Search

The major challenge of cassava cultivation is phytopathogen damage and lack of resistant cultivars. Investigating the defense mechanism in cassava should provide insights into the development of new disease control strategies. Colletotrichum gloeosporioides f. sp. manihotis (CAD) is a fungal pathogen that causes cassava anthracnose disease, which is a serious disease worldwide, affecting cassava production and yield loss. Syntaxins contribute to mediate vesicles fusion in trafficking pathway by specifically forming as ternary SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex to transport defense components to site of microbe infection between the plasma membrane and plant cell wall. The function of syntaxins is largely unknown in resistance to Colletotrichum spp. in cassava. To manipulate resistance against Colletotrichum spp., the role of syntaxin genes and plant hormones; salicylic acid (SA) and jasmonic acid (JA) in triggering various responses to biotic stress was studied. All syntaxin genes in cassava genome were identified, and gene expression was investigated in CAD-resistant (HB60) and -susceptible (HN) cultivars of cassava. MePR1 was highly up-regulated in HB60 upon CAD infection. SA-pretreatment induced MeSYP121 expression and coexisted with delayed development of disease symptoms in HB60. In contrast, the different expression patterns of both genes were demonstrated in HN, and JA-pretreatment could not induce CAD-resistance. Altogether, the results suggest the role of SA-induced syntaxin gene expression to mediate vesicle trafficking and trigger defense-associated factors for more disease resistance in cassava. This investigation could be useful for the development of effective crop protection strategies and plant disease management in modern agriculture.

The proteomic analysis of vascular tissues in rubber tree reveals differentially expressed proteins related to laticifer differentiation in mature phloem and secondary cell wall formation in mature xylem for latex/wood-yield improvement. Rubber tree (Hevea brasiliensis) is a latex-producing plant that has worldwide economic importance for the rubber and wood industries. Natural rubber latex is produced from laticiferous vessels (laticifers) located in the secondary phloem tissue of rubber trees. Improving latex production from rubber tree clones by studying the molecular genetics of vascular development has received much attention, but less information has been obtained from proteomic approaches. This study performed a comparative proteomic analysis of the vascular tissues in high latex-yield and high wood-yield rubber tree clones. One primary vascular tissue (newly developed stem) and two secondary vascular tissues (mature phloem/laticifers and mature xylem/wood) were investigated by qualitative/quantitative proteomic analysis using GeLC-MS/MS. The differentially expressed proteins (DEPs) in the specific vascular tissues were analyzed, and the protein functions in the biological processes related to vascular development and to the specific characteristics of each clone were clarified. The predicted protein–protein interaction network and GO annotation revealed DEPs related to photosynthesis, carbohydrate metabolism, energy production and jasmonic acid-responsive proteins involved in positive regulation of laticifer differentiation in mature phloem of the high latex-yield clone, while DEPs related to cell cytoskeleton maintenance, secondary cell wall components and auxin-, brassinosteroid-, abscisic acid-responsive proteins involved in xylem differentiation were abundant in mature xylem of the high wood-yield clone. This is the first report to demonstrate the correlation and functions of DEPs in phloem/laticifers and xylem/wood differentiation in rubber trees. The regulation of vascular development could be useful in improvement of latex and wood yields from rubber trees.

Hedychium longicornutum Griff. ex Baker is a Zingiberaceous plant that has been used for traditional medicines and ornamental purposes. Though this plant is a vulnerable plant of Thailand, it has a high potential to be promoted as a new economic ornamental plant due to its exotic inflorescences. However, the usage of this valuable plant on a large scale is restricted since the population of H. longicornutum in natural habitat is limited. This current study established a micropropagation protocol to ensure sustainable use of this plant for commercial and conservation purposes. In vitro axenic plants of H. longicornutum were initiated from seeds. Optimum shoot multiplication medium was evaluated by culturing leafy-shoot bases on Murashige and Skoog’s (MS) medium supplemented with 0-35 µM of N6-benzyladenine (BA) or thidiazuron (TDZ). BA supplemented media were better than those TDZ media since no abnormalities of shoot morphology, shoot elongation, and root regeneration were found. MS supplemented with 15 µM BA was revealed to be the most effective medium for shoot multiplication (9.75 leafy-shoots/explant with 9.54 cm height). Regenerated shoots spontaneously rooted on MS medium. The in vitro raised plants were successfully grown ex vitro and showed the highest survival rate (100%) on planting substrate consisting of 2 garden soil mixture: 1 smashed charcoal. The genetic fidelity of the micropropagated plants compared with mother plants grown in the field was assessed by random amplified polymorphic DNA (RAPD) technique. The results consistently demonstrated the genetic uniformity of the regenerants. This is the first report of an established in vitro true-to-true type propagation in H. longicornutum.

