Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill

<p>Abstract</p> <p>Background</p> <p><it>Eucalyptus globulus </it>and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in <it>E. globulus </it>microcuttings.</p> <p>Results</p> <p>By the use of two distinct algorithms, <it>geNorm </it>and <it>NormFinder</it>, we have assessed gene expression stability of eleven candidate reference genes in <it>E. globulus</it>: <it>18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI </it>and <it>33380</it>. The candidate reference genes were evaluated in microccuttings rooted <it>in vitro</it>, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: <it>IDH </it>and <it>SAND </it>for <it>geNorm</it>, and <it>H2B </it>and <it>TUA </it>for <it>NormFinder</it>. Both programs indentified <it>UBI </it>and <it>18S </it>as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the <it>ARGONAUTE1 </it>gene was evaluated in relation to the most stable candidate genes indicated by each algorithm.</p> <p>Conclusion</p> <p>Our study showed that expression stability varied between putative reference genes tested in <it>E. globulus</it>. Based on the <it>AGO1 </it>relative expression profile obtained using the genes suggested by the algorithms, <it>H2B </it>and <it>TUA </it>were considered as the most suitable reference genes for expression studies in <it>E. globulus </it>adventitious rooting. <it>UBI </it>and <it>18S </it>were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.</p

Environmental Modulation of Mini-Clonal Gardens for Cutting Production and Propagation of Hard- and Easy-to-Root <i>Eucalyptus</i> spp

Clonal Eucalyptus propagation is essential for various industry sectors. It requires cuttings to successfully develop adventitious roots (ARs). Environmental conditions are influential on AR development and may be altered to modulate the productivity of hard-to-root clones. The current knowledge gap in research on the physiological patterns underlying commercial-scale propagation results hinders the design of novel strategies. This study aimed to identify patterns of variation in AR-relevant parameters in contrasting seasons and species with distinct rooting performances. E. dunnii and E. ×urograndis (hard- (hardR) and easy-to-root (easyR), respectively) mini-stumps were subjected to light modulation treatments and to mini-tunnel use (MT) for a year. The treatment impact on the branching and rooting rates was recorded. The carbohydrate content, AR-related gene expression, and mineral nutrition profiles of cuttings from the control (Ctrl) and treated mini-stumps were analyzed. Light treatments were often detrimental to overall productivity, while MTs had a positive effect during summer, when it altered the cutting leaf nutrient profiles. Species and seasonality played large roles in all the assessed parameters. E. ×urograndis was particularly susceptible to seasonality, and its overall superior performance correlated with changes in its gene expression profile from excision to AR formation. These patterns indicate fundamental differences between easyR and hardR clones that contribute to the design of data-driven management strategies aiming to enhance propagation protocols

Isolation and characterization of cyclotides from Brazilian Psychotria: significance in plant defense and co-occurrence with antioxidant alkaloids

Plants from the genus Psychotria include species bearing cyclotides and/or alkaloids. The elucidation of factors affecting the metabolism of these molecules. as well as their activities may help to understand their ecological function. In the present study, high concentrations of antioxidant indole alkaloids were found to co-occur with cyclotides in Psychotria leiocarpa and P. brachyceras. The concentrations of the major cyclotides and alkaloids in P. leiocarpa and P. brachyceras were monitored following herbivore- and pathogen associated challenges, revealing a constitutive, phytoanticipin-like accumulation pattern. Psyleio A, the most abundant cyclotide found in the leaves of P. leiocarpa, and also found in P. brachyceras leaves, exhibited insecticidal activity against Helicoverpa armigera larvae. Addition of ethanol in the vehicle for peptide solubilization in larval feeding trials proved deleterious to insecticidal activity and resulted in increased rates of larval survival in treatments containing indole alkaloids. This suggests that plant alkaloids ingested by larvae might contribute to herbivore oxidative stress detoxification, corroborating, in a heterologous system with artificial oxidative stress stimulation, the antioxidant efficiency of Psychotria alkaloids previously observed in planta. Overall, the present study reports data for eight novel cyclotides, the identification of P. leiocarpa as a cyclotide-bearing species, and the absence of these peptides in P. umbellata

Events Associated with Early Age-Related Decline in Adventitious Rooting Competence of Eucalyptus globulus Labill

