Integration analysis of transcriptome and proteome profiles brings new insights of somatic embryogenesis of two eucalyptus species.

BACKGROUND: Somatic embryogenesis (SE) is recognized as a promising technology for plant vegetative propagation. Although previous studies have identified some key regulators involved in the SE process in plant, our knowledge about the molecular changes in the SE process and key regulators associated with high embryogenic potential is still poor, especially in the important fiber and energy source tree - eucalyptus. RESULTS: In this study, we analyzed the transcriptome and proteome profiles of E. camaldulensis (with high embryogenic potential) and E. grandis x urophylla (with low embryogenic potential) in SE process: callus induction and development. A total of 12,121 differentially expressed genes (DEGs) and 3,922 differentially expressed proteins (DEPs) were identified in the SE of the two eucalyptus species. Integration analysis identified 1,353 (131 to 546) DEGs/DEPs shared by the two eucalyptus species in the SE process, including 142, 13 and 186 DEGs/DEPs commonly upregulated in the callus induction, maturation and development, respectively. Further, we found that the trihelix transcription factor ASR3 isoform X2 was commonly upregulated in the callus induction of the two eucalyptus species. The SOX30 and WRKY40 TFs were specifically upregulated in the callus induction of E. camaldulensis. Three TFs (bHLH62, bHLH35 isoform X2, RAP2-1) were specifically downregulated in the callus induction of E. grandis x urophylla. WGCNA identified 125 and 26 genes/proteins with high correlation (Pearson correlation > 0.8 or < -0.8) with ASR3 TF in the SE of E. camaldulensis and E. grandis x urophylla, respectively. The potential target gene expression patterns of ASR3 TF were then validated using qRT-PCR in the material. CONCLUSIONS: This is the first time to integrate multiple omics technologies to study the SE of eucalyptus. The findings will enhance our understanding of molecular regulation mechanisms of SE in eucalyptus. The output will also benefit the eucalyptus breeding program.

Reactive oxygen species act as the key signaling molecules mediating light-induced anthocyanin biosynthesis in Eucalyptus.

Light plays a pivotal role in regulating anthocyanin biosynthesis in plants, and the early light-responsive signals that initiate anthocyanin biosynthesis remain to be elucidated. In this study, we showed that the anthocyanin biosynthesis in Eucalyptus is hypersensitive to increased light intensity. The combined transcriptomic and metabolomic analyses were conducted on Eucalyptus leaves after moderate (ML; 100 µmol m-2 s-1) and high (HL; 300 µmol m-2 s-1) light intensity treatments. The results identified 1940, 1096, 1173, and 2756 differentially expressed genes at 6, 12, 24, and 36 h after HL treatment, respectively. The metabolomic results revealed the primary anthocyanin types, and other differentially accumulated flavonoids and phenylpropane intermediates that were produced in response to HL, which well aligned with the transcriptome results. Moreover, biochemical analysis showed that HL inhibited peroxidase activity and increased the ROS level in Eucalyptus leaves. ROS depletion through co-application of the antioxidants rutin, uric acid, and melatonin significantly reduced, and even abolished, anthocyanin biosynthesis induced by HL treatment. Additionally, exogenous application of hydrogen peroxide efficiently induced anthocyanin biosynthesis within 24 h, even under ML conditions, suggesting that ROS played a major role in activating anthocyanin biosynthesis. A HL-responsive MYB transcription factor EgrMYB113 was identified to play an important role in regulating anthocyanin biosynthesis by targeting multiple anthocyanin biosynthesis genes. Additionally, the results demonstrated that gibberellic acid and sugar signaling contributed to HL-induced anthocyanin biosynthesis. Conclusively, these results suggested that HL triggers multiple signaling pathways to induce anthocyanin biosynthesis, with ROS acting as indispensable mediators in Eucalyptus.

Carbon budget at the individual-tree scale: dominant Eucalyptus trees partition less carbon belowground.