Khon Kaen Agriculture Journal, 50, 4, 1220-1232

In this study, the effects of methyl jasmonate (MeJA) on the phytomass and triterpenoid production of diploid and tetraploid Centella asiatica hairy roots were investigated. Hairy root cultures were obtained from diploid and induced tetraploid plants of C. asiatica infected by Agrobacterium rhizogenes strain ATCC 43057. MeJA triggered triterpenoid production in both ploidy hairy roots, whereas triterpenoids were not produced in the untreated hairy roots. Among the treatments, the 50 µM MeJA treatment yielded the maximum triterpenoid production in diploid hairy roots of 27.25 ± 0.27 µg/mg Dry weight (DW) total triterpenoid at day 21. For the tetraploid hairy root cultures, the 28th-day hairy root culture produced a maximum amount of triterpenoids of 16.29 ± 6.32 µg/mg DW in response to the 50 µM MeJA treatment, whereas the 100 µM MeJA treatment produced a similar triterpenoid amount (16.31 ± 9.24 µg/mg DW) at day 14. Moreover, in response to 50 µM MeJA, we obtained different ratios of aglycone to glycoside, i.e., 1:7 and 1:2, between the diploid and tetraploid hairy root cultures. Asiaticoside was the dominant phytochemical, followed by asiatic acid and madecassic acid. This study provides valuable information for producing triterpenoids for C. asiatica commercial products and preparations by using hairy root cultures.

Cassava storage roots serve as an outstanding source of starch that is commonly utilized for nourishment and industrial applications. Despite the extensive studies, which indicated diverse important roles of miRNAs as post-transcriptional regulators of gene expression, the potential contribution of microRNAs to storage root development in cassava are sparse. Here, we characterized the key miRNAs and auxin content in two main types of cassava roots, fibrous roots and storage roots. The differential expression pattern of miRNAs and their mRNA targets, miR164/NAC and miR167/ARF6, ARF8, revealed the correlation in storage root development. A higher content of indole-3-acetic acid was observed in storage roots in contrast with fibrous roots, and the possible role was discussed. Altogether, this first finding suggested the roles of miR164/miR167 in the molecular mechanism underlying cassava storage root development.

More than 18 wild banana (Musaceae) species and hundreds of cultivars were found in Thailand, many are introduced as ornamentals. The banana family which is classified into three genera: Musa, Ensete and Musella, distributes in the old world tropics and Thailand is among their centers of origin. Many banana species possess attractive bract colors such as orange, pink, bright red, purple, and also, though less frequently found, yellow and green. Several other appealing features of the bananas are dwarf pseudostems, variegated leaves, multiple male inflorescence, uneven colored inflorescence bracts and numerous hands of fruits. Recently, three new banana species, Musa siamensis Hakkinen & Rich. H. Wallace, M. serpentina Swangpol & Somana and M. nanensis Swangpol & Traiperm were discovered in the country. These bananas have decorative potential; taxonomic revision could enhance the selection process of these taxa and bring them into the spot light as economic ornamental crops.

