RESUMO
Natural rubber is an important industrial raw material and is commercially produced by rubber trees (Hevea brasiliensis). The sucrose transporter HbSUT3 plays an essential role in rubber production. Its expression in latex (cytoplasm of rubber-producing laticifers) is induced by bark treatment with Ethrel, an ethylene releaser, and the inducing effect correlates well with Ethrel-stimulated rubber yield increase. However, the mechanisms of ethylene induction on HbSUT3 expression are not known. Here, five Ethylene Response Factor (ERF) genes were identified from the cDNA library of Hevea latex by yeast one-hybrid screening with the promoter of HbSUT3 gene as bait. As revealed in a tobacco (Nicotiana tabacum) protoplast transient expression system, these HbERFs were mainly localized in the nucleus and four of them exhibited apparent transactivation activity. Of the five HbERF genes, HbERF-IXc4 was the most frequently screened in yeast one-hybrid, accounting for 65% of the ERF clones obtained. Moreover, among the five HbERFs, HbERF-IXc4 showed the strongest transactivation capacity when expressed in tobacco protoplast, the highest transcript abundance in latex and a close expressional correlation with its target gene, HbSUT3, in response to the Ethrel treatment. Taken together, our results indicate that ERFs, especially HbERF-IXc4, are critically involved in the activation of HbSUT3 expression in latex after Ethrel treatment on Hevea bark, and thus the stimulated latex yield.
Assuntos
Hevea , Etilenos , Regulação da Expressão Gênica de Plantas , Hevea/genética , Hevea/metabolismo , Látex , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , SacaroseRESUMO
Sucrose-metabolizing enzymes in plant leaves have hitherto been investigated mainly in temperate plants, and rarely conducted in tandem with gene expression and sugar analysis. Here, we investigated the sugar content, gene expression, and the activity of sucrose-metabolizing enzymes in the leaves of Hevea brasiliensis, a tropical tree widely cultivated for natural rubber. Sucrose, fructose and glucose were the major sugars detected in Hevea leaves at four developmental stages (I to IV), with starch and quebrachitol as minor saccharides. Fructose and glucose contents increased until stage III, but decreased strongly at stage IV (mature leaves). On the other hand, sucrose increased continuously throughout leaf development. Activities of all sucrose-cleaving enzymes decreased markedly at maturation, consistent with transcript decline for most of their encoding genes. Activity of sucrose phosphate synthase (SPS) was low in spite of its high transcript levels at maturation. Hence, the high sucrose content in mature leaves was not due to increased sucrose-synthesizing activity, but more to the decline in sucrose cleavage. Gene expression and activities of sucrose-metabolizing enzymes in Hevea leaves showed striking differences compared with other plants. Unlike in most other species where vacuolar invertase predominates in sucrose cleavage in developing leaves, cytoplasmic invertase and sucrose synthase (cleavage direction) also featured prominently in Hevea. Whereas SPS is normally responsible for sucrose synthesis in plant leaves, sucrose synthase (synthesis direction) was comparable or higher than that of SPS in Hevea leaves. Mature Hevea leaves had an unusually high sucrose:starch ratio of about 11, the highest reported to date in plants.
RESUMO
SWEET proteins play an indispensable role as a sugar efflux transporter in plant development and stress responses. The SWEET genes have previously been characterized in several plants. Here, we present a comprehensive analysis of this gene family in the rubber tree, Hevea brasiliensis. There are 36 members of the SWEET gene family in this species, making it one of the largest families in plant genomes sequenced so far. Structure and phylogeny analyses of these genes in Hevea and in other species demonstrated broad evolutionary conservation. RNA-seq analyses revealed that SWEET2, 16, and 17 might represent the main evolutionary direction of SWEET genes in plants. Our results in Hevea suggested the involvement of HbSWEET1a, 2e, 2f, and 3b in phloem loading, HbSWEET10a and 16b in laticifer sugar transport, and HbSWEET9a in nectary-specific sugar transport. Parallel studies of RNA-seq analyses extended to three other plant species (Manihot esculenta, Populus trichocarpa, and Arabidopsis thaliana) produced findings which implicated MeSWEET10a, 3a, and 15b in M. esculenta storage root development, and the involvement of PtSWEET16b and PtSWEET16d in P. trichocarpa xylem development. RT-qPCR results further revealed that HbSWEET10a, 16b, and 1a play important roles in phloem sugar transport. The results from this study provide a foundation not only for further investigation into the functionality of the SWEET gene family in Hevea, especially in its sugar transport for latex production, but also for related studies of this gene family in the plant kingdom.
