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ABSTRACT: Tapping causes the loss of large amounts of latex from laticifers and subsequently enhances latex regeneration, a high carbon- and nitrogen-cost activity in rubber tree. It is suggested that a 67kDa protein associated with protein-storing cells in the inner bark tissues of rubber tree plays an important role in meeting the nitrogen demand for latex regeneration. Here, the 67kDa protein was further characterized by a combination of cell biological, molecular biological and biochemical techniques. Immunogold labeling showed that the 67kDa protein was specifically localized in the central vacuole of protein-storing cells. A full-length cDNA, referred to as HbVSP1, was cloned. The HbVSP1 contained a 1584bp open reading frame encoding a protein of 527 amino acids. The putative protein HbVSP1 shared high identity with the P66 protein from rubber tree and proteins of the linamarase, and bg1A from cassava (Manihot esculenta). HbVSP1 contained the active site sequences of β-glucosidase, TFNEP and I/VTENG. In vitro analysis showed that the 67kDa protein exhibited the activity of both β-glucosidase and linamarase and was thus characterized as a cyanogenic β-glucosidase. Proteins immuno-related to the 67kDa protein were present in leaves and lutoids of laticifers. Tapping down-regulated the expression of HbVSP1, but up-regulated the expression of genes encoding the key enzymes for rubber biosynthesis, while the effect of resting from tapping was the reverse. Taken together, the results suggest that the 67kDa protein is a vacuole-localized cyanogenic β-glucosidase encoded by HbVSP1 and may have a role in nitrogen storage in inner bark tissues of trunk during the leafless periods when rubber tree is rested from tapping.
Journal of plant physiology 03/2013; · 2.50 Impact Factor
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ABSTRACT: To better understand molecular mechanism underlying the difference between self-rooting juvenile clones and donor clones,
a proteomic approach was used to profile protein changes in the latex between self-rooting juvenile clones and donor clones.
Total soluble proteins were extracted from latex in self-rooting juvenile clones and donor clones. Two-dimensional gel electrophoresis
(2-DE) was used to identify proteins that were differentially expressed in self-rooting juvenile clones and donor clones and
image analysis was used to determine which proteins were up- or down-regulated. Twenty-four differentially expressed protein
spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Among the
24 proteins identified, 13 proteins were up-regulated, 11 proteins were decreased in self-rooting juvenile clones. These proteins
were classified as carbohydrate and energy metabolism, secondary metabolism, signal translocation, transcriptional regulation-related,
protein synthesis and degradation, transport, nucleoside acid process, lipid metabolism. Perhaps, the present study contributes
towards an understanding of the molecular mechanism underlying the difference between self-rooting juvenile clones and donor
clones.
Keywords
Hevea brasiliensis
–Differential protein–Self-rooting juvenile clone–Latex
Acta Physiologiae Plantarum 04/2012; 33(5):1853-1859. · 1.64 Impact Factor
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ABSTRACT: AP2/ERF transcription factors play an important role in regulation of the cross-talk between ethylene and jasmonate signaling
pathways mediating defense responses of plants to biotic and abiotic stresses. In this study, an AP2/ERF transcription factor
gene was isolated and characterized from laticifers of rubber tree by using RACE and real time PCR. The full length cDNA,
referred to as HbEREBP1, was 1,095bp in length and contained a 732bp open reading frame encoding a putative protein of 243 amino acid residues.
The molecular mass of the putative protein is 26.4kDa with a pI of 9.46. The deduced amino acid sequence had a specific domain
of AP2 superfamily and an ethylene-responsive element binding factor-associated amphiphilic repression motif, sharing 42.4,
39.1, and 38.0% identity with that of AtERF11, AtERF4, and AtERF8 in Arabidopsis, respectively. HbEREBP1 expression was down-regulated by tapping and mechanical wounding in the laticifers of adult trees. It was also down-regulated
at early stage while up-regulated at late stage upon treatment with exogenous ethephon or methyl jasmonate, which was reverse
to the case of defense genes in laticifers of epicormic shoots of rubber tree. Our results suggest that HbEREBP1 may be a
negative regulator of defense genes in laticifers.
