Rhodamine-Tagged Polymethacrylate Dyes as Alternative Tools for Analysis of Plant Cells.

A rhodamine B (RhB)-based initiator for atom transfer radical polymerization (ATRP) was synthesized and applied for preparation of poly(2-trimethylammoniumethyl methacrylate) (PChMA), poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(2-trimethylsilyloxyethyl methacrylate) (PHEMATMS). Polymer fluorescence was confirmed by determination of quantum yield by comparative method with piroxicam as the standard exhibiting dependency of emission intensity on the polymer chain hydrophilicity and the kind of solvent. The RhB functionalized polymers were used for biological tests in plant materials except for RhB-PHEMATMS because of weak fluorescence. These two polymers slightly differed in cellular localization. RhB-PChMA was mostly observed in cell walls of root tissues and cotyledon epidermis. It was also observed in cytoplasm and cell organelles of root cap cells and rhizodermis, in contrast with cytoplasm of cotyledon epidermis. RhB-PHEMA was also present in apoplast. A strong signal in protoxylem cell walls and a weak signal in cell walls of rhizodermis and cortex were visible. Moreover, it was also present in cell walls of cotyledon epidermis. However, RhB-PHEMA was mostly observed in cytoplasm and cell organelles of all root tissues and epidermis of cotyledons. Both RhB-polymers did not cause cell death which means that they can be used in living plant material.

Analysis of the Distribution of Symplasmic Tracers During Zygotic and Somatic Embryogenesis.

Plasmodesmata (PD) are membraneous channels that span cell walls of adjacent cells to establish the symplasm. These connections are unique to plants and enable the cell-to-cell exchange of information via the symplasm. However, not every plant cell is connected to its neighbor. Absence of PD and lack of communication (symplasmic isolation) are important regulators of cell differentiation. To determine cell-to-cell symplasmic connectivity, the distribution of fluorescent tracers can be analyzed. Here, we describe in detail the entire procedure for conducting such analysis using fluorescence and confocal microscopy to study molecular fluxes in fluorescence recovery after photobleaching (FRAP) experiments. Studies using fluorochromes and fluorescent-labeled dextrans successfully inform the degree of symplasmic connectivity between cells in zygotic and somatic embryos. Small molecules, such as water and ions, travel through PD but also transcription factors and different types of RNA. Studies of symplasmic communication are important to determine the spatio-temporal correlation between cell differentiation and the exchange of information between cells. This information is necessary to determine the role of symplasmic communication during embryogenesis, which is a very important stage in plant development and morphogenesis.

Auxin biosynthesis maintains embryo identity and growth during BABY BOOM-induced somatic embryogenesis.

Somatic embryogenesis is a type of plant cell totipotency where embryos develop from nonreproductive (vegetative) cells without fertilization. Somatic embryogenesis can be induced in vitro by auxins, and by ectopic expression of embryo-expressed transcription factors like the BABY BOOM (BBM) AINTEGUMENTA-LIKE APETALA2/ETHYLENE RESPONSE FACTOR domain protein. These different pathways are thought to converge to promote auxin response and biosynthesis, but the specific roles of the endogenous auxin pathway in somatic embryogenesis induction have not been well-characterized. Here we show that BBM transcriptionally regulates the YUCCA3 (YUC3) and YUC8 auxin biosynthesis genes during BBM-mediated somatic embryogenesis in Arabidopsis (Arabidopsis thaliana) seedlings. BBM induced local and ectopic YUC3 and YUC8 expression in seedlings, which coincided with increased DR5 auxin response and indole-3-acetic acid (IAA) biosynthesis and with ectopic expression of the WOX2 embryo reporter. YUC-driven auxin biosynthesis was required for BBM-mediated somatic embryogenesis, as the number of embryogenic explants was reduced by ca. 50% in yuc3 yuc8 mutants and abolished after chemical inhibition of YUC enzyme activity. However, a detailed YUC inhibitor time-course study revealed that YUC-dependent IAA biosynthesis is not required for the re-initiation of totipotent cell identity in seedlings. Rather, YUC enzymes are required later in somatic embryo development for the maintenance of embryo identity and growth. This study resolves a long-standing question about the role of endogenous auxin biosynthesis in transcription factor-mediated somatic embryogenesis and also provides an experimental framework for understanding the role of endogenous auxin biosynthesis in other in planta and in vitro embryogenesis systems.

Tocopherols mutual balance is a key player for maintaining Arabidopsis thaliana growth under salt stress.