Large trees in plantations generally produce more wood per unit of resource use than small trees. Two processes may account for this pattern: greater photosynthetic resource use efficiency or greater partitioning of carbon to wood production. We estimated gross primary production (GPP) at the individual scale by combining transpiration with photosynthetic water-use efficiency of Eucalyptus trees. Aboveground production fluxes were estimated using allometric equations and modeled respiration; total belowground carbon fluxes (TBCF) were estimated by subtracting aboveground fluxes from GPP. Partitioning was estimated by dividing component fluxes by GPP. Dominant trees produced almost three times as much wood as suppressed trees. They used 25 ± 10% (mean ± SD) of their photosynthates for wood production, whereas suppressed trees only used 12 ± 2%. By contrast, dominant trees used 27 ± 19% of their photosynthate belowground, whereas suppressed trees used 58 ± 5%. Intermediate trees lay between these extremes. Photosynthetic water-use efficiency of dominant trees was c. 13% greater than the efficiency of suppressed trees. Suppressed trees used more than twice as much of their photosynthate belowground and less than half as much aboveground compared with dominant trees. Differences in carbon partitioning were much greater than differences in GPP or photosynthetic water-use efficiency.

Eucalyptus globulus leaf-isolated isorhapontin serves as a natural insecticide via acetylcholinesterase inhibition.

Acetylcholinesterase (AChE) inhibitors cause insect death by preventing the hydrolysis of the neurotransmitter acetylcholine, which overstimulates the nervous system. In this study, isorhapontin, isolated from E. globulus leaves, was evaluated as a natural insecticide with AChE inhibition at 12.5 µM. Using kinetic analyses, we found that isorhapontin acted as a competitive inhibitor that binds to the active site of AChE. The inhibition constant (Ki) was 6.1 µM. Furthermore, isorhapontin and resveratrol, which have basic skeletons, were predicted to bind to the active site of AChE via molecular docking. A comparison of the hydrogen bonding between the two stilbenes revealed characteristic differences in their interactions with amino acids. In isorhapontin, Trp83, Gly149, Tyr162, Tyr324, and Tyr370 interacted with the sugar moiety. These results suggest that with further development, isorhapontin can be used as an insecticide alternative.

EsigPBP3 Was the Important Pheromone-Binding Protein to Recognize Male Pheromones and Key Eucalyptus Volatiles.

Pheromone-binding proteins (PBPs) are specific odorant-binding proteins that can specifically recognize insect pheromones. Through transcriptional analysis of the antennae of adult Endoclita signifer, EsigPBP3 was discovered and identified, and EsigPBP3 was found to be highly expressed in the antennae of male moths. Based on the binding characteristics and ability of EsigPBP3, we can find the key ligands and binding site to consider as a target to control the key wood bore E. signifier. In this study, the fluorescence competitive binding assays (FCBA) showed that EsigPBP3 had a high binding affinity for seven key eucalyptus volatiles. Molecular docking analysis revealed that EsigPBP3 had the strongest binding affinity for the sexual pheromone component, (3E,7E)-4,7,11-trimethyl-1,3,7,10-dodecatetraene. Furthermore, same as the result of FCBA, the EsigPBP3 exhibited high binding affinities to key eucalyptus volatiles, eucalyptol, α-terpinene, (E)-beta-ocimene, (-)-ß-pinene, and (-)-α-pinene, and PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 are key sites. In summary, EsigPBP3 exhibits high binding affinity to male pheromones and key volatile compounds and the crucial binding sites PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 can act as targets in the recognition of E. signifier pheromones.

Integrated Ca, Mg, Cu, and Zn supply upregulates leaf anatomy and metabolic adjustments in Eucalyptus seedlings.

Adaptive responses to abiotic stresses such as soil acidity in Eucalyptus-the most widely planted broad-leaf forest genus globally-are poorly understood. This is particularly evident in physiological and anatomical disorders that inhibit plant development and wood quality. We aimed to explore how the supply of Ca and Mg through liming (lime), combined with Cu and Zn fertilization (CZF), influences physiological and anatomical responses during Eucalyptus grandis seedlings growth in tropical acid soil. Therefore, related parameters of leaf area and leaf anatomy, stomatal size, leaf gas exchange, antioxidant system, nutrient partitioning, and biomass allocation responses were monitored. Liming alone in Eucalyptus increased specific leaf area, stomatal density on the abaxial leaf surface, and Ca and Mg content. Also, Eucalyptus exposed only to CZF increased Cu and Zn content. Lime and CZF increased leaf blade and adaxial epidermal thickness, and improved the structural organization of the spongy mesophyll, promoting increased net CO2 assimilation, and stomatal conductance. Fertilization with Ca, Mg, Cu, and Zn positively affects plant nutrition, light utilization, photosynthetic rate, and antioxidant performance, improving growth. Our results indicate that lime and CZF induce adaptive responses in the physiological and anatomical adjustments of Eucalyptus plantation, thereby promoting biomass accumulation.