KeywordsRubber tree–Laticifer–AP2/ERF transcription factor–Wound signal–Defense
Molecular Biology Reports 04/2012; 39(4):3713-3719. · 2.93 Impact Factor
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ABSTRACT: The cDNA encoding a 14-3-3 protein, designated as Hb14-3-3c, was isolated from Hevea brasiliensis. Hb14-3-3c was 1,269 bp long containing a 795 bp open reading frame encoding a putative protein of 264 amino acids, flanked by a 146 bp 5'UTR and a 328 bp 3' UTR. The predicted molecular mass of Hb14-3-3c is 29.67 kDa, with an isoelectric point of 4.52 and the deduced protein showed high similarity to the 14-3-3 protein from other plant species. Expression analysis revealed more significant accumulation of Hb14-3-3c transcripts in latex than in leaves, buds and flowers. The transcription of Hb14-3-3c in latex was induced by jasmonate and ethephon. Overproduction of recombinant Hb14-3-3c protein gave the Escherichia coli cells more tolerance on Co(2+), Cu(2+) and Zn(2+). Through yeast two-hybrid screening, 11 interaction partners of the Hb14-3-3c, which are involved in rubber biosynthesis, stress-related responses, defence etc., were identified in rubber tree latex. Taking these data together, it is proposed that the Hb14-3-3c may participate in regulation of rubber biosynthesis. Thus, the results of this study provide novel insights into the 14-3-3 signaling related to rubber biosynthesis, stress-related responses in rubber tree.
Molecular Biology Reports 09/2011; 39(4):4491-7. · 2.93 Impact Factor
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ABSTRACT: AP2/ERF transcription factors play an important role in regulation of the cross-talk between ethylene and jasmonate signaling pathways mediating defense responses of plants to biotic and abiotic stresses. In this study, an AP2/ERF transcription factor gene was isolated and characterized from laticifers of rubber tree by using RACE and real time PCR. The full length cDNA, referred to as HbEREBP1, was 1,095 bp in length and contained a 732 bp open reading frame encoding a putative protein of 243 amino acid residues. The molecular mass of the putative protein is 26.4 kDa with a pI of 9.46. The deduced amino acid sequence had a specific domain of AP2 superfamily and an ethylene-responsive element binding factor-associated amphiphilic repression motif, sharing 42.4, 39.1, and 38.0% identity with that of AtERF11, AtERF4, and AtERF8 in Arabidopsis, respectively. HbEREBP1 expression was down-regulated by tapping and mechanical wounding in the laticifers of adult trees. It was also down-regulated at early stage while up-regulated at late stage upon treatment with exogenous ethephon or methyl jasmonate, which was reverse to the case of defense genes in laticifers of epicormic shoots of rubber tree. Our results suggest that HbEREBP1 may be a negative regulator of defense genes in laticifers.
Molecular Biology Reports 07/2011; 39(4):3713-9. · 2.93 Impact Factor
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ABSTRACT: MYC2 transcription factor is a key component of the core module COI1-JAZ-MYC2 of jasmonate signaling in Arabidopsis, but the MYC transcription factor (s) associated with jasmonate signaling in jasmonate-responsive laticifer cells remains to be identified. Two full-length cDNAs, designated HblMYC1 and HblMYC2, were isolated from laticifer cells in Hevea brasiliensis by the method of RACE. HblMYC1 contained 1431bp ORF encoding a putative protein of 476 amino acids while HblMYC2 contained 1428bp ORF encoding a putative protein of 475 amino acids. Bioinformatic analysis showed that the putative proteins, HblMYC1 and HblMYC2, possessed a bHLH domain and were most related to the MYC2 among the selected 27 MYC members with identified functions in Arabidopsis. In addition to the presence of cis-regulatory elements involving jasmonate responsiveness in the promoter regions of HblMYC1 and HblMYC2, the abscisic acid-, salicylic acid- and gibberellin-responsive elements were found in the promoter region of HblMYC1. Transcripts of HblMYC1 and HblMYC2 were most abundant in latex, relatively low in male flowers and nearly undetected in bark tissues and roots by real-time RT-PCR analysis. Regular tapping, mechanical wounding, and ethrel remarkably up-regulated HblMYC1 expression, but had little effect on the expression of HblMYC2 in laticifer cells. Successive tapping, however, significantly down-regulated the expression of HblMYC2 while up-regulating the expression of HblMYC1. The HblMYC2 expression took a mutual ebb and flow relationship with the HblMYC1 expression upon treatment with methyl jasmonate. Characterization of HblMYC1 and HblMYC2 will contribute to the understanding of jasmonate signaling in laticifiers, a kind of specialized tissue for natural rubber biosynthesis in Hevea brasiliensis.
