Elsevier

Plant Physiology and Biochemistry

Volume 111, February 2017, Pages 97-106
Plant Physiology and Biochemistry

Research article
Identification and subcellular localization analysis of two rubber elongation factor isoforms on Hevea brasiliensis rubber particles

https://doi.org/10.1016/j.plaphy.2016.11.006Get rights and content

Highlights

  • Sequential extraction of rubber particle proteins resulted in enrichment of 23.6 and 27.3 kDa rubber elongation factors.

  • The 23.6 and the 27.3 kDa rubber elongation factors are highly similar in amino acid sequences.

  • Subcellular localization results may be helpful to elucidate the function of rubber elongation factors.

  • We suggested that REF/SRPP isoforms are structural proteins in rubber particles of H. brasiliensis.

Abstract

Rubber elongation factor (REF) is the most abundant protein found on the rubber particles or latex from Hevea brasiliensis (the Para rubber tree) and is considered to play important roles in natural rubber (cis-polyisoprene) biosynthesis. 16 BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE separations and mass spectrometric identification had revealed that two REF isoforms shared similar amino acid sequences and common C-terminal sequences. In this study, the gene sequences encoding these two REF isoforms (one is 23.6 kDa in size with 222 amino acid residues and the other is 27.3 kDa in size with 258 amino acid residues) were obtained. Their proteins were relatively enriched by sequential extraction of the rubber particle proteins and separated by 16 BAC/SDS-PAGE. The localization of these isoforms on the surfaces of rubber particles was further verified by western blotting and immunogold electron microscopy, which demonstrated that these two REF isoforms are mainly located on the surfaces of larger rubber particles and that they bind more tightly to rubber particles than the most abundant REF and SRPP (small rubber particle protein).

Introduction

Rubber elongation factor (REF, 138 amino acid residues, 14.6 kDa) and small rubber particle protein (SRPP, 204 amino acid residues, 22.4 kDa) are the most important proteins in Hevea brasiliensis (the Para rubber trees) latex and have attracted the attention of researchers because of their allergenicity in natural rubber products and their high contents in rubber latex. REF and SRPP were first detected as natural latex allergens in natural rubber products and named Hev b 1 and Hev b 3, respectively (Posch et al., 1997). These two proteins account for more than 75% of the proteins found on rubber particles (Dai et al., 2012), which are laticifer-specific organelles accounting for 30%–45% of the latex volume and more than 90% of the latex dry weight in rubber trees (Jacob et al., 1993).

In rubber trees, REF and SRPP are thought to play positive roles in natural rubber biosynthesis (Light et al., 1989). However, the exact functions of these two proteins are unclear. For example, Light et al. (1989) suggested that REF helps a “rubber transferase” in the rubber latex to switch between cis-prenyl transferase and trans-prenyl transferase activities, but Cornish (1993) refuted this.

The amino acid sequences for REF and SRPP share 72% similarity (Oh et al., 1999). Many REF and SRPP analogues in rubber trees have been reported. Rahman et al. (2013) found 10 REF genes and 12 SRPP genes in the genome of rubber tree, Tang et al. (2016) recently characterized 8 and 10, and Lau et al. (2016) found 9 and 8, respectively. They constitute the REF superfamily and are usually divided into REF or SRPP subfamilies (Chow et al., 2007, Rahman et al., 2013). However, when more REF/SRPP protein isoforms in H. brasiliensis were found through genome sequencing and analyzed phylogenetically, no distinct REF and SRPP subfamilies were found (Tang et al., 2016).

Laibach et al. (2015) found that TbREF, a HbREF analogue, contained two conserved REF domains in Taraxacum brevicorniculatum, and that TbREF-silenced plants showed lower TbCPT protein levels and less TbCPT activity in the latex, and therefore lower rubber yielding, but the molecular weight of rubber and colloidal stability of rubber particles was not affected. According to these results, Laibach et al. (2015) suggested that TbREF may play a role in rubber particle biogenesis and affect the biosynthesis of natural rubber. SRPP analogues have been found in other plant species, including non-rubber-producing plants, such as Arabidopsis (Kim et al., 2016) and Capsicum annuum (Kim et al., 2010), and rubber-producing plants, such as guayule (Parthenium argentatum Gray) (Kim et al., 2004), Russian dandelion (Taraxacum kok-saghyz) (Schmidt et al., 2010), and lettuce (Lactuca sativa) (Chakrabarty et al., 2015). Previous investigations demonstrated that SRPP homologues in Arabidopsis (Kim et al., 2016) and in Capsicum annuum (Kim et al., 2010) were associated with tissue growth and development, and with drought stress responses, and that an SRPP homolog in guayule can enhance the biosynthesis of natural rubber (Kim et al., 2004).