Transcriptome analysis of the transition from primary to secondary growth of vertical stem in Eucalyptus grandis.

Eucalyptus was one of the most cultivated hardwood species worldwide, with rapid growth, good wood properties and a wide range of adaptability. Eucalyptus stem undergoes primary growth (longitudinal growth) followed by secondary growth (radial growth), which produces biomass that is an important source of energy worldwide. In order to better understand the genetic regulation of secondary growth in Eucalyptus grandis, Transcriptome analyses in stem segments along a developmental gradient from the third internode to the eleventh internode of E. grandis that spanned primary to secondary growth were carried out. 5,149 genes that were differentially expressed during stem development were identified. Combining the trend analysis by the Mfuzz method and the module-trait correlation analysis by the Weighted Gene Co-expression Network Analysis method, a total of 70 differentially expressed genes (DEGs) selected from 868 DEGs with high connectivity were found to be closely correlated with secondary growth. Results revealed that the differential expression of these DEGs suggests that they may involve in the primary growth or secondary growth. AP1, YAB2 TFs and EXP genes are highly expressed in the IN3, whereas NAC, MYB TFs are likely to be important for secondary growth. These results will expand our understanding of the complex molecular and cellular events of secondary growth and provide a foundation for future studies on wood formation in Eucalyptus.

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa.

Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways.

Proteome profiling of vascular sap regarding Eucalyptus grandis, Eucalyptus urophylla, and Eucalyptus camaldulensis.

The plant vascular system is a key element for long-distance communication. Understanding its composition may provide valuable information on how plants grow and develop themselves. In this study, a quantitative proteome dataset of the vascular sap proteome of three commercially important Eucalyptus species was shown. Protein extraction was carried out using a pressure bomb, whereas only in silico predicted extracellular proteins were considered as part of the sap proteome. A total of 132 different proteins were identified in all three Eucalyptus species and the most abundant proteome subset within all three species was comprised of proteins involved in the carbohydrate metabolic process, proteolysis, components of membrane, and defense response. The sap proteome of the species E. grandis and E. urophylla revealed the highest similarities. Functional classification indicated that the sap proteome of E. grandis and E. urophylla are mostly comprised of proteins involved in defense response and proteolysis; whereas no prominent functional class was observed for the E. camaldulensis species. Quantitative comparison highlighted characteristic sap proteins in each of the Eucalyptus species. The results that could be found in this study can be used as a reference for the proteome sap analysis of Eucalyptus plants grown under different conditions.

Cytokinin promotes anthocyanin biosynthesis via regulating sugar accumulation and MYB113 expression in Eucalyptus.

Anthocyanins are flavonoid-like substances that play important roles in plants' adaptation to various environmental stresses. In this research, we discovered that cytokinin (CK) alone could effectively induce the anthocyanin biosynthesis in Eucalyptus and many other perennial woody plant species, but not in tobacco and Arabidopsis, suggesting a diverse role of CK in regulating anthocyanin biosynthesis in different species. Transcriptomic and metabolomic strategies were used to further clarify the specific role of CK in regulating anthocyanin biosynthesis in Eucalyptus. The results showed that 801 and 2241 genes were differentially regulated at 6 and 24 h, respectively, after CK treatment. Pathway analysis showed that most of the differentially expressed genes were categorized into pathways related to cellular metabolism or transport of metabolites, including amino acids and sugars. The metabolomic results well supported the transcriptome data, which showed that most of the differentially regulated metabolites were related to the metabolism of sugar, amino acids and flavonoids. Moreover, CK treatment significantly induced the accumulation of sucrose in the CK-treated leaves, while sugar starvation mimicked by either defoliation or shading treatment of the basal leaves significantly reduced the sugar increase of the CK-treated leaves and thus inhibited CK-induced anthocyanin biosynthesis. The results of in vitro experiment also suggested that CK-induced anthocyanin in Eucalyptus was sugar-dependent. Furthermore, we identified an early CK-responsive transcription factor MYB113 in Eucalyptus, the expression of which was significantly upregulated by CK treatment in Eucalyptus, but was inhibited in Arabidopsis. Importantly, the overexpression of EgrMYB113 in the Eucalyptus hairy roots was associated with significant anthocyanin accumulation and upregulation of most of the anthocyanin biosynthetic genes. In conclusion, our study demonstrates a key role of CK in the regulation of anthocyanin biosynthesis in Eucalyptus, providing a molecular basis for further understanding the regulatory mechanism and diversity of hormone-regulated anthocyanin biosynthesis in different plant species.