Journal of plant physiology 04/2011; 168(14):1649-58. · 2.50 Impact Factor
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ABSTRACT: The cDNA code of thioredoxin h, designated as HbTRX1, was isolated from Hevea brasiliensis by rapid amplification of cDNA ends. HbTRX1 contained a 542-bp open reading frame encoding 123 amino acids. The deduced HbTRX1 protein showing high identity to thioredoxin h of other plant species was predicted to possess the conserved catalytic site WCXPC. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that HbTRX1 was constitutively expressed in all tested tissues. HbTRX1 transcripts accumulated at relatively low levels in the flower, somatic embryo, and leaves, while HbTRX1 transcripts accumulated at relatively high levels in the callus and latex. The HbTRX1 transcript was expressed at different levels, with higher levels in self-rooting juvenile clones than in their donor clones. HbTRX1 was expressed in Escherichia coli, and its activity was demonstrated using the dithiothreitol-dependent insulin assay. This work provides a basis for studying the biological function of thioredoxin h in rubber tree.
Molecular Biology Reports 03/2011; 38(3):1989-94. · 2.93 Impact Factor
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ABSTRACT: Three MADS-box genes, designated HbMADS1, HbMADS2 and HbMADS3, were isolated from Hevea brasiliensis. HbMADS1, HbMADS2 and HbMADS3 encode polypetides consisting of 245, 217 and 239 amino acids, respectively, containing conserved MADS-box motifs at N-terminus. Transcription pattern analysis revealed that three MADS-box genes had highly transcription in the laticifer cells. The transcriptions of HbMADS1and HbMADS3 were induced in the laticifer cells by jamonic acid, while HbMADS2 was not induction by jamonic acid. Ethephone is not effective in inducing their expression. The three genes were differentially expressed during somatic embryogenesis of rubber tree. Characterization of HbMADSs will attribute to understand their possible function in rubber tree.
Molecular Biology Reports 11/2010; 38(6):4045-52. · 2.93 Impact Factor
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ABSTRACT: Calcium-dependent protein kinases (CDPKs), as major primary Ca(2+) sensors, have been implicated in the regulation of stress and developmental signals in plants. In this study, a novel CDPK gene, designated HbCDPK1, was isolated from Hevea brasiliensis. The HbCDPK1 cDNA had 2,400 bp with an open reading frame of 1,671 bp encoding 556 amino acids, and the deduced HbCDPK1 protein contained four characteristic domains identified in CDPKs, showing a high level of sequence similarity to CDPKs from other plants. Expression analysis revealed more significant accumulation of the transcripts of HbCDPK1 in latex than in the leaves, bark, and roots in H. brasiliensis. In addition, transcription of HbCDPK1 was strongly induced by mechanical wounding, jasmonic acid (JA), and ethephon. Recombinant HbCDPK1 was expressed in E. coli, and its activity was assayed. The assay indicated that HbCDPK1 had the kinase and Ca(2+)-binding activity in vitro as a calcium-dependent protein. The potential roles of the HbCDPK1 are discussed as to latex production and rubber biosynthesis.
Bioscience Biotechnology and Biochemistry 11/2010; 74(11):2183-8. · 1.28 Impact Factor
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ABSTRACT: A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions: nitrogen storage and defense.
