RESUMO

Latex flow in Hevea brasiliensis (the Para rubber tree), the sole commercial source of natural rubber (cis-1,4-polyisoprene, NR), renders it uniquely suited for the study of plant stress responses. Calcineurin B-like interacting protein kinases (CIPK) serving as calcium-sensor protein kinases react with calcineurin B-like proteins (CBL) to play crucial roles in hormone signaling transduction and response to abiotic stress in plant developmental processes. However, little is known about their functions in Hevea. In this study, a total of twelve CBL (HbCBL) and thirty CIPK (HbCIPK) genes were identified from the Hevea genome. Structure and phylogenetic analysis assigned these CIPKs to five groups and CBLs to four groups, and mapped onto fourteen of the eighteen Hevea chromosomes. RNA-seq and qPCR analysis showed that the expressions of HbCBL and HbCIPK genes varied in the seven Hevea tissues examined, i.e., latex (cytoplasm of rubber-producing laticifers), bark, leaf, root, seed, female flower, and male flower. The expressions of two HbCBL and sixteen HbCIPK genes showed upward trends during leaf development. Following ethylene yield stimulation and the latex tapping treatment, both practices invoking stress, the expression levels of most latex-expressed genes were significantly altered. Yeast two-hybrid test revealed interactions for multiple combinations of HbCBLs and HbCIPKs with substantial gene expression in latex or other Hevea tissues. However, all the HbCBL-HbCIPK complexes examined did not recruit HbSOS1 or AtSOS1 to form functional salt tolerance SOS pathway in yeast cells. Taken together, the results suggested a role of the Hevea CBL-CIPK network as a point of convergence for several different signaling pathways in growth, development, and stress responses in relation to latex production.

RESUMO

Fructokinase (FRK) mediates fructose phosphorylation to regulate the carbon flow and its assignment to sink tissues. Out of five HbFRKs in the genome of the rubber tree, three (HbFRK1-3) that were highly expressed in latex (cytoplasm of laticifers) were isolated and examined. According to phylogenetic analysis and intracellular location experiment, both HbFRK2 and HbFRK3 were highly possible to be expressed in cytosol, while HbFRK1 was in plastid. As the predominant isoform in laticifers, HbFRK2 had the highest transcripts, followed by HbFRK3 and HbFRK1. In enzymatic function, HbFRK2 also showed the highest affinity for fructose. To examine the roles of FRKs in latex yield and regeneration, changes in HbFRKs were examined when latex outflow from the trees were increased through two experimental interventions. In the first approach, tapping was initiated on previously untapped trees, resulting in latex yield increasing with consecutive tapping at the initial stage before it stabilized. In the second approach, latex yield from trees that were already in regular tapping was stimulated by treatment with the ethylene-based yield stimulant, ethephon. Using either method to induce an increase in latex yield, the abundance of HbFRK2 and HbFRK3 in transcripts, was increased. This development, which was especially marked in HbFRK2, may reflect a strengthening of glycolysis to meet the carbon flux and energy demands for increased rubber biosynthesis to replace rubber lost in the increased latex yield. Our results, therefore, suggest that HbFRK2 plays a critical role in fructose catabolism to facilitate rubber regeneration in the commercially exploited rubber tree.

Assuntos

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

RESUMO

BACKGROUND: Sucrose (Suc), as the precursor molecule for rubber biosynthesis in Hevea brasiliensis, is transported via phloem-mediated long-distance transport from leaves to laticifers in trunk bark, where latex (cytoplasm of laticifers) is tapped for rubber. In our previous report, six Suc transporter (SUT) genes have been cloned in Hevea tree, among which HbSUT3 is verified to play an active role in Suc loading to the laticifers. In this study, another latex-abundant SUT isoform, HbSUT5, with expressions only inferior to HbSUT3 was characterized especially for its roles in latex production. RESULTS: Both phylogenetic analysis and subcellular localization identify HbSUT5 as a tonoplast-localized SUT protein under the SUT4-clade (=type III). Suc uptake assay in baker's yeast reveals HbSUT5 to be a typical Suc-H+ symporter, but its high affinity for Suc (Km = 2.03 mM at pH 5.5) and the similar efficiency in transporting both Suc and maltose making it a peculiar SUT under the SUT4-clade. At the transcript level, HbSUT5 is abundantly and preferentially expressed in Hevea barks. The transcripts of HbSUT5 are conspicuously decreased both in Hevea latex and bark by two yield-stimulating treatments of tapping and ethephon, the patterns of which are contrary to HbSUT3. Under the ethephon treatment, the Suc level in latex cytosol decreases significantly, but that in latex lutoids (polydispersed vacuoles) changes little, suggesting a role of the decreased HbSUT5 expression in Suc compartmentalization in the lutoids and thus enhancing the Suc sink strength in laticifers. CONCLUSIONS: Our findings provide insights into the roles of a vacuolar sucrose transporter, HbSUT5, in Suc exchange between lutoids and cytosol in rubber-producing laticifers.

Assuntos

RESUMO

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

Along with changes in morphology in the course of maturation, leaves of Hevea brasiliensis become more resistant to leaf diseases, including the South American Leaf Blight (SALB), a devastating fungal disease of this economically important tree species. To understand the underlying mechanisms of this defense, and to identify the candidate genes involved, we sequenced the Hevea leaf transcriptome at four developmental stages (I to IV) by Illumina sequencing. A total of 62.6 million high-quality reads were generated, and assembled into 98,796 unique transcripts. We identified 3,905 differentially expressed genes implicated in leaf development, 67.8% (2,651) of which were during the transition to leaf maturation. The genes involved in cyanogenic metabolism, lignin and anthocyanin biosynthesis were noteworthy for their distinct patterns of expression between developing leaves (stages I to III) and mature leaves (stage IV), and the correlation with the change in resistance to SALB and the Oidium/Colletotrichum leaf fall. The results provide a first profile of the molecular events that relate to the dynamics of leaf morphology and defense strategies during Hevea leaf development. This dataset is beneficial to devising strategies to engineer resistance to leaf diseases as well as other in-depth studies in Hevea tree.

Assuntos

RESUMO

As a key enzyme in the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) provides nicotinamide adenine dinucleotide phosphate (NADPH) and intermediary metabolites for rubber biosynthesis, and plays an important role in plant development and stress responses. In this study, four Hevea brasiliensis (Para rubber tree) G6PDH genes (HbG6PDH1 to 4) were identified and cloned using a genome-wide scanning approach. All four HbG6PDH genes encode functional G6PDH enzymes as shown by heterologous expression in E. coli. Phylogeny analysis and subcellular localization prediction show that HbG6PDH3 is a cytosolic isoform, while the other three genes (HbG6PDH1, 2 and 4) are plastidic isoforms. The subcellular locations of HbG6PDH3 and 4, two latex-abundant isoforms were further verified by transient expression in rice protoplasts. Enzyme activity assay and expression analysis showed HbG6PDH3 and 4 were implicated in PPP during latex regeneration, and to influence rubber production positively in rubber tree. The cytosolic HbG6PDH3 is a predominant isoform in latex, implying a principal role for this isoform in controlling carbon flow and NADPH production in the PPP during latex regeneration. The expression pattern of plastidic HbG6PDH4 correlates well with the degree of tapping panel dryness, a physiological disorder that stops the flow of latex from affected rubber trees. In addition, the four HbG6PDHs responded to temperature and drought stresses in root, bark, and leaves, implicating their roles in maintaining redox balance and defending against oxidative stress.