Histochemical Localization of Phenolic Compounds and Reactive Oxygen Species in Eucalypt Microcuttings.

Among the main features of plant specialized metabolism are cell- and tissue- specific expression and responsiveness to oxidative stress conditions. Although quantitative techniques have significantly improved over time, allowing higher levels of resolution in plant metabolic studies, such analyses are often expensive and/or require relatively large amounts of starting material. The following protocols offer a relatively simple way to survey specialized and related metabolites (total phenolics, flavonoids, lignins), as well as reactive oxygen species (superoxide and hydrogen peroxide), using light and fluorescence microscopy. Also, a step-by-step guide on how to quantify stained areas is provided. Through the association of qualitative and quantitative data, general patterns of molecule distribution across plant tissues may be inferred, allowing for the solving of biological questions, and contributing to hypothesis refinement. Eucalyptus microcuttings were chosen as plant material to exemplify how these protocols can provide useful data for the understanding of complex developmental processes, such as adventitious root formation, which may be influenced by specialized metabolites and redox conditions.

Microcutting Redox Profile and Anatomy in Eucalyptus spp. With Distinct Adventitious Rooting Competence.

Adventitious root (AR) development takes place in an intricate cellular environment. Reactive oxygen species (ROS) and antioxidant defenses, triggered by wounding in cuttings, can modulate this process. A comparative assessment of biochemical and anatomical parameters at critical rooting stages in hard- (Eucalyptus globulus Labill.) and easy- (Eucalyptus grandis W.Hill ex Maiden) to-root species was carried out. Microcuttings from seedlings were inoculated in auxin-free AR induction medium and, after 96 h, transferred to AR formation medium for a period of 24 h. Samples were collected upon excision (Texc) and at the 5th day post excision (Tform). Delayed xylem development, with less lignification, was recorded in E. globulus, when compared to E. grandis, suggesting lower activity of the cambium layer, an important site for AR development. Superoxide was more densely present around the vascular cylinder at both sampled times, and in greater quantity in E. globulus than E. grandis, declining with time in the former. Hydrogen peroxide was localized primarily along cell walls, more intensely in the primary xylem and phloem, and increased significantly at Tform in E. globulus. Ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT) activities were generally higher in E. grandis and varied as a function of time in E. globulus. Soluble guaiacol peroxidase (GPRX) activity increased from Texc to Tform in both species, whereas cell wall-bound GPRX activity increased with time in E. grandis, surpassing E. globulus. Flavonoid content increased with time in E. grandis and was higher than E. globulus at Tform. Principal component analysis showed that species- and time-derived differences contributed to almost 80% of the variance. Overall, data indicate that E. grandis shows higher cambium activity and tighter modulation of redox conditions than E. globulus. These features may influence ROS-based signaling and phytohormone homeostasis of cuttings, thereby impacting on AR development. Besides being players in the realm of AR developmental differences, the specific features herein identified could become potential tools for early clone selection and AR modulation aiming at improved clonal propagation of this forest crop.

Characterization of the nucellus-specific dehydrin MdoDHN11 demonstrates its involvement in the tolerance to water deficit.

KEY MESSAGE: MdoDHN11 acts in the nucellus layer to protect the embryo and the endosperm from limited water availability during apple seed development. Dehydrins (DHNs) are protective proteins related to several plant developmental responses that involve dehydration such as seed desiccation and abiotic stresses. In apple (Malus × domestica Borkh.), the seed-specific MdoDHN11 was suggested to play important roles against dehydration during seed development. However, this hypothesis has not yet been evaluated. Within this context, several experiments were performed to functionally characterize MdoDHN11. In situ hybridization analysis during apple seed development showed that MdoDHN11 expression is confined to a maternal tissue called nucellus, a central mass of parenchyma between the endosperm and the testa. The MdoDHN11 protein was localized in the cytosol and nucleus. Finally, transgenic Arabidopsis plants expressing MdoDHN11 were generated and exposed to a severe water-deficit stress, aiming to mimic a situation that can occurs during seed development. All transgenic lines showed increased tolerance to water deficit in relation to wild-type plants. Taken together, our results provide evidences that MdoDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from limited water availability, and the mechanism of action probably involves the interaction of MdoDHN11 with proteins and other components in the cell.

Cytological differentiation and cell wall involvement in the growth mechanisms of articulated laticifers in Tabernaemontana catharinensis A.DC. (Apocynaceae).

The cellular mechanisms of laticifer growth are of particular interest in plant biology but are commonly neglected. Using transmission electron microscopy and immunocytochemical methods, we recorded cytological differentiation and evaluated the cell wall involvement in the growth of articulated laticifers with intrusive growth in the mature embryo and plant shoot apex of Tabernaemontana catharinensis. The incorporation of adjacent meristematic cells into the laticifer system occurred in the embryo and plant shoot apex, and the incorporated cells acquired features of laticifer, confirming the laticifers' action-inducing mechanism. In the embryo, this was the main growth mechanism, and began with enlargement of the plasmodesmata and the formation of pores between laticifers and meristematic cells. In the plant shoot apex, it began with loose and disassembled walls and the reorientation of the cortical microtubules of the incorporated cell. Plasmodesmata were absent in these laticifers. There was stronger evidence of intrusive growth in undifferentiated portions of the plant shoot apex than in the embryo. The numerous plasmodesmata in laticifers of the embryo may have been related to the lower frequency of intrusive growth. Intrusive growth was associated with presence of arabinan (increasing wall flexibility and fluidity), and absence of galactan (avoiding wall stiffness), and callose (as a consequence of reduction in symplastic connections) in the laticifer walls. The abundance of low de-methyl-esterified homogalacturonan in the middle lamella and corners may reestablish cell-cell bonding in the laticifers. The cell wall features differed between embryo and plant shoot apex and are directly associated to laticifer growth mechanisms.

Somatic embryogenesis from immature cotyledons of soybean ( Glycine max ( L. ) Merr. ): ontogeny of somatic embryos

Embriões somáticos de soja em diferentes estádios de desenvolvimento, obtidos a partir de embriões zigóticos imaturos, foram estudados histologicamente com o objetivo de caracterizar as possíveis rotas ontogenéticas seguidas por esses embriões. Foram encontrados embriões nos estádios de proembrião, globular, cordiforme, torpedo e cotiledonar, revelando uma ontogenia similar àquela encontrada nos embriões zigóticos. Entretanto, a ausência de um suspensor definido, bem como o retardo no aparecimento de organização interna são diferenças marcantes entre esses dois processos embriogênicos. Além dessa rota de desenvolvimento, prováveis rotas alternativas de diferenciação são apresentadas.