Journal of Integrative Plant Biology 04/2009; 51(4):352-9. · 2.53 Impact Factor
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ABSTRACT: The full-length cDNA encoding a coronatine insensitive-1 (COI1) protein, designated HbCOI1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCOI1 contained a 2,187 bp open reading frame encoding 597 amino acids. The deduced HbCOI1 protein, which showed high identity to COI1 protein of other plant species, was predicted to possess F-box and LRRs domains. The promoter region of HbCOI1 was isolated by the PCR-based DNA walking method. TATA box and other core configurations were found in the promoter. Several sequences similar to the eukaryotic cis regulatory element were found in the 5'-untranslated region (UTR) proximal 5' flanking sequence of HbCOI1. Southern blot analysis indicated that the HbCOI1 is present as a single copy in Hevea brasiliensis. Transcription pattern analysis revealed that HbCOI1 had high transcription in laticifer, low in barks and leaf. Transcription of HbCOI1 in latex was induced by jasmonate and tapping.
Bioscience Biotechnology and Biochemistry 04/2009; 73(3):665-70. · 1.28 Impact Factor
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ABSTRACT: The full-length cDNA encoding a cysteine protease,designated HbCP1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCP1 contained a 1371 bp open reading frame encoding 457 amino acids.The deduced HbCP1 protein,which showed high identity to cysteine proteases of other plant species,was predicted to possess a putative repeat in toxin (RTX) domain at the N-terminal and a granulin (GRAN) domain at the C-terminal.Southern blot analysis indicated that the HbCP1 gene is present as a single copy in the rubber tree.Transcription pattern analysis revealed that HbCP1 had high transcription in laticifer,and low transcription in bark and leaf.The transcription of HbCP1 in latex was induced by ethylene and tapping.Cloning of the HbCP1 gene will enable us to further understand the molecular characterization of cysteine protease and its possible function in the rubber tree.
Journal of Biosciences 01/2009; 33(5):681-90. · 1.65 Impact Factor
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ABSTRACT: Vegetative storage proteins (VSPs) are commonly bioactive in herbaceous plants but few VSPs with bioactivity have been identified in trees. In addition, information on the characterization of VSPs in evergreen trees is limited. The objective of this study was to characterize the VSPs with bioactivity in evergreen trees. Methods The VSP in lychee (Litchi chinensis), an evergreen fruit tree, was characterized by a combination of cytological, biochemical and molecular biological techniques.
The VSP in lychee was a 22-kDa protein. It accumulated in the large central vacuoles of protein-storing cells (PSCs) in two distinguishable forms, granular and floccular. The PSCs were of a novel type. The 22-kDa protein is distributed in mature leaves, bark tissues of branches, trunk and large roots, paralleling the distribution of PSCs. Its homologues were present in mature seed. During young shoot development and fruiting, the 22-kDa protein decreased apparently, suggesting a nitrogen-storage function. The 22-kDa protein had several isoforms encoded by a small multigene family. One gene member, LcVSP1, was cloned. The LcVSP1 had no intron and contained a 675 bp open reading frame encoding a putative protein of 225 amino acids. LcVSP1 was homologous to Kunitz trypsin inhibitors. The 22-kDa protein inhibited trypsin and chymotrypsin, but had no inhibitory effect on subtilisin.
Lychee is rich in a 22-kDa VSP with trypsin inhibitor activity. The VSP plays an important role in nitrogen storage while its possible defensive function remains to be elucidated.
Annals of Botany 01/2008; 100(6):1199-208. · 4.03 Impact Factor
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ABSTRACT: In order to identify appropriate plant materials for studying the gene expression and biological function of vegetative storage proteins (VSPs) in woody plants, the VSPs in the seedlings of Swietenia macrophylla King were investigated by using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blotting. The seed of S. macrophylla was rich in storage proteins that accumulated in the vacuoles of cotyledon parenchyma cells in appearance of compact spherical grains. The growth and development of S. macrophylla seedlings were characterized by an obvious growth rhythm. The storage proteins in seeds disappeared during seedling growth while VSPs appeared in the stem 2 weeks after seedling leaves matured. Thereafter, the VSPs in the seedling stem almost exhausted during new shoot growth, and when the leaves of new shoot just matured, both the stem beneath the new shoot of seedlings and the stem of new shoot started to accumulate VSPs. Nitrogen application dramatically increased the level of VSPs, but had little influence on the dynamics of VSP consumption and accumulation in seedling stem. Together with these data, the fluctuation of VSPs in seedlings was very similar to that in the branches of the adult trees. In addition, seedlings are easy to be treated due to their small size. Our results suggested that S. macrophylla seedlings were suitable for investigating the biological roles of VSPs and the mechanism of nitrogen storage in trees.