Reduction of methane and nitrogen emission and improvement of feed efficiency, rumen fermentation, and milk production through strategic supplementation of eucalyptus (Eucalyptus citriodora) leaf meal in the diet of lactating buffalo (Bubalus bubalis).

Buffalo plays a compelling role in reducing malnutrition and ensuring food to the people of Asian countries by its major contribution to milk and meat pool of the livestock agriculture farming system in the region. As Asia is the home for more than 90% of world buffalo population, they are also one of the largest emitters of greenhouse gasses. Eucalyptus (Eucalyptus sp.) leaves are rich sources of naturally occurring essential oils and phenolic compounds, which could modulate rumen fermentation through mitigation of methanogenesis and nitrogen excretion along with stimulation of immune system and production performances of animals. Therefore, the present study investigated the impact of dietary inclusion of eucalyptus (Eucalyptus citriodora) leaf meal (ELM) on voluntary feed intake, rumen functions, methane emission, nutrient utilization, milk yield and fatty acids profile, and immune response in lactating buffalo (Bubalus bubalis). An in vitro experiment conducted with graded dose (10-40 g/kg) inclusion of ELM into the total mixed ration to select ideal level for feeding to lactating buffaloes, an improvement (P < 0.05) in feed degradability (IVDMD), microbial biomass and ruminal volatile fatty acids concentration with reduced (P < 0.05) methane and ammonia-N production were evidenced when ELM was added at 10-20 g/kg DM, beyond which negative effects on rumen fermentation were pronounced. An in vivo experimentation was conducted with sixteen Murrah (Bubalus bubalis) buffaloes of mean live weight, 544.23 ± 10.02 kg; parity, 2-4 at initial stage (~60 days) of lactation with average milk yield of 11.43 ± 1.32 kg and were divided into two groups (CON, ELM) of eight each in a completely randomized design. All the animals were kept individually on wheat straw-based diet with required quantity of concentrate mixture and green fodder. The control group buffaloes were fed a total mixed ration; however, the treatment group (ELM) was supplemented with 10 g/kg DM diet of dry grounded eucalyptus (Eucalyptus citriodora) leaves by mixing with the concentrate mixture. The feeding experiment was conducted for 120 days, including 15 days for adaptation to the experimental diets and 105 days for data recording. The nutrient digestibility (DM, OM, CP, and EE) was improved (P < 0.05) without affecting feed intake (P > 0.05) and fiber digestibility (NDF and ADF) in ELM supplemented buffaloes. Increased (P < 0.05) milk production and rumenic acid concentration (cis 9 trans 11 C18:2 CLA) were demonstrated with comparable (P > 0.05) milk composition and major fatty acids profile of milk in the supplemented buffaloes. Dietary inclusion of ELM reduced (P < 0.05) enteric methane production and fecal excretion of nitrogen. The health status of buffaloes fed ELM improved throughout the experimental period was improved by enhancing cell mediated (P = 0.09) and humoral (P < 0.01) immune responses without affecting (P > 0.05) major blood metabolites. The study described feeding ELM at 10 g/kg diet to lactating Murrah buffaloes as a natural source of phenols and essential oils to increase milk production and CLA content, reduce methane and nitrogen emissions, and improve health status. Thus, feeding of ELM could be beneficial for climate smart buffalo production system for enhancing milk production with lesser impact on environment.

Identification of WUSCHEL-related homeobox gene and truncated small peptides in transformation efficiency improvement in Eucalyptus.

BACKGROUND: The WUSCHEL-related Homeobox (WOX) genes, which encode plant-specific homeobox (HB) transcription factors, play crucial roles in regulating plant growth and development. However, the functions of WOX genes are little known in Eucalyptus, one of the fastest-growing tree resources with considerable widespread cultivation worldwide. RESULTS: A total of nine WOX genes named EgWOX1-EgWOX9 were retrieved and designated from Eucalyptus grandis. From the three divided clades marked as Modern/WUS, Intermediate and Ancient, the largest group Modern/WUS (6 EgWOXs) contains a specific domain with 8 amino acids: TLQLFPLR. The collinearity, cis-regulatory elements, protein-protein interaction network and gene expression analysis reveal that the WUS proteins in E. grandis involve in regulating meristems development and regeneration. Furthermore, by externally adding of truncated peptides isolated from WUS specific domain, the transformation efficiency in E. urophylla × E. grandis DH32-29 was significant enhanced. The transcriptomics data further reveals that the use of small peptides activates metabolism pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis. CONCLUSIONS: Peptides isolated from WUS protein can be utilized to enhance the transformation efficiency in Eucalyptus, thereby contributing to the high-efficiency breeding of Eucalyptus.

