Márcia Rodrigues de Almeida

When stress and development go hand in hand: main hormonal controls of adventitious rooting in cuttings.

Adventitious rooting (AR) is a multifactorial response leading to new roots at the base of stem cuttings, and the establishment of a complete and autonomous plant. AR has two main phases: (a) induction, with a requirement for higher auxin concentration; (b) formation, inhibited by high auxin and in which anatomical changes take place. The first stages of this process in severed organs necessarily include wounding and water stress responses which may trigger hormonal changes that contribute to reprogram target cells that are competent to respond to rooting stimuli. At severance, the roles of jasmonate and abscisic acid are critical for wound response and perhaps sink strength establishment, although their negative roles on the cell cycle may inhibit root induction. Strigolactones may also inhibit AR. A reduced concentration of cytokinins in cuttings results from the separation of the root system, whose tips are a relevant source of these root induction inhibitors. The combined increased accumulation of basipetally transported auxins from the shoot apex at the cutting base is often sufficient for AR in easy-to-root species. The role of peroxidases and phenolic compounds in auxin catabolism may be critical at these early stages right after wounding. The events leading to AR strongly depend on mother plant nutritional status, both in terms of minerals and carbohydrates, as well as on sink establishment at cutting bases. Auxins play a central role in AR. Auxin transporters control auxin canalization to target cells. There, auxins act primarily through selective proteolysis and cell wall loosening, via their receptor proteins TIR1 (transport inhibitor response 1) and ABP1 (Auxin-Binding Protein 1). A complex microRNA circuitry is involved in the control of auxin response factors essential for gene expression in AR. After root establishment, new hormonal controls take place, with auxins being required at lower concentrations for root meristem maintenance and cytokinins needed for root tissue differentiation.

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

BackgroundEucalyptus globulus 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 E. globulus microcuttings.ResultsBy the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, 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: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs indentified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm.ConclusionOur study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression profile obtained using the genes suggested by the algorithms, H2B and TUA were considered as the most suitable reference genes for expression studies in E. globulus adventitious rooting. UBI and 18S 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.

Pre and post-severance effects of light quality on carbohydrate dynamics and microcutting adventitious rooting of two Eucalyptus species of contrasting recalcitrance

Adventitious rooting is a complex developmental response affected by genetic and environmental factors. Radiation quality effects on adventitious rooting depend on characteristics such as species, growth stage, irradiance, spectral quality, and time of exposure. Eucalyptus is an essential genus for the paper industry, and high yield plantations depend on adventitious rooting of selected genotypes. This work addressed two hypotheses: (1) radiation quality equally affects adventitious rooting in Eucalyptus species of different recalcitrance; (2) adventitious rooting outcome depends on both donor plant and cutting radiation quality treatments. To that end, the easy-to-root Eucalyptus grandis and the recalcitrant Eucalyptus globulus were evaluated. The effect of white, blue, red and far-red radiation enrichment on microcuttings and donor plants of both species was evaluated in relation to rooting. There was no effect of radiation quality on adventitious rooting of E. grandis or when radiation treatments were applied to E. globulus microcuttings. In contrast, donor plants of E. globulus, grown in medium devoid of sucrose and exposed to far-red radiation, yielded microcuttings showing higher rooting percentage, even in the absence of exogenous auxin in the rooting medium. Sucrose in donor plant medium abolished the positive effect of far-red radiation. An increase in endogenous soluble sugars and starch contents in basal microcuttings was associated with far-red radiation treatment of donor plants. These results underline the importance of appropriate carbohydrate partitioning in donor plants for adventitious rooting of cuttings and provide a basis for understanding and overcoming rooting recalcitrance in E. globulus clones.

Expression of auxin carrier genes during adventitious rooting in Eucalyptus globulus

Background Eucalyptus globulus and its hybrids are important for the cellulose and paper industry, mainly due to their relatively low lignin content. However, rooting of cuttings of this species is often recalcitrant and exogenous auxin application is necessary for adventitious root development. Auxin plays a central role in rooting capacity, which is particularly affected by its endogenous content and transport rate. The shoot apex is a major source of endogenous auxin, which is mainly transported by both influx (AUX1) and efflux (PIN) carriers in a specific basipetal active transport through the vascular parenchyma in stems. As part of a larger study to investigate the causes of low rooting in E. globulus microcuttings without exogenous auxin, we evaluated the expression profiles of AUX1 andPIN1 during the process of adventitious rooting using qPCR.

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.

Nitrogen Sources and Adventitious Root Development in Eucalyptus globulus Microcuttings

In previous studies, it has been shown that nitrate supply may favor adventitious rooting in the rooting recalcitrant Eucalyptus globulus. Herein, the impact of various N sources on adventitious rooting and root branching in microcuttings of E. globulus was investigated. The positive effect of nitrate on adventitious root development was confirmed and extended to root branching. Urea yielded a rooting response comparable to that observed in presence of nitrate. Urease activity was observed, displaying two peaks: one at the root induction and another at the root formation step. The use of glutamic acid, glutamine or asparagine promoted higher root number, but yielded shorter roots. Rooted microcuttings derived from all nitrogen (N) sources were successfully acclimated to ex vitro conditions. The manipulation of N sources in adventitious rooting media can be a tool for improving new root density, length and branching in this species.

Laser Capture Microdissection: Avoiding Bias in Analysis by Selecting Just What Matters

Laser capture microdissection (LCM) is a powerful technique for harvesting specific cells from a heterogeneous population. As each cell and tissue has its unique genetic, proteomic, and metabolic profile, the use of homogeneous samples is important for a better understanding of complex processes in both animal and plant systems. In case of plants, LCM is very suitable as the highly regular tissue organization and stable cell walls from these organisms enable visual identification of various cell types without staining of tissue sections, which can prevent some downstream analysis. Considering the applicability of LCM to any plant species, here we provide a step-by-step protocol for selecting specific cells or tissues through this technology.