RESUMO
Calcium-dependent protein kinases (CDPKs or CPKs) play important roles in various physiological processes of plants, including growth and development, stress responses and hormone signaling. Although the CDPK gene family has been characterized in several model plants, little is known about this gene family in Hevea brasiliensis (the Para rubber tree). Here, we characterize the entire H. brasiliensis CDPK and CDPK-related kinase (CRK) gene families comprising 30 CDPK genes (HbCPK1 to 30) and nine CRK genes (HbCRK1 to 9). Structure and phylogeny analyses of these CDPK and CRK genes demonstrate evolutionary conservation in these gene families across H. brasiliensis and other plant species. The expression of HbCPK and HbCRK genes was investigated via Solexa sequencing in a range of experimental conditions (different tissues, phases of leaf development, ethylene treatment, and various abiotic stresses). The results suggest that HbCPK and HbCRK genes are important components in growth, development, and stress responses of H. brasiliensis. Parallel studies on the CDPK and CRK gene families were also extended to five other plant species (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Manihot esculenta, and Ricinus communis). The CDPK and CRK genes from different plant species that exhibit similar expression patterns tend to cluster together, suggesting a coevolution of gene structure and expression behavior in higher plants. The results serve as a foundation to further functional studies of these gene families in H. brasiliensis as well as in the whole plant kingdom.
RESUMO
In Hevea brasiliensis, an alkaline/neutral invertase (A/N-Inv) is responsible for sucrose catabolism in latex (essentially the cytoplasm of rubber-producing laticifers, the source of natural rubber) and implicated in rubber yield. However, neither the gene encoding this enzyme nor its molecular and biochemical properties have been well documented. Three Hevea A/N-Inv genes, namely HbNIN1, 2 and 3, were first cloned and characterized in planta and in Escherichia coli. Cellular localizations of HbNIN2 mRNA and protein were probed. From latex, active A/N-Inv proteins were purified, identified, and explored for enzymatic properties. HbNIN2 was identified as the major A/N-Inv gene functioning in latex based on its functionality in E. coli, its latex-predominant expression, the conspicuous localization of its mRNA and protein in the laticifers, and its expressional correlation with rubber yield. An active A/N-Inv protein was partially purified from latex, and determined as HbNIN2. The enhancement of HbNIN2 enzymatic activity by pyridoxal is peculiar to A/N-Invs in other plants. We conclude that HbNIN2, a cytosolic A/N-Inv, is responsible for sucrose catabolism in rubber laticifers. The results contribute to the studies of sucrose catabolism in plants as a whole and natural rubber synthesis in particular.