Genes expression profiles in vascular cambium of Eucalyptus urophylla × Eucalyptus grandis at different ages.

BACKGROUND: Wood is a secondary xylem generated by vascular cambium. Vascular cambium activities mainly include cambium proliferation and vascular tissue formation through secondary growth, thereby producing new secondary phloem inward and secondary xylem outward and leading to continuous tree thickening and wood formation. Wood formation is a complex biological process, which is strictly regulated by multiple genes. Therefore, molecular level research on the vascular cambium of different tree ages can lead to the identification of both key and related genes involved in wood formation and further explain the molecular regulation mechanism of wood formation. RESULTS: In the present study, RNA-Seq and Pac-Bio Iso-Seq were used for profiling gene expression changes in Eucalyptus urophylla × Eucalyptus grandis (E. urograndis) vascular cambium at four different ages. A total of 59,770 non-redundant transcripts and 1892 differentially expressed genes (DEGs) were identified. The expression trends of the DEGs related to cell division and differentiation, cell wall biosynthesis, phytohormone, and transcription factors were analyzed. The DEGs encoding expansin, kinesin, cycline, PAL, GRP9, KNOX, C2C2-dof, REV, etc., were highly expressed in E. urograndis at three years old, leading to positive effects on growth and development. Moreover, some gene family members, such as NAC, MYB, HD-ZIP III, RPK, and RAP, play different regulatory roles in wood formation because of their sophisticated transcriptional network and function redundantly. CONCLUSIONS: These candidate genes are a potential resource to further study wood formation, especially in fast-growing and adaptable eucalyptus. The results may also serve as a basis for further research to unravel the molecular mechanism underlying wood formation.

Genome-wide identification and expression profile analysis of metal tolerance protein gene family in Eucalyptus grandis under metal stresses.

Metal tolerance proteins (MTPs) as Me2+/H+(K+) antiporters participate in the transport of divalent cations, leading to heavy metal stress resistance and mineral utilization in plants. In the present study, to obtain better knowledge of the biological functions of the MTPs family, 20 potential EgMTPs genes were identified in Eucalyptus grandis and classified into seven groups belonging to three cation diffusion facilitator groups (Mn-CDFs, Zn/Fe-CDFs, and Zn-CDFs) and seven groups. EgMTP-encoded amino acids ranged from 315 to 884, and most of them contained 4-6 recognized transmembrane domains and were clearly prognosticated to localize into the cell vacuole. Almost all EgMTP genes experienced gene duplication events, in which some might be uniformly distributed in the genome. The numbers of cation efflux and the zinc transporter dimerization domain were highest in EgMTP proteins. The promoter regions of EgMTP genes have different cis-regulatory elements, indicating that the transcription rate of EgMTP genes can be a controlled response to different stimuli in multiple pathways. Our findings provide accurate perception on the role of the predicted miRNAs and the presence of SSR marker in the Eucalyptus genome and clarify their functions in metal tolerance regulation and marker-assisted selection, respectively. Gene expression profiling based on previous RNA-seq data indicates a probable function for EgMTP genes during development and responses to biotic stress. Additionally, the upregulation of EgMTP6, EgMTP5, and EgMTP11.1 to excess Cd2+ and Cu2+ exposure might be responsible for metal translocation from roots to leaves.

Tolerance of microorganisms to residual herbicides found in eucalyptus plantations.