The development of adventitious roots is affected by several factors, including the age of the cutting donor plant, which negatively affects rooting capacity. Eucalyptus globulus quickly loses rooting capacity of cuttings as the donor plant ages, although the molecular and biochemical mechanisms behind this process are still unclear. To better understand the bases of rooting competence loss in E. globulus, the time required for a significant decline in rhizogenic ability without exogenous auxin was determined in microcuttings derived from donor plants of different ages after sowing. Tip cuttings of donor plants were severed before and after loss of rooting competence of microcuttings to test the hypothesis that auxin and carbohydrate homeostasis regulate rooting competence decline. There were no significant changes in concentration of carbohydrates, flavonoids, or proteins before and after the loss of rooting capacity. Peroxidase (EC 1.11.1.7) total activity increased with loss of rooting competence. Auxin concentration showed the opposite pattern. In good agreement, TAA1, a key gene in auxin biosynthesis, had lower expression after loss of rooting capacity. The same applied to the auxin receptor gene TIR1, suggesting reduced auxin sensitivity. On the other hand, genes associated with auxin response repression (TPL, IAA12) or with the action of cytokinins, the rhizogenesis inhibitor-related ARR1, showed higher expression in plants with lower rooting competence. Taken together, data suggest that age negatively affects E. globulus rooting by a combination of factors. Decreased endogenous auxin concentration, possibly caused by less biosynthesis, lower auxin sensitivity, higher expression of genes inhibiting auxin action, as well as of genes related to the action of cytokinins, appear to play roles in this process

Reference Genes for qPCR Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis

Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes) and rosin (diterpenes) fractions. Higher oleoresin yields are of economic interest, since oleoresin derivatives make up a valuable source of materials for chemical industries. Oleoresin can be extracted from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface. To better understand the molecular basis of chemically-stimulated and wound induced oleoresin production, we evaluated the stability of 11 putative reference genes for the purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis. Samples for RNA extraction were collected from field-grown adult trees under tapping operations using stimulant pastes with different compositions and at various time points after paste application. Statistical methods established by geNorm, NormFinder, and BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3 and UBI. To confirm expression stability of the candidate reference genes, expression profiles of putative P. elliottii orthologs of resin biosynthesis-related genes encoding Pinus contorta β-pinene synthase [PcTPS-(−)β-pin1], P. contorta levopimaradiene/abietadiene synthase (PcLAS1), Pinus taeda α-pinene synthase [PtTPS-(+)αpin], and P. taeda α-farnesene synthase (PtαFS) were examined following stimulant paste application. Increased oleoresin yields observed in stimulated treatments using phytohormone-based pastes were consistent with higher expression of pinene synthases. Overall, the expression of all genes examined matched the expected profiles of oleoresin-related transcript changes reported for previously examined conifers

Phytotoxicity of Quillaja lancifolia Leaf Saponins and Their Bioherbicide Potential

Weeds are major threats to the integrity of agricultural and natural environments due to their invasive and competing potential. Bioherbicides are substances based on natural compounds that are biodegradable and often have low residual effects. Plant species able to produce and release phytotoxic compounds may represent effective bioherbicide sources. Leaves of Quillaja lancifolia D.Don (formerly Q. brasiliensis (A.St.-Hil. &amp; Tul.) Mart.) produce water-soluble specialized metabolites of the saponin class that could be evaluated for phytotoxic activity and potential as natural herbicides. This study was conducted to examine the impacts of Q. lancifolia total saponins aqueous extract (AE) at 4 and 10% (w/v) and of two combined reverse-phase chromatography purified saponin fractions (QB) at 1 and 2% (w/v) on morpho-physiological parameters of Lactuca sativa (lettuce) and Echinochloa crus-galli (barnyardgrass) in pre- and post-emergence bioassays. QB was only tested in pre-emergence assays. In pre-emergence bioassays, the germination rate and germination kinetics were determined. Post-emergence evaluations included effects on seedling morphology, root and shoot length, dry mass, and chlorophyll content. Osmotic potential and pH analyses ruled out roles for these factors in the observed responses. AE had a high inhibitory impact on the germination of both lettuce and barnyardgrass. QB at 1% and 2% (w/v) significantly decreased the growth of lettuce seedlings germinated in its presence by more than 10-fold. Phytotoxic effects on the post-emergence growth of lettuce, especially at the highest concentration tested of AE (10% w/v), was also observed. The presence of quillaic acid-based triterpene saponins in AE and QB was confirmed using different analytical methods. Therefore, both saponin-enriched fraction and aqueous extracts of Q. lancifolia inhibited tested plant growth and development. The water solubility of saponins and the availability of a sustainable source of these molecules from the leaves of cultivated young Q. lancifolia plants make them attractive candidates for use as bioherbicides