GARP transcription factors repress Arabidopsis nitrogen starvation response via ROS-dependent and -independent pathways.

Plants need to cope with strong variations of nitrogen availability in the soil. Although many molecular players are being discovered concerning how plants perceive NO3- provision, it is less clear how plants recognize a lack of nitrogen. Following nitrogen removal, plants activate their nitrogen starvation response (NSR), which is characterized by the activation of very high-affinity nitrate transport systems (NRT2.4 and NRT2.5) and other sentinel genes involved in N remobilization such as GDH3. Using a combination of functional genomics via transcription factor perturbation and molecular physiology studies, we show that the transcription factors belonging to the HHO subfamily are important regulators of NSR through two potential mechanisms. First, HHOs directly repress the high-affinity nitrate transporters, NRT2.4 and NRT2.5. hho mutants display increased high-affinity nitrate transport activity, opening up promising perspectives for biotechnological applications. Second, we show that reactive oxygen species (ROS) are important to control NSR in wild-type plants and that HRS1 and HHO1 overexpressors and mutants are affected in their ROS content, defining a potential feed-forward branch of the signaling pathway. Taken together, our results define the relationships of two types of molecular players controlling the NSR, namely ROS and the HHO transcription factors. This work (i) up opens perspectives on a poorly understood nutrient-related signaling pathway and (ii) defines targets for molecular breeding of plants with enhanced NO3- uptake.

Evolutionary transcriptomics reveals the origins of olives and the genomic changes associated with their domestication.

The olive (Olea europaea L. subsp. europaea) is one of the oldest and most socio-economically important cultivated perennial crop in the Mediterranean region. Yet, its origins are still under debate and the genetic bases of the phenotypic changes associated with its domestication are unknown. We generated RNA-sequencing data for 68 wild and cultivated olive trees to study the genetic diversity and structure both at the transcription and sequence levels. To localize putative genes or expression pathways targeted by artificial selection during domestication, we employed a two-step approach in which we identified differentially expressed genes and screened the transcriptome for signatures of selection. Our analyses support a major domestication event in the eastern part of the Mediterranean basin followed by dispersion towards the West and subsequent admixture with western wild olives. While we found large changes in gene expression when comparing cultivated and wild olives, we found no major signature of selection on coding variants and weak signals primarily affected transcription factors. Our results indicated that the domestication of olives resulted in only moderate genomic consequences and that the domestication syndrome is mainly related to changes in gene expression, consistent with its evolutionary history and life history traits.

Structural and Functional Annotation of Transposable Elements Revealed a Potential Regulation of Genes Involved in Rubber Biosynthesis by TE-Derived siRNA Interference in Hevea brasiliensis.

The natural rubber biosynthetic pathway is well described in Hevea, although the final stages of rubber elongation are still poorly understood. Small Rubber Particle Proteins and Rubber Elongation Factors (SRPPs and REFs) are proteins with major function in rubber particle formation and stabilization. Their corresponding genes are clustered on a scaffold1222 of the reference genomic sequence of the Hevea brasiliensis genome. Apart from gene expression by transcriptomic analyses, to date, no deep analyses have been carried out for the genomic environment of SRPPs and REFs loci. By integrative analyses on transposable element annotation, small RNAs production and gene expression, we analysed their role in the control of the transcription of rubber biosynthetic genes. The first in-depth annotation of TEs (Transposable Elements) and their capacity to produce TE-derived siRNAs (small interfering RNAs) is presented, only possible in the Hevea brasiliensis clone PB 260 for which all data are available. We observed that 11% of genes are located near TEs and their presence may interfere in their transcription at both genetic and epigenetic level. We hypothesized that the genomic environment of rubber biosynthesis genes has been shaped by TE and TE-derived siRNAs with possible transcriptional interference on their gene expression. We discussed possible functionalization of TEs as enhancers and as donors of alternative transcription start sites in promoter sequences, possibly through the modelling of genetic and epigenetic landscapes.

Overexpression of Hevea brasiliensis ethylene response factor HbERF-IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation.