During growth of woody plant-trunk, the secondary meristem functions in giving rise the xylem and phloem. Rubber tree (Hevea brasiliensis Muell. Arg.), in addition, contains laticifers (latex producing vessels) in the vicinity of phloem. Insights into regulatory mechanisms of gene networks underlying laticifer proliferation in rubber tree has remained very limited. The candidate vascular development-related genes were selected to investigate for expression profile in phloem and xylem tissues of high latex yield- and high wood yield-clones of rubber tree. The differential gene expression between the mature branch-xylem and -phloem tissues was clearly observed. The cis-regulatory motif analysis revealed the existent of putative jasmonic acid (JA)- and brassinosteroid (BR)-responsive regulatory motifs in promoter regions of these genes, and consequently the effect of exogenous application of JA, BR or their respective signaling inhibitors, on the formation of laticifers in rubber tree was demonstrated. Interestingly, the laticifer numbers were significantly increased in JA-treatment, correlated with up-regulation of phloem development-related genes in both rubber tree clones. On the contrary, the laticifers were decreased in BR-treatment accompanying by up-regulation of xylem development-related genes, especially in high wood yield-rubber tree clone. BR-inhibitor treatment also enhanced laticifer numbers, while JA-inhibitor suppressed laticifer differentiation. Taken together, this study unveils the molecular interplay between JA/BR on vascular development in rubber tree and how this impacts the appearance of laticifers in this plant. This process is vital for a better understanding on laticifer differentiation and its impact in the manipulation of wood and latex yield in rubber tree improvement program. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12298-019-00686-0) contains supplementary material, which is available to authorized users.

Natural rubber (cis-1,4-polyisoprene) is a product of the isoprenoid biosynthesis pathway which requires an allylic pyrophosphate and isopentenyl pyrophosphate (IPP) to initiate and elongate the rubber molecule. The biosynthesis of IPP occurs via two distinct routes: the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways. In this study, the expression of 34 genes related to rubber biosynthesis were compared between high and low latex yielding trees of two rubber tree clones, PB 217 and PB 260. Almost all tested genes revealed no significantly differential expression related to latex yield. Only mitochondrial pyruvate dehydrogenase (PDCE1) showed specific up-regulation in the high latex yielding trees of both tested clones. Interestingly, the expression of PDCE1 involving in the production of acetyl-CoA in mitochondria was also significantly induced by latex loss upon tapping. The increasing of acetyl-CoA and energy production may favor rubber tree to produce more latex. The in silico analysis showed that HbPDCE1 promoter contained ethylene and copper-responsive elements. Ethylene is worldwide used rubber stimulant while copper sulfate was also reported to be able to stimulate the latex yield. This suggested that HbPDCE1 may be transcriptionally regulated by these two compounds however the in vivo regulation of this gene should be further investigated.

Rubber tree (Hevea brasiliensis) is a pivotal source for natural rubber production. Polyisoprene is synthesized in laticifer vessels, which are developed from vascular cambium in the phloem area of the inner bark tissue. The transcription factors and phytohormones were reported to be involved in network regulation of plant vascular tissue differentiation. Methyl jasmonate (MeJA) was reported to act as a stimulator for laticifer vessel formation in rubber tree, but the regulatory mechanism remains largely unknown. In this study, the correlation of laticifer vessel proliferation in phloem tissue upon MeJA treatment and the MeJA-responsive gene expression was investigated. Histochemical study of the laticifer revealed the circle and diffused structure around the secondary phloem of the stem. The number of laticifer vessels was increased from the top shoot to the lower stem part. The higher average number of laticifer vessels in stems of high latex-producing clones than in low latex/high wood yield clones suggested the correlation of laticifer vessel number and latex yield potential. This study demonstrated the temporal differential expression of MeJA-responsive genes upon MeJA treatment and adjusted back to a normal level after 3 months in two high latex-producing clones. The increasing number of laticifer vessels in MeJA-treated plants confirmed the consequent effect of MeJA treatment, and the possible roles of these genes in relation with laticifer vessel proliferation are discussed. A better understanding of gene function in laticifer development would be beneficial in rubber tree improvement and exploitation.