Enhanced channeling carbon through pathways: shikimate/chorismate, benzenoid-phenylopropanoid or 2-C-methyl-D-erythritol 4-phosphate (MEP) provides a multitude of secondary metabolites and cell wall components and allows plants response to environmental stresses. Through the biosynthetic pathways, different secondary metabolites, like tocopherols (TCs), are bind to mutual dependencies and metabolic loops, that are not yet fully understood. We compared, in parallel, the influence of α- and γ-TCs on metabolites involved in osmoprotective/antioxidative response, and physico-chemical modification of plasma membrane and cell wall. We studied Arabidopsis thaliana Columbia ecotype (WT), mutant vte1 deficient in α- and γ-TCs, mutant vte4 over-accumulating γ-TC instead of α-TC, and transgenic line tmt over-accumulating α-TC; exposed to NaCl. The results indicate that salt stress activates ß-carboxylation processes in WT plants and in plants with altered TCs accumulation. In α-TC-deficient plants, NaCl causes ACC decrease, but does not change SA, whose concentration remains higher than in α-TC accumulating plants. α/γ-TCs contents influence carbohydrates, poliamines, phenolic (caffeic, ferrulic, cinnamic) acids accumulation patterns. Salinity results in increased detection of the LM5 galactan and LM19 homogalacturonan epitopes in α-TC accumulating plants, and the LM6 arabinan and MAC207 AGP epitopes in α-TC deficient mutants. Parallel, plants with altered TCs composition show decreased both the cell turgor and elastic modulus determined at the individual cell level. α-TC deficient plants reveal lower values of cell turgor and elastic modulus, but higher cell hydraulic conductivity than α-TC accumulating plants. Under salt stress, α-TC shows stronger regulatory effect than γ-TC through the impact on chloroplastic biosynthetic pathways and ROS/osmotic-modulating compounds.

Symplasmic Isolation Contributes to Somatic Embryo Induction and Development in the Tree Fern Cyathea delgadii Sternb.

In this report, we describe studies on symplasmic communication and cellular rearrangement during direct somatic embryogenesis (SE) in the tree fern Cyathea delgadii. We analyzed changes in the symplasmic transport of low-molecular-weight fluorochromes, such as 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) and fluorescein (delivered to cells as fluorescein diacetate, FDA), within stipe explants and somatic embryos originating from single epidermal cells and developing during 16-d long culture. Induction of SE is preceded by a restriction in fluorochrome distribution between certain explant cells. Microscopic analysis showed a series of cellular changes like a decrease in vacuole size, increase in vacuole numbers, and increased density of cytoplasm and deposition of electron-dense material in cell walls that may be related with embryogenic transition. In somatic embryos, the limited symplasmic communication between cells was observed first in linear tri-cellular embryos. Further development of the fern embryo was associated with the formation of symplasmic domains corresponding to the four segments of the plant body. Using symplasmic tracers, we provided evidence that the changes in plasmodesmata permeability are corelated with somatic-to-embryogenic transition and somatic embryo development.

Development of Embryo Suspensors for Five Genera of Crassulaceae with Special Emphasis on Plasmodesmata Distribution and Ultrastructure.

The suspensor in the majority of angiosperms is an evolutionally conserved embryonic structure functioning as a conduit that connects ovule tissues with the embryo proper for nutrients and growth factors flux. This is the first study serving the purpose of investigating the correlation between suspensor types and plasmodesmata (PD), by the ultrastructure of this organ in respect of its full development. The special attention is paid to PD in representatives of Crassulaceae genera: Sedum, Aeonium, Monanthes, Aichryson and Echeveria. The contribution of the suspensor in transporting nutrients to the embryo was confirmed by the basal cell structure of the suspensor which produced, on the micropylar side of all genera investigated, a branched haustorium protruding into the surrounding ovular tissue and with wall ingrowths typically associated with cell transfer. The cytoplasm of the basal cell was rich in endoplasmic reticulum, mitochondria, dictyosomes, specialized plastids, microtubules, microbodies and lipid droplets. The basal cell sustained a symplasmic connection with endosperm and neighboring suspensor cells. Our results indicated the dependence of PD ultrastructure on the type of suspensor development: (i) simple PD are assigned to an uniseriate filamentous suspensor and (ii) PD with an electron-dense material are formed in a multiseriate suspensor. The occurrence of only one or both types of PD seems to be specific for the species but not for the genus. Indeed, in the two tested species of Sedum (with the distinct uniseriate/multiseriate suspensors), a diversity in the structure of PD depends on the developmental pattern of the suspensor. In all other genera (with the multiseriate type of development of the suspensor), the one type of electron-dense PD was observed.

Identification of symplasmic domains in the embryo and seed of Sedum acre L. (Crassulaceae).

MAIN CONCLUSION: Our study demonstrated that symplasmic communication between Sedum acre seed compartments and the embryo proper is not uniform. The presence of plasmodesmata (PD) constitutes the structural basis for information exchange between cells, and symplasmic communication is involved in the regulation of cell differentiation and plant development. Most recent studies concerning an analysis of symplasmic communication between seed compartments and the embryo have been predominantly performed on Arabidopsis thaliana. The results presented in this paper describe the analysis of symplasmic communication on the example of Sedum acre seeds, because the ultrastructure of the seed compartments and the embryo proper, including the PD, have already been described, and this species represents an embryonic type of development different to Arabidopsis. Moreover, in this species, an unusual electron-dense dome associated with plasmodesmata on the border between the basal cell/chalazal suspensor cells and the basal cell/the endosperm has been described. This prompted the question as to whether these plasmodesmata are functional. Thus, the aim of this study was to describe the movement of symplasmic transport fluorochromes between different Sedum seed compartments, with particular emphasis on the movement between the basal cell and the embryo proper and endosperm, to answer the following questions: (1) are seeds divided into symplasmic domains; (2) if so, are they stable or do they change with the development? The results have shown that symplasmic tracers movement: (a) from the external integument to internal integument is restricted; (b) from the basal cell to the other part of the embryo proper and from the basal cell to the endosperm is also restricted;