Ethylene response factor 1 (ERF1) is an essential integrator of the jasmonate and ethylene signalling pathways coordinating a large number of genes involved in plant defences. Its orthologue in Hevea brasiliensis, HbERF-IXc5, has been assumed to play a major role in laticifer metabolism and tolerance to harvesting stress for better latex production. This study sets out to establish and characterize rubber transgenic lines overexpressing HbERF-IXc5. Overexpression of HbERF-IXc5 dramatically enhanced plant growth and enabled plants to maintain some ecophysiological parameters in response to abiotic stress such as water deficit, cold and salt treatments. This study revealed that HbERF-IXc5 has rubber-specific functions compared to Arabidopsis ERF1 as transgenic plants overexpressing HbERF-IXc5 accumulated more starch and differentiated more latex cells at the histological level. The role of HbERF-IXc5 in driving the expression of some target genes involved in laticifer differentiation is discussed.

Chemotherapy and radiotherapy in nasopharyngeal carcinoma: an update of the MAC-NPC meta-analysis.

BACKGROUND: A previous individual patient data meta-analysis by the Meta-Analysis of Chemotherapy in Nasopharynx Carcinoma (MAC-NPC) collaborative group to assess the addition of chemotherapy to radiotherapy showed that it improves overall survival in nasopharyngeal carcinoma. This benefit was restricted to patients receiving concomitant chemotherapy and radiotherapy. The aim of this study was to update the meta-analysis, include recent trials, and to analyse separately the benefit of concomitant plus adjuvant chemotherapy. METHODS: We searched PubMed, Web of Science, Cochrane Controlled Trials meta-register, ClinicalTrials.gov, and meeting proceedings to identify published or unpublished randomised trials assessing radiotherapy with or without chemotherapy in patients with non-metastatic nasopharyngeal carcinoma and obtained updated data for previously analysed studies. The primary endpoint of interest was overall survival. All trial results were combined and analysed using a fixed-effects model. The statistical analysis plan was pre-specified in a protocol. All data were analysed on an intention-to-treat basis. FINDINGS: We analysed data from 19 trials and 4806 patients. Median follow-up was 7·7 years (IQR 6·2-11·9). We found that the addition of chemotherapy to radiotherapy significantly improved overall survival (hazard ratio [HR] 0·79, 95% CI 0·73-0·86, p<0·0001; absolute benefit at 5 years 6·3%, 95% CI 3·5-9·1). The interaction between treatment effect (benefit of chemotherapy) on overall survival and the timing of chemotherapy was significant (p=0·01) in favour of concomitant plus adjuvant chemotherapy (HR 0·65, 0·56-0·76) and concomitant without adjuvant chemotherapy (0·80, 0·70-0·93) but not adjuvant chemotherapy alone (0·87, 0·68-1·12) or induction chemotherapy alone (0·96, 0·80-1·16). The benefit of the addition of chemotherapy was consistent for all endpoints analysed (all p<0·0001): progression-free survival (HR 0·75, 95% CI 0·69-0·81), locoregional control (0·73, 0·64-0·83), distant control (0·67, 0·59-0·75), and cancer mortality (0·76, 0·69-0·84). INTERPRETATION: Our results confirm that the addition of concomitant chemotherapy to radiotherapy significantly improves survival in patients with locoregionally advanced nasopharyngeal carcinoma. To our knowledge, this is the first analysis that examines the effect of concomitant chemotherapy with and without adjuvant chemotherapy as distinct groups. Further studies on the specific benefits of adjuvant chemotherapy after concomitant chemoradiotherapy are needed. FUNDING: French Ministry of Health (Programme d'actions intégrées de recherche VADS), Ligue Nationale Contre le Cancer, and Sanofi-Aventis.

Development of a new pCAMBIA binary vector using Gateway® technology.

pCAMBIA vectors have become popular for their easy handling, stability and the existence of a range of selection and reporter genes. However, these vectors have yet to integrate the Gateway® cloning system, which has enabled site-specific recombination without the need for restriction enzymes and ligases. This paper sets out to convert the pCambia2300 binary vector into a destination vector with the Gateway® cassette driven by the CaMV35S promoter. The destination vector, pCamway35S, was then evaluated using the uidA reporter gene. Transient and stable transformation experiments were successfully assayed, either by particle bombardment or by Agrobacterium tumefaciens in Allium cepa and Hevea embryogenic calli. After counting the transformation units, the statistical analysis performed on the data showed that the pCamway 35S::uidA vector was as efficient as pCambia2301, a pCAMBIA2300 containing the uidA reporter gene under the CaMV 35S promoter.