Cassava (Manihot esculenta Crantz) is one of the most important crops of Thailand. Its storage roots are used as food, feed, starch production, and be the important source for biofuel and biodegradable plastic production. Despite the importance of cassava storage roots, little is known about the mechanisms involved in their formation. This present study has focused on comparison of the expression profiles of cassava root proteome at various developmental stages using two-dimensional gel electrophoresis and LC-MS/MS. Based on an anatomical study using Toluidine Blue, the secondary growth was confirmed to be essential during the development of cassava storage root. To investigate biochemical processes occurring during storage root maturation, soluble and membrane proteins were isolated from storage roots harvested from 3-, 6-, 9-, and 12-month-old cassava plants. The proteins with differential expression pattern were analysed and identified to be associated with 8 functional groups: protein folding and degradation, energy, metabolism, secondary metabolism, stress response, transport facilitation, cytoskeleton, and unclassified function. The expression profiling of membrane proteins revealed the proteins involved in protein folding and degradation, energy, and cell structure were highly expressed during early stages of development. Integration of these data along with the information available in genome and transcriptome databases is critical to expand knowledge obtained solely from the field of proteomics. Possible role of identified proteins were discussed in relation with the activities during storage root maturation in cassava.

About 90% of natural rubber is obtained from tapping of the rubber tree (Hevea brasiliensis) for latex, which is circulated in the laticifers. Nowadays, the world supply of natural rubber is not sufficient for global demand, thus the increased latex yield was significantly underlined. In addition, the demand for wood (as a xylem part) from rubber tree was also increased as renewable resource for various applications. Laticifers are found in the secondary phloem containing tissue of rubber tree trunk-inner soft bark. The number of laticifers varies in consistent with latex yield and in responses to jasmonic acid level. This present study was committed to comparative transcriptome analysis in laticifers containing mature phloem, mature xylem and newly developed stem tissues of high latex-yield clone (RRIT251, with more laticifers) and high wood-yield clone (RRIT402, with less laticifers) of rubber tree to classify the genes and pathways involved with phloem (with laticifers) and xylem cell differentiation. There were 49, 54, 46 and 50 of vascular development-related genes in primary and secondary tissues of phloem and xylem, respectively. Differentially expressed genes in jasmonic acid signaling pathway was established with their highest expression in phloem tissue with laticifer cells of RRIT251, while genes in auxin signaling and secondary cell wall biosynthetic pathways were up-regulated in xylem tissue of RRIT402 for high wood yield. Promoter analysis of candidate-differentially expressed genes suggested the related pathway and putative regulatory elements for gene regulation. This genome-wide exploration of vascular development-related genes unraveled a largely unknown gap of this special vascular development containing laticifers in rubber tree.

Sets of polymorphic expressed sequence tag–simple sequence repeat (EST-SSR) markers from the rubber tree (Hevea brasiliensis) have been published by many researchers, but none has been specifically developed to study latex and wood yield traits. In this study, a total 10,321 rubber tree EST sequences, generated from suppression subtractive hybridization-cDNA libraries of bark and latex of high- and low-yielding clones, were used as sources for SSR searching. A total of 432 EST-SSR loci were identified and it was possible to design primer pairs for a subset of 298 EST-SSRs. The highest proportion of EST-SSRs was represented by dinucleotide repeats (46.6 %), followed by trinucleotide repeats (44.3 %). Based on BLASTX analysis, 234 ESTs (80 %) showed similarity to genes in NCBI databases and could be divided into 120 putative proteins with known function and 114 unknown proteins. To enhance the resolution of an existing linkage map from previous work on a rubber tree RRIM600 × PB217 population, 69 EST-SSR markers from the above set were tested to be integrated into the reference genetic map. The enriched map of 18 linkage groups spanned 2054.2 cM in length, showed an average genetic distance of 4.3 cM between adjacent markers, and included 63 new EST-SSR markers. The enhanced map from this study provides a basis for comparative mapping using PCR-based markers and identification of expressed genes possibly affecting important traits of interest.

