Genome-Wide Identification and Analysis of Small Nucleolar RNAs and Their Roles in Regulating Latex Regeneration in the Rubber Tree (Hevea brasiliensis)

Small nucleolar RNAs (snoRNAs) are a class of conserved nuclear RNAs that play important roles in the modification of ribosomal RNAs (rRNAs) in plants. In rubber trees, rRNAs are run off with latex flow during tapping and need to be regenerated for maintaining the functions of the laticifer cells. SnoRNAs are expected to play essential roles in the regeneration of rRNAs. However, snoRNAs in the rubber tree have not been sufficiently characterized thus far. In this study, we performed nuclear RNA sequencing (RNA-seq) to identify snoRNAs globally and investigate their roles in latex regeneration. We identified a total of 3,626 snoRNAs by computational prediction with nuclear RNA-seq data. Among these snoRNAs, 50 were highly expressed in latex; furthermore, the results of reverse transcription polymerase chain reaction (RT-PCR) showed the abundant expression of 31 of these snoRNAs in latex. The correlation between snoRNA expression and adjusted total solid content (TSC/C) identified 13 positively yield-correlated snoRNAs. To improve the understanding of latex regeneration in rubber trees, we developed a novel insulated tapping system (ITS), which only measures the latex regenerated in specific laticifers. Using this system, a laticifer-abundant snoRNA, HbsnoR28, was found to be highly correlated with latex regeneration. To the best of our knowledge, this is the first report to globally identify snoRNAs that might be involved in latex regeneration regulation and provide new clues for unraveling the mechanisms underlying the regulation of latex regeneration.

Transcript Profiling Provides Insights into the Molecular Mechanisms of Harvesting-activated Latex Regeneration in Virgin Rubber Trees

Background: Natural rubber, an important industrial raw material with wide applications, is harvested in the form of latex (cytoplasm of rubber-producing laticifers) from Hevea brasiliensis (para rubber tree) by the way of tapping, i.e. removing a slice of trunk bark by a special knife. In regularly tapped rubber trees, latex regeneration consists of one of the main yield-limiting factors for rubber productivity. Conspicuous stimulation on latex production for the first few tappings makes virgin (untapped before) rubber trees an ideal model to investigate the regulatory mechanisms of latex regeneration. To understand the underlying mechanisms, genome-wide transcript profiling was conducted with a silver-staining cDNA-AFLP technology against the latex samples for the first five tappings.Results: A total of 505 non-redundant differentially expressed (DE) transcript-derived fragments (TDFs) were identified, of which 217 were up-regulated, 180 down-regulated, and 108 bell type-regulated among the five tappings. About 72.5% of these DE-TDFs were functionally annotated, and classified into 11 functional categories, which were discussed with reference to harvesting-stimulated latex regeneration. The importance of sugar metabolism and rubber biosynthesis was highlighted, due to the fact that most of the DE-TDFs annotated in sucrose transport, sugar catabolism, glycolysis, tricarboxylic acid cycle and pentose-phosphate pathway and nine of the ten rubber biosynthesis pathway DE-TDFs were up-regulated by the tapping treatment. More than one tenth of the total DE-TDFs were randomly selected for expression validation by semi-quantitative RT-PCR, and 83.8% showed patterns consistent with their original cDNA-AFLP gel profiles. Moreover, quantitative RT-PCR analysis revealed an 89.7% consistency for the 29 latex-regeneration related DE-TDFs examined.Conclusions: In brief, our results indicate the tapping treatment incurs extensive physiological and molecular changes in the laticifers of virgin rubber trees. The vast numbers of tapping-responsive DE-TDFs identified here provide a basis for unravelling the gene regulatory network for latex regeneration in regularly harvested rubber trees.