Involvement of Ethylene in the Latex Metabolism and Tapping Panel Dryness of Hevea brasiliensis.

Ethephon, an ethylene releaser, is used to stimulate latex production in Hevea brasiliensis. Ethylene induces many functions in latex cells including the production of reactive oxygen species (ROS). The accumulation of ROS is responsible for the coagulation of rubber particles in latex cells, resulting in the partial or complete stoppage of latex flow. This study set out to assess biochemical and histological changes as well as changes in gene expression in latex and phloem tissues from trees grown under various harvesting systems. The Tapping Panel Dryness (TPD) susceptibility of Hevea clones was found to be related to some biochemical parameters, such as low sucrose and high inorganic phosphorus contents. A high tapping frequency and ethephon stimulation induced early TPD occurrence in a high latex metabolism clone and late occurrence in a low latex metabolism clone. TPD-affected trees had smaller number of laticifer vessels compared to healthy trees, suggesting a modification of cambial activity. The differential transcript abundance was observed for twenty-seven candidate genes related to TPD occurrence in latex and phloem tissues for ROS-scavenging, ethylene biosynthesis and signalling genes. The predicted function for some Ethylene Response Factor genes suggested that these candidate genes should play an important role in regulating susceptibility to TPD.

Transcriptional and post-transcriptional regulation of the jasmonate signalling pathway in response to abiotic and harvesting stress in Hevea brasiliensis.

BACKGROUND: Latex harvesting in Hevea brasiliensis amounts to strong abiotic stress that can cause a halt in production in the most susceptible clones. Although the role of jasmonic acid has been suggested in laticifer differentiation, its role in latex production and in the response to harvesting stress has received very little attention. Only a few key genes acting in the COI-JAZ-MYC module have been isolated and studied at transcriptional level. RESULTS: Use of a reference transcriptome obtained on rubber clone PB 260 covering a large number of tissues under different environmental conditions enabled us to identify 24 contigs implicated in the jasmonate signalling pathway in the rubber tree. An analysis of their expression profile by qPCR, combined with hierarchical clustering, suggested that the jasmonate signalling pathway is highly activated in laticifer cells and, more particularly, in the response to harvesting stress. By comparison with their genomic sequences, the existence of regulation by alternative splicing was discovered for JAZ transcripts in response to harvesting stress. Lastly, positive transcriptional regulation of the HbJAZ_1405 gene by MYC was demonstrated. CONCLUSION: This study led to the identification of all actors of jasmonate signalling pathway and revealed a specific gene expression pattern in latex cells. In-depth analysis of this regulation showed alternative splicing that has been previously shown in Arabidopsis. Interestingly, genotypic variation was observed in Hevea clones with contrasting latex metabolism. This result suggests an involvement of jasmonate signalling pathway in latex production. The data suggest that specific variability of the JA pathway may have some major consequences for resistance to stress. The data support the hypothesis that a better understanding of transcriptional regulations of jasmonate pathway during harvesting stress, along with the use of genotypic diversity in response to such stress, can be used to improve resistance to stress and rubber production in Hevea.

Identification of the Hevea brasiliensis AP2/ERF superfamily by RNA sequencing.

BACKGROUND: Rubber tree (Hevea brasiliensis) laticifers are the source of natural rubber. Rubber production depends on endogenous and exogenous ethylene (ethephon). AP2/ERF transcription factors, and especially Ethylene-Response Factors, play a crucial role in plant development and response to biotic and abiotic stresses. This study set out to sequence transcript expressed in various tissues using next-generation sequencing and to identify AP2/ERF superfamily in the rubber tree. RESULTS: The 454 sequencing technique was used to produce five tissue-type transcript libraries (leaf, bark, latex, embryogenic tissues and root). Reads from all libraries were pooled and reassembled to improve mRNA lengths and produce a global library. One hundred and seventy-three AP2/ERF contigs were identified by in silico analysis based on the amino acid sequence of the conserved AP2 domain from the global library. The 142 contigs with the full AP2 domain were classified into three main families (20 AP2 members, 115 ERF members divided into 11 groups, and 4 RAV members) and 3 soloist members. Fifty-nine AP2/ERF transcripts were found in latex. Alongside the microRNA172 already described in plants, eleven additional microRNAs were predicted to inhibit Hevea AP2/ERF transcripts. CONCLUSIONS: Hevea has a similar number of AP2/ERF genes to that of other dicot species. We adapted the alignment and classification methods to data from next-generation sequencing techniques to provide reliable information. We observed several specific features for the ERF family. Three HbSoloist members form a group in Hevea. Several AP2/ERF genes highly expressed in latex suggest they have a specific function in Hevea. The analysis of AP2/ERF transcripts in Hevea presented here provides the basis for studying the molecular regulation of latex production in response to abiotic stresses and latex cell differentiation.