Journal of Integrative Plant Biology 03/2007; 49(3):362 - 367. · 2.53 Impact Factor
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ABSTRACT: Using the information about the sequence from a differentially expressed clone (designated as HbSSH10) encodes a protein specifying a cysteine-rich sequence containing a putative "RING finger" or "C3HC4" consensus motif that was cloned recently by the subtractive hybridization between latex and leaves from rubber tree (Hevea brasiliensis). A full-length cDNA encoding C3HC4 type zinc-finger protein was isolated and characterized from rubber tree. Sequence analysis revealed that the ORFs of HbRZF encode 156 amino acid residues with a total predicted molecular mass of 17.2 kD, HbRZF protein having a putative "RING finger" segment (amino acid residues 100-144). The deduced amino acid sequences of HbRZF showed high identities of 48%, 52% and 50% to those of the ring zinc protein from Poncirus trifoliata, Arabidopsis thaliana, Thellungiella halophila. The result of Northern blot analysis indicated that the transcripts of the HbRZF were expressed more in the latex than in the leaves, whereas little expression was detected in roots and flowers. The transcription of HbRZF was induced by jasmonic acid, whereas ethylene had little effect.
Zhi wu sheng li yu fen zi sheng wu xue xue bao = Journal of plant physiology and molecular biology 01/2007; 32(6):627-33.
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ABSTRACT: It was generally assumed that the accumulation of vegetative storage protein (VSP) in poplar trees and/or temperate hardwoods did not occur in spring. To test this assumption, the accumulation of the 32-kDa VSP and the differential expression of a gene encoding for the protein in poplars were investigated using light and electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reverse transcription-polymerase chain reaction (RT-PCR). We report, for the first time, that poplar trees initiated VSP accumulation in new shoots during the development of new shoots in spring under conditions of high temperature and long days. The amount of 32-kDa VSP increased gradually in the stem of new shoots and in two-year-old branches, but there were no detectable changes in its abundance in the bark tissues of trunks during new shoot development. Based on the presence of a 286-bp DNA fragment that is identical to the VSP gene bspA, encoding for the 32-kDa VSP in Populus deltoids Bartr. ex Marsh., the differential expression of the 32-kDa VSP gene in P. canadensis Moench was revealed by RT-PCR. The results indicated that the 32-kDa VSP gene was expressed strongly in new shoots, relative weakly in two-year-old branches and was not expressed in the trunk during new shoot development. This pattern of VSP accumulation and VSP gene space-time differential expression may be an important mechanism by which stored nitrogen compounds are used preferentially to exogenously available nitrogen and, in addition, the dynamic pattern may also have a role in the regulation of nitrogen metabolism, especially nitrogen uptake by the roots.(Managing editor: Wei WANG)
Journal of Integrative Plant Biology 05/2005; 47(6):717 - 725. · 2.53 Impact Factor
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ABSTRACT: The distribution and ultrastructure of vegetative storage proteins in 44 species and one variety of 31 genera of Leguminosae were investigated by light-and electron microscopy and SDS-PAGE. Leguminosae are as a whole a vegetative storage protein-rich family, abundant with vacuolar protein inclusions in deciduous trees while much less so in evergreen trees. Several prominent proteins with molecular weights ranging from 15 to 45 kDa were isolated and identified to be vegetative storage proteins on the basis of their association with vacuolar protein inclusions and seasonal fluctuation. Vacuolar protein inclusions were present in protein body-like organelles in temperate species while localized in large central vacuoles in tropical ones during leafless periods. The inclusions varied in forms among species or in the same species, but the different forms were present in different cells, suggesting that vegetative storage pro-teins may be cell-type specific to some extent.
IAWA Journal. 01/2004; 25:459-469.