Plant microRNAs (miRNAs) have been demonstrated to play an important regulatory role in a variety of biological processes ranging from plant growth and development, defensive pathways, as well as biotic and abiotic stress responses. MiRNAs have been extensively studied in plant models with available genome sequence data. The Euphorbiaceae family is a large flowering plant family, which is of considerable economic importance, but relatively limited genome sequence data. The four Euphorbiaceous species; rubber tree, cassava, castor bean and leafy spurge; were selected for this study. Using conserved plant miRNAs sequences aligned to the GenBank expressed sequenced tag database of each target plants, and further potential precursor miRNA-secondary structure analysis, the 526 potential miRNAs belonging to 17 miRNA families and their targets were identified. The majority of miRNA families was able to be identified in cassava. The minimal folding free energy index of precursor miRNAs was significantly higher than other non-coding or coding RNAs. The largest group of miRNA target genes consists of regulatory genes predicted to encode transcription factors, while non-regulatory genes involving in various functions. Some miRNAs contain different targets, suggesting the diverse functions of miRNAs depending on plant species and environmental conditions. The 6 expressed Hbr-miRNAs were verified in rubber tree. Gaining insight into the miRNA targets will help us to understand the range of miRNA expression regulation and to more coherently describe the functional importance of miRNAs. These findings considerably facilitate the basis for broaden future investigation on the role of these miRNAs in the economic important Euphorbiaceous species.

Cassava (Manihot esculenta Crantz) is a root crop that accumulates large quantities of starch, and it is an important source of carbohydrate. Study on gene expressions during storage root development provides important information on storage root formation and starch accumulation as well as unlock new traits for improving of starch yield. cDNA-Amplified Fragment Length Polymorphism (AFLP) was used to compare gene expression profiles in fibrous and storage roots of cassava cultivar Kasetsart 50. Total of 155 differentially expressed transcript-derived fragments with undetectable or low expression in leaves were characterized and classified into 11 groups regarding to their functions. The four major groups were no similarity (20%), hypothetical or unknown proteins (17%), cellular metabolism and biosynthesis (17%) and cellular communication and signaling (14%). Interestingly, sulfite reductase (MeKD82), calcium-dependent protein kinase (CDPK) (MeKD83), ent-kaurene synthase (KS) (MeKD106) and hexose transporter (HT) (MeKD154) showed root-specific expression patterns. This finding is consistent with previously reported genes involved in the initiation of potato tuber. Semi-quantitative reverse transcription polymerase chain reaction of early-developed root samples confirmed that those four genes exhibited significant expression with similar pattern in the storage root initiation and early developmental stages. We proposed that KS and HT may involve in transient induction of CDPK expression, which may play an important role in the signaling pathway of storage root initiation. Sulfite reductase, on the other hand, may involve in storage root development by facilitating sulfur-containing protein biosynthesis or detoxifying the cyanogenic glucoside content through aspartate biosynthesis.

The rubber tree is an economically important plant that produces natural rubber for various industrial uses. The application of ethylene contributes to increased latex production in rubber trees; however, the molecular biology behind the effects of ethylene on latex yield remains to be elucidated. Recently, the intersection between microRNA (miRNA) regulation and phytohormone responses has been revealed. Insight into the regulation of miRNAs and their target genes should help to determine the functional importance of miRNAs as well as the role of miRNAs in signaling under ethylene stimulation in the rubber tree. In this study, hbr-miR159 and hbr-miR166 were down-regulated in bark under ethylene treatment. The ethylene also down-regulated ATHB15-like (Class III Homeodomain Leucine Zipper, HD-ZIP III) which have been extensively implicated in the regulation of primary and secondary vascular tissue pattern formation. The strong negative-regulation of ARF6/ARF8 caused by hbr-miR167 involved in an attenuation of vascular development and may gradually lead to bark dryness syndrome in the long term ethylene treatment. The negative correlation of hbr-miR172 and its target REF3 in the inner soft bark under ethylene treatment results in dramatic increases in latex yield in the ethylene-sensitive clone of the rubber tree. The overall results suggested that the differential expression of HD-ZIP III, miR167/ARF6, ARF8, and miR172/REF3 and related genes may play possible roles in the response to ethylene treatment, resulting in longer latex flow and increased latex yield.