Regulation of MIR genes in response to abiotic stress in Hevea brasiliensis.

Increasing demand for natural rubber (NR) calls for an increase in latex yield and also an extension of rubber plantations in marginal zones. Both harvesting and abiotic stresses lead to tapping panel dryness through the production of reactive oxygen species. Many microRNAs regulated during abiotic stress modulate growth and development. The objective of this paper was to study the regulation of microRNAs in response to different types of abiotic stress and hormone treatments in Hevea. Regulation of MIR genes differs depending on the tissue and abiotic stress applied. A negative co-regulation between HbMIR398b with its chloroplastic HbCuZnSOD target messenger is observed in response to salinity. The involvement of MIR gene regulation during latex harvesting and tapping panel dryness (TPD) occurrence is further discussed.

Immunogenicity and Safety of a Hexavalent DTwP-IPV-HB-PRP~T Vaccine Versus Separate DTwP-HB-PRP~T, bOPV, and IPV Vaccines Administered at 2, 4, 6 Months of Age Concomitantly With Rotavirus and Pneumococcal Conjugate Vaccines in Healthy Infants in Thailand.

BACKGROUND: This study investigated the immunogenicity and safety of a fully liquid, hexavalent, diphtheria (D)-tetanus (T)-whole-cell pertussis (wP)-inactivated poliovirus (IPV)-hepatitis B (HB)- Haemophilus influenzae b (PRP-T) vaccine compared to licensed DTwP-HB-PRP~T, IPV, and bivalent oral poliovirus (bOPV) vaccines following co-administration with other pediatric vaccines [pneumococcal conjugate vaccine (PCV13) and rotavirus vaccine]. METHODS: Phase III, randomized, open-label study in Thailand. Healthy infants received DTwP-IPV-HB-PRP~T at 2, 4 and 6 months of age (N = 228), or DTwP-HB-PRP~T and bOPV (2, 4 and 6 months of age) and IPV (4 months of age) (N = 231). All participants received PCV13 (2, 4 and 6 months of age) and rotavirus vaccine (2 and 4 months of age). Immunogenicity for all antigens was assessed using validated assays, and noninferiority post-third dose was evaluated for anti-D, anti-T, anti-pertussis [anti-pertussis toxin (anti-PT) and anti-fimbriae 2/3 (anti-FIM)], anti-polio 1, 2, 3, anti-HB, and anti-PRP~T. Safety was assessed using parental reports. RESULTS: Noninferiority was demonstrated for each antigen, and overall noninferiority of DTwP-IPV-HB-PRP~T versus DTwP-HB-PRP~T+bOPV+IPV was concluded. Similarity in each group was observed for the GMC ratio for antirotavirus antibodies (20.9 and 17.3, respectively) and anti-PCV13 antibodies (range: 8.46-32.6 and 7.53-33.1, respectively). Two serious adverse events were related to DTwP-IPV-HB-PRP~T (febrile convulsion and acute febrile illness) and 1 was related to DTwP-HB-PRP~T+bOPV+IPV (febrile seizure), but overall there were no safety concerns with similar rates of participants experiencing solicited (99.1% and 98.3%) and unsolicited (19.3% and 19.5%) adverse events in each group. CONCLUSIONS: This study confirmed the suitability of DTwP-IPV-HB-PRP~T primary series vaccination in combination with rotavirus and PCV13 vaccines.

A multi-level approach reveals key physiological and molecular traits in the response of two rice genotypes subjected to water deficit at the reproductive stage.

