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

Carbon Sequestration in Resin-Tapped Slash Pine (Pinus elliottii Engelm.) Subtropical Plantations.

Every year more than 150,000 tons of resin used in a myriad of industrial applications are produced by Brazilian plantations of Pinus elliottii Engelm. (slash pine), which are also used for timber. A pine tree can be tapped for resin over a period of several years. Resin is a complex mixture of terpenes, which are carbon-rich molecules, presumably influencing pine plantation carbon budgets. A total of 270 trees (overall mean DBH of 22.93 ± 0.11 cm) of 14-, 24-, and 26-year-old stands had their C content measured. Three different treatments (intact, wounded panels, and wounded + chemically stimulated panels, 30 trees each) were applied per site. Above- and belowground biomass, as well as resin yield, were quantified for two consecutive years. Data were statistically evaluated using normality distribution tests, analyses of variance, and mean comparison tests (p &#8804; 0.05). The highest resin production per tree was recorded in the chemically stimulated 14-year-old stand. Tree dry wood biomass, a major stock of carbon retained in cell wall polysaccharides, ranged from 245.69 ± 11.73 to 349.99 ± 16.73 kg among the plantations. Variations in carbon concentration ranged from 43% to 50% with the lowest percentages in underground biomass. There was no significant difference in lignin concentrations. Soils were acidic (pH 4.3 ± 0.10?5.83 ± 0.06) with low C (from 0.05% to 1.4%). Significantly higher C stock values were recorded in pine biomass compared to those reported for temperate zones. Resin-tapping biomass yielded considerable annual increments in C stocks and should be included as a relevant component in C sequestration assessments of planted pine forests

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

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