Cassava is a starchy root crop for food and industrial applications in many countries around the world. Among the factors that affect cassava production, diseases remain the major cause of yield loss. Cassava anthracnose disease is caused by the fungus Colletotrichum gloeosporioides. Severe anthracnose attacks can cause tip die-backs and stem cankers, which can affect the availability of planting materials especially in large-scale production systems. Recent studies indicate that plants over- or under-express certain microRNAs (miRNAs) to cope with various stresses. Understanding how a disease-resistant plant protects itself from pathogens should help to uncover the role of miRNAs in the plant immune system. In this study, the disease severity assay revealed different response to C. gloeosporioides infection in two cassava cultivars. Quantitative RT-PCR analysis uncovered the differential expression of the two miRNAs and their target genes in the two cassava cultivars that were subjected to fungal infection. The more resistant cultivar revealed the up-regulation of miR160 and miR393, and consequently led to low transcript levels in their targets, ARF10 and TIR1, respectively. The more susceptible cultivar exhibited the opposite pattern. The cis-regulatory elements relevant to defense and stress responsiveness, fungal elicitor responsiveness and hormonal responses were the most prevalent present in the miRNAs gene promoter regions. The possible dual role of these specific miRNAs and their target genes associated with cassava responses to C. gloeosporioides is discussed. This is the first study to address the molecular events by which miRNAs which might play a role in fungal-infected cassava. A better understanding of the functions of miRNAs target genes should greatly increase our knowledge of the mechanism underlying susceptibility and lead to new strategies to enhance disease tolerance in this economically important crop.

Trunk phloem necrosis (TPN), a physiological bark disorder of the rubber tree (Hevea brasiliensis), is a serious problem that affects the yield of natural rubber. The resultant bark dryness occurs in up to half of a plantation's trees in almost every rubber tree plantation region, causing a great annual loss of dry rubber for natural rubber production. Different types of injury and physical damage caused by mechanical activation as well as environmental stresses cause physiological bark disorder in tree. Due to the essential role of miR166, miR393 and miR167 in vascular development and abiotic stress response in diverse plant species, it was interesting to investigate the role of these miRNAs in rubber trees, particularly during development of a physiological bark disorder. In this study, the expression pattern of miR166, miR393 and miR167; and their target genes, HD-ZIP III; TIR1 and ARF8, respectively; was demonstrated in healthy tree and different TPN trees. Their existence and function in vivo was validated using RNA ligase-mediated 5′ rapid amplification of cDNA ends. Taken together, the results suggest a possible dual role of these three miRNAs in maintaining normal bark regeneration in healthy trees, coping with overtapping by affecting the wound healing system leading to abnormal bark regeneration in overtapped-TPN trees, and act as additional forces that enhance the attenuation of vascular development resulting in bark necrosis and cell death in the natural-TPN tree. This is the first study to address the molecular events of miRNAs involved in the physiological bark disorder TPN in rubber tree. Further study will open the possibility to better understanding of physiological and molecular perspectives during TPN development, and lead to improvement of monitoring the exploitation of rubber tree plantations.

MicroRNAs (miRNAs) are a newly discovered class of noncoding endogenous small RNAs involved in plant growth and development as well as response to environmental stresses. miRNAs have been extensively studied in various plant species, however, only few information are available in cassava, which serves as one of the staple food crops, a biofuel crop, animal feed and industrial raw materials. In this study, the 169 potential cassava miRNAs belonging to 34 miRNA families were identified by computational approach. Interestingly, mes-miR319b was represented as the first putative mirtron demonstrated in cassava. A total of 15 miRNA clusters involving 7 miRNA families, and 12 pairs of sense and antisense strand cassava miRNAs belonging to six different miRNA families were discovered. Prediction of potential miRNA target genes revealed their functions involved in various important plant biological processes. The cis-regulatory elements relevant to drought stress and plant hormone response were identified in the promoter regions of those miRNA genes. The results provided a foundation for further investigation of the functional role of known transcription factors in the regulation of cassava miRNAs. The better understandings of the complexity of miRNA-mediated genes network in cassava would unravel cassava complex biology in storage root development and in coping with environmental stresses, thus providing more insights for future exploitation in cassava improvement.

Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to liber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbTIP1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2;1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbTIP1;1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1;1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbTIP1;1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1;1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2;1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow.

