. Our findings not only provide new directions for studying the molecular events involved in rubber biosynthesis and TPD syndrome and contribute to rubber management strategies, but also broaden our knowledge of plant isoprenoid metabolism and its regulatory networks.

Keywords: Hevea brasiliensis; MYB transcription factor; farnesyl diphosphate synthase; iTRAQ; protein-protein interaction; rubber biosynthesis; tapping panel dryness; transcriptome sequencing.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Latex yield and physiological parameters of healthy and TPD‐affected rubber trees. (a) Latex yield. (b) Sucrose content. (c) Pi content. (d) Mg2+ content. (e) RSH content. (f) Average RP size. (g) Percentage of SRPs (VMD < 0.3 μm). (h) Percentage of LRPs (VMD > 1.0 μm). (i) Rubber biosynthesis activity of latex. (j) Rubber average molecular weight. One‐way analysis of variance and the Student–Newman–Keuls test were performed to identify significant differences between groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 12. TPD, tapping panel dryness; RP, rubber particle; SRPs, small rubber particles; LRPs, large rubber particles; VMD, volume mean diameter; APC13C, atom percentage of 13C.
Figure 2
Figure 2
KEGG pathway analysis of DAPs and DEGs and expression profiles of rubber biosynthesis‐related DAPs and DEGs. KEGG pathway annotations for DAPs and DEGs in comparisons of (a) T2_H and (b) T4_H. The numbers of DAPs (left) and DEGs (right) associated with a pathway are indicated. (c) KEGG enrichment‐based cluster analysis of DAPs and DEGs (P < 0.05). (d) Expression profiles of DAPs and DEGs involved in rubber biosynthesis. DAPs, differentially abundant proteins; DEGs, differentially expressed genes; MVA, mevalonate; A‐CoA, acetyl‐CoA; ACAT, acetyl‐CoA C‐acetyltransferase; AA‐CoA, acetoacetyl‐CoA; HMGS, HMG‐CoA synthase; HMG‐CoA, 3‐hydroxy‐3‐methyl‐glutaryl‐CoA; HMGR, HMG‐CoA reductase; MK, mevalonate kinase; MevP, mevalonate‐5‐phosphate; PMK, MevP kinase; MevPP, mevalonate‐5‐pyrophosphate; MDC, mevalonate‐5‐pyrophosphate decarboxylase; G3P, glyceraldehyde‐3‐phosphate; DXS, DXP synthase; DXP, 1‐deoxy‐d‐xylulose‐5‐phosphate; DXR, DXP reductoisomerase; MEP, 2‐C‐methyl‐d‐erythritol‐4‐phosphate; MCT, CDP‐ME synthase; CDP‐ME, 4‐(cytidine‐5′‐diphospho)‐2‐C‐methyl‐d‐erythritol; CMK, CDP‐ME kinase; CDP‐MEP, 2‐phospho‐4‐(cytidine‐5′‐diphospho)‐2‐C‐methyl‐d‐erythritol; MDS, ME‐cPP synthase; ME‐cPP, 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate; HDS, HMBPP synthase; HMBPP, 4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate; HDR, HMBPP reductase; IPP, isopentenyl pyrophosphate; IDI, IPP isomerase; DMAPP, dimethylallyl pyrophosphate; GPS, geranyl pyrophosphate synthase; GPP, geranyl pyrophosphate; FPS, farnesyl pyrophosphate synthase; FPP, farnesyl pyrophosphate; GGPS, GGPP synthase; GGPP, geranylgeranyl pyrophosphate; CPT, cis‐prenyltransferase; REF, rubber elongation factor; SRPP, small rubber particle protein.
Figure 3
Figure 3
Relative gene expression and protein abundance of HbFPS1, FPS content, and relative FPP content in the latex of healthy and TPD‐affected trees. (a) Relative gene expression of HbFPS1. (b) Relative protein abundance of HbFPS1. Data were derived from the RNA‐seq transcriptome data FPKM values (*FDR < 0.05, n = 3) and the fold change values from the iTRAQ‐based proteome data (Student's t‐test, *P < 0.05, n = 3), respectively. (c) FPS content and (d) relative FPP content. One‐way analysis of variance and the Student–Newman–Keuls test were performed to identify significant differences between groups. *P < 0.05, **P < 0.01, ****P < 0.0001, n = 6–12. FPS, farnesyl pyrophosphate synthase; FPP, farnesyl pyrophosphate; TPD, tapping panel dryness.
Figure 4
Figure 4
Effects of FPS and FPP concentrations on rubber biosynthesis efficiency and molecular weight in vitro. The effects of FPS and FPP concentrations on rubber biosynthesis efficiency and molecular weight were assessed using WRPs as cofactor in (a) and (c) and latex as cofactor in (b) and (d). The symbols + and − indicate differences in reaction buffer components. Data are presented as means ± SE. One‐way analysis of variance and the Student–Newman–Keuls test were performed to identify significant differences between groups. Different letters indicate significant differences (P < 0.05, n = 3). FPS, farnesyl pyrophosphate synthase; FPP, farnesyl pyrophosphate; WRPs, washed rubber particles; APC13C, atom percentage of 13C.