Competition with weeds is one of the main factors that limit the development of forest species. Some herbicides used to control these plants have a residual effect on the soil. Bioremediation is an alternative to decontaminate these areas. The aim of this study was to evaluate the tolerance of Aspergillus niger, Penicillium pinophilum and Trichoderma sp. and its degrading potential on residual effect herbicides. The tolerance of Bacillus subtilis, Pseudomonas sp. and Azospirillum brasilense to herbicides was also evaluated. The herbicides used in this study were indaziflam, sulfentrazone, sulfentrazone + diuron, clomazone and glyphosate + s-metolachlor. The analysis of the tolerance and degradation potential of fungi was carried out in Czapek Dox medium and the growth was evaluated by determining the biomass. Bacterial tolerance analysis was performed in Luria Bertani medium and growth monitored by optical density. The data were applied to the Gompertz model to evaluate the behavior of bacteria. Bacterial growth parameters were not influenced by the presence of herbicides. All fungi were tolerant to the herbicides tested and there was an increase in the growth of Trichoderma sp. Thus, the analysis of the degrading potential was performed only for Trichoderma sp. in the presence of herbicides that potentiated its growth. In this analysis, there was no effect of herbicides on fungal growth; the fungus was unable to use the carbon present in the herbicide to enhance its growth; and there was no significant effect of nitrogen in the presence of the herbicide. It is concluded, therefore, that the tested residual herbicides do not interfere with the development of the evaluated microorganisms.

Transcriptome-Based Construction of the Gibberellin Metabolism and Signaling Pathways in Eucalyptus grandis × E. urophylla, and Functional Characterization of GA20ox and GA2ox in Regulating Plant Development and Abiotic Stress Adaptations.

Gibberellins (GAs) are the key regulators controlling plant growth, wood production and the stress responses in perennial woody plants. The role of GA in regulating the above-mentioned processes in Eucalyptus remain largely unclear. There is still a lack of systematic identification and functional characterization of GA-related genes in Eucalyptus. In this study, a total of 59,948 expressed genes were identified from the major vegetative tissues of the E. grandis × E. urophylla using transcriptome sequencing. Then, the key gene families in each step of GA biosynthesis, degradation and signaling were investigated and compared with those of Arabidopsis, rice, and Populus. The expression profile generated using Real-time quantitative PCR showed that most of these genes exhibited diverse expression patterns in different vegetative organs and in response to abiotic stresses. Furthermore, we selectively overexpressed EguGA20ox1, EguGA20ox2 and EguGA2ox1 in both Arabidopsis and Eucalyptus via Agrobacterium tumefaciens or A. rhizogenes-mediated transformation. Though both Arabidopsis EguGA20ox1- and EguGA20ox2-overexpressing (OE) lines exhibited better vegetative growth performance, they were more sensitive to abiotic stress, unlike EguGA2ox1-OE plants, which exhibited enhanced stress resistance. Moreover, overexpression of EguGA20ox in Eucalyptus roots caused significantly accelerated hairy root initiation and elongation and improved root xylem differentiation. Our study provided a comprehensive and systematic study of the genes of the GA metabolism and signaling and identified the role of GA20ox and GA2ox in regulating plant growth, stress tolerance, and xylem development in Eucalyptus; this could benefit molecular breeding for obtaining high-yield and stress-resistant Eucalyptus cultivars.

Physiological responses of three field-grown species (Ceratonia siliqua, Eucalyptus camaldulensis, and Moringa oleifera) to water deficits in a Mediterranean semi-arid climate.

Reforestation of degraded drylands calls for the selection of species with the capacity to withstand water scarcity. In this current study we have assessed, the physiological responses of three field-grown species (Ceratonia siliqua, Eucalyptus camaldulensis and Moringa oleifera) to water deficits in semi-arid regions in order to suggest a potential species for rehabilitation programs. The physiological behavior of the given species was studied in three irrigation schemes: subsurface drip irrigation (applied weekly), tank irrigation (applied monthly), and unirrigated plants. In a stressed state, an assessment of relative water content (RWC), water potential (pre-dawn water potential PWP and midday water potential MWP) and stomatal conductance revealed three contrasting physiological responses. First, C. siliqua stomata remained open with a high RWC at low water potentials. Consequently, this species tolerated water deficits by decreasing its leaf water potential, primarily associated with osmotic adjustment. On the other hand, E. camaldulensis was found to be a drought-avoider species, mutated to a water-saving strategy by complete stomatal closure. Finally, for the extreme case, M. oleifera showed leaf shedding under water deficit conditions. These different physiological responses allowed these species to survive water deficits, and consequently, could be considered suitable candidates for rehabilitating degraded semi-arid areas.

High-throughput miRNA sequencing and identification of a novel ICE1-targeting miRNA in response to low temperature stress in Eucalyptus camaldulensis.