Assuntos
Hevea/enzimologia , Sacarose/metabolismo , beta-Frutofuranosidase/metabolismo , Sequência de Aminoácidos , Citosol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hevea/citologia , Hevea/genética , Látex/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/citologia , Caules de Planta/enzimologia , Caules de Planta/genética , Alinhamento de Sequência , beta-Frutofuranosidase/genéticaRESUMO
ROP (Rho of plants) proteins are plant-specific Rho-type small GTPases which play important roles in cellular processes and stress responses. This study explores the characteristics and possible functions of ROPs that are expressed primarily in laticifers of the rubber tree (Hevea brasiliensis). The work serves as a preliminary step to determining their involvement in latex flow and regeneration, laticifers formation and tapping panel dryness (TPD, a physiological disorder in rubber trees that result in the stoppage of latex flow). In this connection, we (i) identified five HbROPs (HbROP1-HbROP5) by searching latex transcripts database and the genome databases, (ii) characterized molecular and phylogenic aspects of the HbROPs and examined the cis-regulatory elements in their promoter regions; (iii) analyzed by Real-time Quantitative PCR (QPCR) the tissue specificity of the HbROPs and their expression patterns in response to tapping, bark wounding and growth regulator treatments. All five HbROP genes were strongly expressed in the latex, with HbROP1, 3, 4 and 5 showing the highest expression among the six Hevea tissues examined, viz. latex, bud, mature leaf, bark, male flower and seed. When tapping was initiated on previously untapped trees, HbROP3 transcription was substantially down-regulated whereas HbROP5 expression was markedly up-regulated. Transcripts of HbROP3 rose gradually with the development of TPD. Except for the cytokinin 6-benzyl aminopurine that induced a rise in HbROP5 transcripts by more than 2-fold, the other growth regulators tested had little effect on HbROPs expression. The roles of HbROPs in rubber tree are discussed in relation to the diverse functions of ROP homologs reported in other plant species.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hevea/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Citocininas/fisiologia , Primers do DNA , DNA Complementar/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Increasing demand for natural rubber prompts studies into the mechanisms governing the productivity of rubber tree (Heveabrasiliensis). It is very interesting to notice that a rubber tree of clone PR107 in Yunnan, China is reported to yield more than 20 times higher than the average rubber tree. This super-high-yielding (SHY) rubber tree (designated as SY107), produced 4.12 kg of latex (cytoplasm of rubber producing laticifers, containing about 30% of rubber) per tapping, more than 7-fold higher than that of the control. This rubber tree is therefore a good material to study how the rubber production is regulated at a molecular aspect. A comprehensive cDNA-AFLP transcript profiling was performed on the latex of SY107 and its average counterparts by using the 384 selective primer pairs for two restriction enzyme combinations (ApoI/MseI and TaqI/MseI). A total of 746 differentially expressed (DE) transcript-derived fragments (TDFs) were identified, of which the expression patterns of 453 TDFs were further confirmed by RT-PCR. These RT-PCR confirmed TDFs represented 352 non-redundant genes, of which 215 had known or partially known functions and were grouped into 10 functional categories. The top three largest categories were transcription and protein synthesis (representing 24.7% of the total genes), defense and stress (15.3%), and primary and secondary metabolism (14.0%). Detailed analysis of the DE-genes suggests notable characteristics of SHY phenotype in improved sucrose loading capability, rubber biosynthesis-preferred sugar utilization, enhanced general metabolism and timely stress alleviation. However, the SHY phenotype has little correlation with rubber-biosynthesis pathway genes.
Assuntos
Hevea/genética , Hevea/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Látex/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Borracha/metabolismo , TranscriptomaRESUMO
Isolating high-quality RNA from latex of H. brasiliensis is a prerequisite to elucidating the molecular mechanisms of rubber biosynthesis and its regulation. Here, an improved protocol was developed for latex collection, transportation, storage, and RNA isolation. Compared with existing ones, our protocol eliminated liquid nitrogen for latex collection and subsequent low-temperature (-70 degrees C) condition for latex storage, making it more convenient and feasible when latex was collected in remote sampling sites, and latex storage and RNA isolation were conducted in poorly-equipped laboratories. Different methods (UV absorbance scans, denaturing gel electrophoresis, autoradiograph monitoring of cDNA synthesis) were used to confirm the high quality of the RNA prepared with this protocol, whose usefulness was further verified by several practical applications, including construction of one high-quality cDNA library, cloning of the full-length cDNAs of 3 novel Hevea sucrose transporter genes, and semi-quantitative RT-PCR analysis of two rubber-biosynthesis essential genes and one sucrose transporter gene.