In situ 13CO2 labelling of rubber trees reveals a seasonal shift in the contribution of the carbon sources involved in latex regeneration

Rubber trees (Hevea brasiliensis) are the main source of natural rubber, extracted from latex, which exudes from the trunk after tapping. Tapped trees require large amounts of carbon (C) to regenerate the latex after its collection. Knowing the contribution of C sources involved in latex biosynthesis will help in understanding how rubber trees face this additional C demand. Whole crown 13CO2 pulse labelling was performed on 4-year-old rubber trees in June, when latex production was low, and in October, when it was high. 13C content was quantified in the foliage, phloem sap, wood, and latex. In both labelling periods, 13C was recovered in latex just after labelling, indicating that part of the carbohydrate was directly allocated to latex. However, significant amounts of 13C were still recovered in latex after 100 d and the peak was reached significantly later than in phloem sap, demonstrating the contribution of a reserve pool as a source of latex C. The contribution of new photosynthates to latex regeneration was faster and higher when latex metabolism was well established, in October, than in June. An improved understanding of C dynamics and the source–sink relationship in rubber tree is crucial to adapt tapping system practices and ensure sustainable latex production.

Contribution of the carbon sources involved in latex regeneration in rubber trees (Hevea brasiliensis): an in situ 13CO2 labelling experiment

<p>Rubber trees (<em>Hevea brasiliensis</em>) are the main source of natural rubber, extracted from latex, which exudes from the trunk after tapping. Tapped trees require large amounts of carbon (C) to regenerate the latex after its collection. Knowing the contribution of C sources involved in latex biosynthesis will help understand how rubber trees face this additional C demand. Whole crown<sup> 13</sup>CO<sub>2 </sub>pulse labelling was performed on 4-year-old rubber trees in June when latex production was low and in October, when it was high. <sup>13</sup>C contents were quantified in the foliage, phloem sap, wood and latex. In both labelling periods, <sup>13</sup>C was recovered in latex just after labelling, indicating that part of the carbohydrates was directly allocated to latex. However, significant <sup>13</sup>C amounts were still recovered in latex after 100 days and the peak was reached significantly later than in phloem sap, demonstrating the contribution of a reserve pool as a source of latex C. The contribution of new photosynthates to latex regeneration was faster and higher when latex metabolism was well established, in October than in June. An improved understanding of C dynamics and source-sink relationship in rubber tree is crucial to adapt tapping system practices and ensure sustainable latex production.</p>

Starch Synthesis and Mobilization in Wood and Bark of Rubber Tree, in Relation with Latex Production, (1) Methodological Approach

In rubber tree, starch reserves are necessary for growth and latex regeneration when the demand exceeds supply from photosynthesis. It tends to accumulate in the wood near the tapping cut [1,2] whereas sucrose remains rather stable in the wood and decreases in the latex vessels where it is used to regenerate the exported latex [3].Thus higher starch ability could sustain higher latex yield. However the enzymatic processes driving the dynamics of starch synthesis and hydrolysis as related to tapping are not known. The objective of the study is to analyze the effects of tapping on the enzymes involved in starch and sucrose metabolism in the wood of rubber trees. The first approach of this study was to set up the most adapted methodology on measurement of total nonstructural carbohydrates (NSC) and related enzymes activities. The experiment was conducted in Heveabrasiliensis (rubber tree), clone RRIM600. Treatments include untapped trees (Control) and yielding trees tapped with Ethephon stimulation (ET). Each treatment includes 6 trees. Samples have been collected along the trunk and separated into 2 parts, wood and bark, from both side of the tree tapped and untapped panel. The activity of acid invertase (AI), amylase (AMY), sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) were assessed in the part of soft bark with amethod of sample preparation recoveringmore protein and concentrated enzyme with acetone precipitation. The method has increased enzyme activities of SuSy and SPS. On another hand, starch, sucrose, fructose and glucose concentrations have been enzymatically measured.The results showed that starch was the major component in wood and sucrose was mostly found in bark. There was no difference between the former drainage area and resting area after 2 untapped years before restarting tapping.