In addition to the stress-related functions associated with REF superfamily proteins, the relationship between the proteins and the integrity of rubber particles has been noticed by several researchers. Dai et al. (2012) suggested the REF superfamily proteins might act as structural proteins in rubber particles in a way similar to that oleosin does in lipid droplets. Hillebrand et al. (2012) found that in T. brevicorniculatum, down-regulation of SRPP homologues affected rubber particle integrity and consequently rubber biosynthesis. However, Chakrabarty et al. (2015) found that silencing the lettuce (Lactuca sativa) SRPP homologues did not interfere with natural rubber biosynthesis. Biophysical studies on the interactions between REF/SRPP and lipid monolayers were carried out (Berthelot et al., 2014a, Berthelot et al., 2014b). Interestingly, lipid-droplet associated proteins (Gidda et al., 2013, Horn et al., 2013), which are SRPP homologues, act as structural proteins in avocado mesocarp lipid droplets, whereas oleosins are structural proteins in plant seed lipid droplets.

Immunogold electron microscopy has shown that REF and SRPP locate on the surface of rubber particles (Shamsul Bahri and Hamzah, 1996), but there have been no similar studies to show the localization of other REF/SRPP analogues in H. brasiliensis. In our previous study, 16 BAC/SDS-PAGE separations and mass spectrometry revealed two REF isoforms that had not been reported in the literature at that time, and they had common C-terminal sequences (Dai et al., 2012). However, it was uncertain whether the difference between their apparent molecular weights was due to the different amino acid sequences or merely some kind of post-translational modification, such as glycosylation. In this study, the individual gene sequences encoding these two REF isoforms were obtained, the two REF isoforms were enriched by sequential extraction and separated by 16 BAC/SDS-PAGE, and their localizations on the surfaces of rubber particles were verified by western blotting and immunogold electron microscopy.

Section snippets

Plant materials

Ten-year-old regularly tapped rubber trees (H. brasiliensis Reyan 7-33-97 clones) from an experimental farm operated by the Chinese Academy of Tropical Agricultural Sciences (CATAS) in Hainan, China, were used for the experiments. Fresh latex was collected and preserved as previously described (Dai et al., 2016).

Extraction, electrophoretic separation, and mass spectrometric identification of rubber particle proteins

The fresh latex was centrifuged at 11,000 rpm (9880×g) and 4 °C for 1 h, and the creamy rubber particle layer was removed and washed three times with a solution of 10 mM Tris and 250 mM

16 BAC/SDS PAGE separations and mass spectrometric analysis of rubber particle proteins extracted with Triton X-100 and SDS containing buffers

The rubber particle sample collected at 11,000 rpm was first extracted with a Triton X-100 containing buffer and then an SDS containing buffer. Triton X-100 is a weak non-ionic detergent and SDS is a strong anionic detergent. The proteins extracted were separated by 16 BAC/SDS PAGE (Fig. 1), and then the protein spots were in-gel digested with trypsin and analyzed with an AB SCIEX 5800 MALDI TOF/TOF MS system.

Fig. 1 shows that different gel profiles were obtained from protein samples that had

Discussion

In recent years, a considerable amount of transcriptomic data on H. brasiliensis has been published. However, not all genes can be found in these data sets, and the gene encoding the 27.3 kDa REF isoform is an example. Therefore, direct cloning of some genes is still necessary, and this study provides information about the gene cloning of two H. brasiliensis REF isoforms, although partial sequences for them have been previously published (Dai et al., 2012). The success in separating and

Conclusion

Through sequential extraction, western blotting, and immunogold electron microscopy, we found that the 23.6 and 27.3 kDa rubber elongation factors in H. brasiliensis are bound more tightly to rubber particles than the most abundant REF and SRPP isoforms are, and that they locate mainly on large rubber particles. These findings may support the suggestion that REF/SRPP family proteins are structural proteins in H. brasiliensis rubber particles.

Contributions

Longjun Dai planned the experiments, analyzed the data, and wrote the manuscript. Longjun Dai, Zhiyi Nie, Guijuan Kang, and Yu Li conducted the experiments. Rizhong Zeng supervised the progress of this study and revised the manuscript.

Acknowledgements

This work was financially supported by the Natural Science Foundation of Hainan Province (grant No. 312028) and the National Natural Science Foundation of China (grant No. 31270713). We thank Dr. Dejun Li. for allowing us to use his transcriptome database before its official publication, Dr. Yongjun Fang and Dr. Caorong Tang for providing the REF/SRPP sequences, and Xinwen Zhou from Fudan University, for assistance with database searches. We also thank the reviewers for their valuable

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