Figure 5
Figure 5
Rubber biosynthesis activity and FPS and HbSRPP1 abundance in RPs of different sizes. (a) Rubber biosynthesis activity of the RPs with different sizes. (b) FPS abundance in the RPs with different sizes. (c) SDS‐PAGE profiles of different size RPs. (d) Western blot analyses with monoclonal antibodies raised against HbSRPP1. The HbSRPP1 protein is indicated by the arrow. RP1, RPs with VMD < 0.15 μm; RP2, RPs with VMD about 0.3 μm; RP3, RPs with VMD about 0.5 μm; RP4, RPs with VMD about 0.7 μm; RP5, RPs with VMD > 1.0 μm. Control, the reaction was performed in 400 μl normal reaction buffer containing 30 μl resuspended RPs with 25 mm EDTA added for immediate reaction termination. M, molecular markers. Data are presented as means ± SE. One‐way analysis of variance and the Student–Newman–Keuls test were performed to identify significant differences between groups. Different letters indicate significant differences (P < 0.05, n = 3). FPS, farnesyl pyrophosphate synthase; RPs, rubber particles; VMD, volume mean diameter; APC13C, atom percentage of 13C.
Figure 6
Figure 6
Subcellular localization of HbFPS1 and HbSRPP1 in N. benthamiana leaf epidermal cells. (a–c) HbSRPP1‐GFP is shown in green and OsSPP1‐RFP is shown in pink. (d–f) HbFPS1‐GFP is shown in green and OsSPP1‐RFP is shown in pink. (g–i) HbFPS1‐GFP is shown in green and chlorophyll autofluorescence is shown in red. (j–l) HbFPS1‐GFP is shown in green and nuclei stained with DAPI are shown in blue. Scale bars: 25 μm.
Figure 7
Figure 7
Co‐expression and physical interaction analyses of HbFPS1 with HbSRPP1. (a) Co‐expression analyses of HbFPS1 with HbSRPP1. HbSRPP1‐GFP is shown in green and HbSRPP1‐RFP is shown in pink. The nuclear envelope and the ER are indicated by yellow and red arrows, respectively. IV–VI represent magnified images of the areas identified by the squares in I–III. Scale bars: 25 μm. (b) Physical interaction analyses between HbREF1, HbSRPP1, and HbFPS1 by individual Y2H assays. (I) SD/−Leu−Trp. (II) QDO. (III) QDO/X‐α‐gal. (c) HbFPS1–HbSRPP1 interaction analysis by BiFC assay. The bZIP63 transcription factor was used as a positive control. The empty pairs (n‐YFP and c‐YFP) were used as negative controls. YFP fluorescence was detected under a confocal laser microscope. Scale bar = 25 μm. (d) Possible model for RP biogenesis and protein‐protein interacting pattern among the rubber biosynthesis‐related proteins. QDO, quadruple dropout plates (SD/−Ade−His−Leu−Trp); RP, rubber particle; ER, endoplasmic reticulum; SRP, small rubber particle; CPT, cis‐prenyltransferase; SRPP, small rubber particle protein; FPS, farnesyl pyrophosphate synthase; FPP, farnesyl pyrophosphate; IPP, isopentenyl pyrophosphate; NR, natural rubber.
Figure 8
Figure 8
Validation of HbFPS1 transcription regulators. (a) Activation of the HbFPS1 promoter in yeast by the HbEREBP1 and MYB2‐like proteins. (1) SD/−Leu/−Trp. (2) TDO/80 mm 3‐AT. (3) TDO/120 mm 3‐AT. (4) TDO/160 mm 3‐AT. (b) HbFPS1, HbEREBP1, and MYB2‐like FPKM values in the latex of TPD‐affected and healthy trees. *FDR < 0.05. (c) Physical binding of HbEREBP1 (1) and MYB2‐like (2) to the HbFPS1 promoter was shown by electrophoretic mobility shift assays. The symbols + and − indicate differences in reaction buffer components. (d) Schematic representation of the vector structure for the Dual‐LUC assays. (e) Transient transcriptional activity assays of HbEREBP1 and MYB2‐like on the HbFPS1 promoter. Data are presented as means ± SE. One‐way analysis of variance and the Student–Newman–Keuls test were performed to identify significant differences between groups. Different letters indicate significant differences (P < 0.05, n = 6). TDO, triple dropout plates (SD/−His−Leu−Trp); TPD, tapping panel dryness.
Figure 9
Figure 9
Relative expression of HbFPS1 and the transcription factor‐encoding genes MYB2‐like and HbEREBP1 in (a) different rubber tree tissues, (b) the latex of TPD‐affected and healthy rubber trees, and (c) the barks of TPD‐affected and healthy rubber trees. Data are presented as means ± SE. One‐way analysis of variance and the Student–Newman–Keuls test were performed to identify significant differences between groups. *P < 0.05, **P < 0.01, ***P < 0.001, n = 3. TPD, tapping panel dryness.
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