MicroRNAs (miRNAs) play a crucial role in the growth, development, morphogenesis, signal transduction, and stress response in plants. The ICE (Inducer of CBF expression)-CBF (C-repeat binding factor)-COR (Cold-regulated gene) regulatory cascade is an important signalling pathway in plant response to low temperature stress, and it remains unknown whether this pathway is regulated by miRNAs. In this study, high-throughput sequencing was employed for predicting and identifying the miRNAs that were likely to target the ICE-CBF-COR pathway in Eucalyptus camaldulensis. A novel ICE1-targeting miRNA, eca-novel-miR-259-5p (nov-miR259), was further analysed. A total of 392 conserved miRNAs and 97 novel miRNAs were predicted, including 80 differentially expressed miRNAs. Of these, 30 miRNAs were predicted to be associated with the ICE-CBF-COR pathway. The full-length of mature nov-miR259 was 22 bp and its precursor gene was 60 bp in length, with a typical hairpin structure. The RNA ligase-mediated 5' amplification of cDNA ends (5'-RLM-RACE) and Agrobacterium-mediated tobacco transient expression assays demonstrated that nov-miR259 could cleave EcaICE1 in vivo. Moreover, qRT-PCR and Pearson's correlation analysis further revealed that the expression levels of nov-miR259 were almost significantly negatively correlated with those of its target gene, EcaICE1, and the other genes in the ICE-CBF-COR pathway. We first identified the nov-miR259 as a novel ICE1-targeting miRNA, and the nov-miR259-ICE1 module may be involved in regulating the cold stress response in E. camaldulensis.

Upregulation of PCSK9, rho kinase and cardiac troponin by Eucalyptus globulus leaf extract improves fructose-streptozotocin-induced diabetic cardiac dysfunction in rats.

CONTEXT: The effect of Eucalyptus globulus in diabetic cardiac dysfunction and the possible mechanisms involved have not been explored. OBJECTIVE: To evaluate the effect of ethanol leaf extract of E. globulus (NEE) on the cardiac function of fructose/streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Type-2 diabetes was induced in rats with 10% fructose feeding for 14 days and an intraperitoneal administration of 40 mg/kg streptozotocin. Diabetic animals were treated with NEE (100-400 mg/kg) or 5 mg/kg glibenclamide orally for 21 days. Biochemical assays, histopathological examination and analyses of PCSK9, Rho kinase and Cardiac troponin expression were performed. RESULTS: The untreated diabetic group showed decreased expression of the genes, oxidative stress, dyslipidemia, increased activities of creatine kinase MB and lactate dehydrogenase, reduced nitric oxide level, and depletion of cardiomyocytes, which were reversed in NEE treated groups. CONCLUSIONS: Eucalyptus globulus ameliorated diabetic cardiac dysfunction through increased PCSK9, Rho kinase and Cardiac troponin expression.

Recruitment of distinct UDP-glycosyltransferase families demonstrates dynamic evolution of chemical defense within Eucalyptus L'Hér.

The economic and ecologically important genus Eucalyptus is rich in structurally diverse specialized metabolites. While some specialized metabolite classes are highly prevalent across the genus, the cyanogenic glucoside prunasin is only produced by c. 3% of species. To investigate the evolutionary mechanisms behind prunasin biosynthesis in Eucalyptus, we compared de novo assembled transcriptomes, together with online resources between cyanogenic and acyanogenic species. Identified genes were characterized in vivo and in vitro. Pathway characterization of cyanogenic Eucalyptus camphora and Eucalyptus yarraensis showed for the first time that the final glucosylation step from mandelonitrile to prunasin is catalyzed by a novel UDP-glucosyltransferase UGT87. This step is typically catalyzed by a member of the UGT85 family, including in Eucalyptus cladocalyx. The upstream conversion of phenylalanine to mandelonitrile is catalyzed by three cytochrome P450 (CYP) enzymes from the CYP79, CYP706, and CYP71 families, as previously shown. Analysis of acyanogenic Eucalyptus species revealed the loss of different ortholog prunasin biosynthetic genes. The recruitment of UGTs from different families for prunasin biosynthesis in Eucalyptus demonstrates important pathway heterogeneities and unprecedented dynamic pathway evolution of chemical defense within a single genus. Overall, this study provides relevant insights into the tremendous adaptability of these long-lived trees.