Rice is more vulnerable to drought than maize, wheat, and sorghum because its water requirements remain high throughout the rice life cycle. The effects of drought vary depending on the timing, intensity, and duration of the events, as well as on the rice genotype and developmental stage. It can affect all levels of organization, from genes to the cells, tissues, and/or organs. In this study, a moderate water deficit was applied to two contrasting rice genotypes, IAC 25 and CIRAD 409, during their reproductive stage. Multi-level transcriptomic, metabolomic, physiological, and morphological analyses were performed to investigate the complex traits involved in their response to drought. Weighted gene network correlation analysis was used to identify the specific molecular mechanisms regulated by each genotype, and the correlations between gene networks and phenotypic traits. A holistic analysis of all the data provided a deeper understanding of the specific mechanisms regulated by each genotype, and enabled the identification of gene markers. Under non-limiting water conditions, CIRAD 409 had a denser shoot, but shoot growth was slower despite better photosynthetic performance. Under water deficit, CIRAD 409 was weakly affected regardless of the plant level analyzed. In contrast, IAC 25 had reduced growth and reproductive development. It regulated transcriptomic and metabolic activities at a high level, and activated a complex gene regulatory network involved in growth-limiting processes. By comparing two contrasting genotypes, the present study identified the regulation of some fundamental processes and gene markers, that drive rice development, and influence its response to water deficit, in particular, the importance of the biosynthetic and regulatory pathways for cell wall metabolism. These key processes determine the biological and mechanical properties of the cell wall and thus influence plant development, organ expansion, and turgor maintenance under water deficit. Our results also question the genericity of the antagonism between morphogenesis and organogenesis observed in the two genotypes.

Antibody Persistence Following Administration of a Hexavalent DTwP-IPV-HB-PRP~T Vaccine Versus Separate DTwP-HB-PRP~T and IPV Vaccines and Safety and Immunogenicity of a Booster Dose of DTwP-IPV-HB-PRP~T Administered With an MMR Vaccine in Healthy Infants in India.

BACKGROUND: Antibody persistence of a whole-cell pertussis-containing hexavalent vaccine (DTwP-IPV-HB-PRP~T) and its co- or sequential administration with measles, mumps, rubella (MMR) vaccine were evaluated. METHODS: Phase III, open-label, randomized, multicenter study in India. Healthy toddlers 12-24 months of age who had received DTwP-IPV-HB-PRP~T or separate DTwP-HB-PRP~T+IPV primary vaccination at 6-8, 10-12 and 14-16 weeks of age received a DTwP-IPV-HB-PRP~T booster concomitantly with MMR (N = 336) or 28 days before MMR (N = 340). Participants had received a first dose of measles vaccine. Immunogenicity assessment used validated assays and safety was by parental reports. All analyses were descriptive. RESULTS: All participants had prebooster anti-T ≥0.01 IU/mL and anti-polio 1 and 3 ≥8 1/dil, and ≥96.5% had anti-D ≥0.01 IU/mL, anti-HBs ≥10 mIU/mL, anti-polio 2 ≥8 1/dil and anti-PRP ≥0.15 µg/mL; for pertussis, antibody persistence was similar in each group. Postbooster immunogenicity for DTwP-IPV-HB-PRP~T was similar for each antigen in each group: ≥99.5% of participants had anti-D ≥0.01 IU/mL, anti-T ≥0.01 IU/mL, anti-polio 1, 2 and 3 >8 1/dil, anti-HBs ≥10 mIU/mL and anti-PRP ≥1 µg/mL; for pertussis, vaccine response was similar in each group [72.0%-75.9% (anti-PT), 80.8%-81.4% (anti-FIM), 77.6%-79.5% (anti-PRN), 78.2%-80.8% (anti-FHA)]. There was no difference in MMR immunogenicity between groups, and no difference in DTwP-IPV-HB-PRP~T booster immunogenicity based on the primary series. There were no safety concerns. CONCLUSIONS: DTwP-IPV-HB-PRP~T antibody persistence was similar to licensed comparators. Booster immunogenicity was robust after DTwP-IPV-HB-PRP~T with or without MMR, and MMR immunogenicity was not affected by coadministration with DTwP-IPV-HB-PRP~T. CLINICAL TRIALS REGISTRY INDIA NUMBER: CTRI/2020/04/024843.