† Background and Aims The major economic product of Hevea brasiliensis is a rubber-containing cytoplasm (latex), which flows out of laticifers (latex cells) when the bark is tapped. The latex yield is stimulated by ethylene. Sucrose, the unique precursor of rubber synthesis, must cross the plasma membrane through specific sucrose transporters before being metabolized in the laticifers. The relative importance of sucrose transporters in determining latex yield is unknown. Here, the effects of ethylene (by application of Ethrel w ) on sucrose transporter gene expression in the inner bark tissues and latex cells of H. brasiliensis are described. † Methods Experiments, including cloning sucrose transporters, real time RT-PCR and in situ hybridization, were carried out on virgin (untapped) trees, treated or untreated with the latex yield stimulant Ethrel. † Key Results Seven putative full-length cDNAs of sucrose transporters were cloned from a latex-specific cDNA library. These transporters belong to all SUT (sucrose transporter) groups and differ by their basal gene expression in latex and inner soft bark, with a predominance of HbSUT1A and HbSUT1B. Of these sucrose transporters, only HbSUT1A and HbSUT2A were distinctly increased by ethylene. Moreover, this increase was shown to be specific to laticifers and to ethylene application. † Conclusion The data and all previous information on sucrose transport show that HbSUT1A and HbSUT2A are related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees.

Natural rubber is synthesized in specialized articulated cells (laticifers) located in the inner liber of Hevea brasiliensis. Upon bark tapping, the laticifer cytoplasm (latex) is expelled due to liber tissue turgor pressure. In mature virgin (untapped) trees, short-term kinetic studies confirmed that ethylene, the rubber yield stimulant used worldwide, increased latex yield, with a concomitant decrease in latex total solid content, probably through water influx in the laticifers. As the mature laticifers are devoid of plasmodesmata, the rapid water exchanges with surrounding liber cells probably occur via the aquaporin pathway. Two full-length aquaporin cDNAs (HbPIP2;1 and HbTIP1;1, for plasma membrane intrinsic protein and tonoplast intrinsic protein, respectively) were cloned and characterized. The higher efficiency of HbPIP2;1 than HbTIP1;1 in increasing plasmalemma water conductance was verified in Xenopus laevis oocytes. HbPIP2;1 was insensitive to HgCl2 . In situ hybridization demonstrated that HbPIP2;1 was expressed in all liber tissues in the young stem, including the laticifers. HbPIP2;1 was up-regulated in both liber tissues and laticifers, whereas HbTIP1;1 was down-regulated in liber tissues but up-regulated in laticifers in response to bark Ethrel treatment. Ethylene-induced HbPIP2;1 up-regulation was confirmed by western-blot analysis. The promoter sequences of both genes were cloned and found to harbor, among many others, ethylene-responsive and other chemical-responsive (auxin, copper, and sulfur) elements known to increase latex yield. Increase in latex yield in response to ethylene was emphasized to be linked with water circulation between the laticifers and their surrounding tissues as well as with the probable maintenance of liber tissue turgor, which together favor prolongation of latex flow.

The latex of Hevea brasiliensis, expelled upon bark tapping, is the cytoplasm of anastomosed latex cells in the inner bark of the rubber tree. Latex regeneration between two tappings is one of the major limiting factors of rubber yield. Hevea species contain high amounts of cyanogenic glucosides from which cyanide is released when the plant is damaged providing an efficient defense mechanism against herbivores. In H. brasiliensis, the cyanogenic glucosides mainly consist of the monoglucoside linamarin (synthesized in the leaves), and its diglucoside transport-form, linustatin. Variations in leaf cyanide potential (CNp) were studied using various parameters. Results showed that the younger the leaf, the higher the CNp. Leaf CNp greatly decreased when leaves were directly exposed to sunlight. These results allowed us to determine the best leaf sampling conditions for the comparison of leaf CNp. Under these conditions, leaf CNp was found to vary from less than 25 mM to more than 60 mM. The rubber clones containing the highest leaf CNp were those with the highest yield potential. In mature virgin trees, the CNp of the trunk inner bark was shown to be proportional to leaf CNp and to decrease on tapping. However, the latex itself exhibited very low (if any) CNp, while harboring all the enzymes (beta-D-diglucosidase, linamarase and beta-cyanoalanine synthase) necessary to metabolize cyanogenic glucosides to generate non-cyanogenic compounds, such as asparagine. This suggests that in the rubber tree bark, cyanogenic glucosides may be a source of buffering nitrogen and glucose, thereby contributing to latex regeneration/production.