Identification of novel microRNAs in Hevea brasiliensis and computational prediction of their targets.

BACKGROUND: Plants respond to external stimuli through fine regulation of gene expression partially ensured by small RNAs. Of these, microRNAs (miRNAs) play a crucial role. They negatively regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs (mRNAs). In Hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. This study set out to identify abiotic stress-related miRNAs in Hevea using next-generation sequencing and bioinformatic analysis. RESULTS: Deep sequencing of small RNAs was carried out on plantlets subjected to severe abiotic stress using the Solexa technique. By combining the LeARN pipeline, data from the Plant microRNA database (PMRD) and Hevea EST sequences, we identified 48 conserved miRNA families already characterized in other plant species, and 10 putatively novel miRNA families. The results showed the most abundant size for miRNAs to be 24 nucleotides, except for seven families. Several MIR genes produced both 20-22 nucleotides and 23-27 nucleotides. The two miRNA class sizes were detected for both conserved and putative novel miRNA families, suggesting their functional duality. The EST databases were scanned with conserved and novel miRNA sequences. MiRNA targets were computationally predicted and analysed. The predicted targets involved in "responses to stimuli" and to "antioxidant" and "transcription activities" are presented. CONCLUSIONS: Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs when the complete genome is not yet available. Our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in Hevea.

Some ethylene biosynthesis and AP2/ERF genes reveal a specific pattern of expression during somatic embryogenesis in Hevea brasiliensis.

BACKGROUND: Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. RESULTS: The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. CONCLUSIONS: This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult.

An Epigenetic Alphabet of Crop Adaptation to Climate Change.

Crop adaptation to climate change is in a part attributed to epigenetic mechanisms which are related to response to abiotic and biotic stresses. Although recent studies increased our knowledge on the nature of these mechanisms, epigenetics remains under-investigated and still poorly understood in many, especially non-model, plants, Epigenetic modifications are traditionally divided into two main groups, DNA methylation and histone modifications that lead to chromatin remodeling and the regulation of genome functioning. In this review, we outline the most recent and interesting findings on crop epigenetic responses to the environmental cues that are most relevant to climate change. In addition, we discuss a speculative point of view, in which we try to decipher the "epigenetic alphabet" that underlies crop adaptation mechanisms to climate change. The understanding of these mechanisms will pave the way to new strategies to design and implement the next generation of cultivars with a broad range of tolerance/resistance to stresses as well as balanced agronomic traits, with a limited loss of (epi)genetic variability.

Variation in GUS activity in vegetatively propagated Hevea brasiliensis transgenic plants.

Hevea brasiliensis transgenic plants are regenerated from transgenic callus lines by somatic embryogenesis. Somatic embryogenesis is not yet available for commercial propagation of Hevea clones, which requires conventional grafting of buds on rootstock seedlings (budding). The stability of transgene expression in budded plants is therefore necessary for further development of genetic engineering in rubber trees. Transgene expression was assessed by fluorimetric beta-glucuronidase (GUS) activity in fully developed leaves of in vitro plants from transgenic lines and their sub-lines obtained by budding. A large variation in GUS activity was found in self-rooted in vitro plants of five transgenic lines, and the absence of activity in one line suggested transgene silencing. Beyond confirming transmissibility of the reporter gene by budding and long-term expression, a quantification of GUS activity revealed that greater variability existed in budded plants compared to self-rooted mother in vitro plants for three transgenic lines. Although somatic embryogenesis provided more stable GUS activity, budding remained an efficient way of propagating transgenic plants but transgene expression in budded plants should be verified for functional analysis and further development.

Groundwater quality changes in peri-urban areas of the Walloon region of Belgium.

An extensive survey of groundwater quality was performed at the regional scale in peri-urban and industrial contexts of the Walloon Region (Belgium). To this end, 243 sampling locations from 8 areas located in different geological contexts and different peri-urban areas of the region were sampled outside pollution hotspots related to contaminated sites. Each groundwater sample was analyzed for 19 inorganic trace elements, 59 organic micro-pollutants and 8 major and minor elements. Five physico-chemical parameters were measured in the field, at the time of sampling to determine the environmental conditions prevailing in groundwater. Existing groundwater chemical data available outside such peri-urban and industrial areas were also compiled from existing groundwater quality databases for comparison. Most of the organic and inorganic pollutants are detected and their levels of occurrence are described statistically. Cumulative distribution function plots allow comparing the distributions of inorganic trace elements in and outside the urbanized and industrialized areas and among the different prevailing geological contexts. Two indicators are introduced, one quantifying the impact on inorganic trace elements of diffuse pollution in urban and industrial areas, the other reflecting the influence of lithology on concentrations in groundwater. Results show that the composition of groundwater is altered in peri-urban and industrial areas with increased concentrations for several organic pollutants and for most inorganic trace elements. However, the results clearly indicate that for the inorganic trace elements, anthropogenic influence is not limited to increased discharge of pollutants in the urban and industrial areas but also to significant changes in environmental conditions, with more reducing and acidic conditions observed in groundwater. The influence of lithology is also marked in the data set. From a more pragmatic point of view, the data set has been also used to derive upper limits of pollutant background concentrations to be used in decision-making related to the management of contaminated groundwater in urban and industrial environments in the Walloon Region.

The Identification of Small RNAs Differentially Expressed in Apple Buds Reveals a Potential Role of the Mir159-MYB Regulatory Module during Dormancy.

Winter dormancy is an adaptative mechanism that temperate and boreal trees have developed to protect their meristems against low temperatures. In apple trees (Malus domestica), cold temperatures induce bud dormancy at the end of summer/beginning of the fall. Apple buds stay dormant during winter until they are exposed to a period of cold, after which they can resume growth (budbreak) and initiate flowering in response to warmer temperatures in spring. It is well-known that small RNAs modulate temperature responses in many plant species, but however, how small RNAs are involved in genetic networks of temperature-mediated dormancy control in fruit tree species remains unclear. Here, we have made use of a recently developed ARGONAUTE (AGO)-purification technique to isolate small RNAs from apple buds. A small RNA-seq experiment resulted in the identification of 17 micro RNAs (miRNAs) that change their pattern of expression in apple buds during dormancy. Furthermore, the functional analysis of their predicted target genes suggests a main role of the 17 miRNAs in phenylpropanoid biosynthesis, gene regulation, plant development and growth, and response to stimulus. Finally, we studied the conservation of the Arabidopsis thaliana regulatory miR159-MYB module in apple in the context of the plant hormone abscisic acid homeostasis.

Gene expression pattern in response to wounding, methyl jasmonate and ethylene in the bark of Hevea brasiliensis.

Natural rubber production in Hevea brasiliensis is determined by both tapping and ethephon frequencies. It is affected by a complex physiological disorder called tapping panel dryness. This syndrome is likely to be induced by environmental and latex harvesting stresses. Defence responses, including rubber biosynthesis, are dramatically mediated by wounding, jasmonate and ethylene (ET), among other factors. Using real-time RT-PCR, the effects of wounding, methyl jasmonate (MeJA) and ET on the relative transcript abundance of a set of 25 genes involved in their signalling and metabolic pathways were studied in the bark of 3-month-old epicormic shoots. Temporal regulation was found for 9 out of 25 genes. Wounding treatment regulated the transcript abundance of 10 genes. Wounding-specific regulation was noted for the HbMAPK, HbBTF3b, HbCAS1, HbLTPP and HbPLD genes. MeJA treatment regulated the transcript abundance of nine genes. Of these, the HbMYB, HbCAS2, HbCIPK and HbChi genes were shown to be specifically MeJA inducible. ET response was accompanied by regulation of the transcript abundance of eight genes, and six genes, HbETR2, HbEIN2, HbEIN3, HbCaM, HbPIP1 and HbQM, were specifically regulated by ET treatment. Additionally, the transcript level of the HbGP and HbACR genes was enhanced by all three treatments simultaneously. Overall, a large number of genes were found to be regulated 4 h after the treatments were applied. This study nevertheless revealed some jasmonic acid-independent wound signalling pathways in H. brasiliensis, provided a general characterization of signalling pathways and will serve as a new base from which to launch advanced studies of the network of pathways operating in H. brasiliensis.