Reproductive biology of the "Brazilian pine" (Araucaria angustifolia-Araucariaceae): the pollen tube growth and the seed cone development.

KEY MESSAGE: In Araucaria angustifolia, the seed scale is part of the ovule, the female gametophyte presents a monosporic origin and arises from a coenocytic tetrad, and the pollen tube presents a single axis. The seed cone of conifers has many informative features, and its ontogenetic data may help interpret relationships among function, development patterns, and homology among seed plants. We reported the seed cone development, from pollination to pre-fertilization, including seed scale, ovule ontogeny, and pollen tube growth in Araucaria angustifolia. The study was performed using light microscopy, scanning electron microscopy, and X-ray microcomputed tomography (µCT). During the pollination period, the ovule arises right after the seed scale has emerged. From that event to the pre-fertilization period takes about 14 months. Megasporogenesis occurs three weeks after ovule formation, producing a coenocytic tetrad. At the same time as the female gametophyte's first nuclear division begins, the pollen tube grows through the seed scale adaxial face. Until maturity, the megagametophyte goes through the free nuclei stage, cellularization stage, and cellular growth stage. Along its development, many pollen tubes develop in the nucellar tissue extending straight toward the female gametophyte. Our observations show that the seed scale came out of the same primordia of the ovule, agreeing with past studies that this structure is part of the ovule itself. The formation of a female gametophyte with a monosporic origin that arises from a coenocytic tetrad was described for the first time in conifers, and the three-dimensional reconstruction of the ovule revealed the presence of pollen tubes with only one axis and no branches, highlighting a new pattern of pollen tube growth in Araucariaceae.

Neither lysigenous nor just oil: Demystifying myrtaceous secretory cavities.

PREMISE: Leaf subepidermal secretory cavities are a notable trait in Myrtaceae, but their formation is still controversial because of the lack of consensus on their ontogeny among authors. Knowledge about the compounds present in these cavities has grown over the last few years, demonstrating that terpenoid-rich oils are not their unique content. These two points are the focus of this study on the ontogeny, structure, and contents of secretory cavities in neotropical Myrtaceae. METHODS: We used histochemical tests and Raman analysis to verify the basic chemical composition of the cavity contents of nine species. We studied the ontogeny of glands in one species, comparing aldehyde-fixed tissues and fresh sections mounted in an inert medium. RESULTS: We observed schizogenous development and appearance of the secretory cavities and found that sample processing may induce cell breakdown, which can be misinterpreted as lysigeny. The content of these cavities contains putative terpenes, resins, carbonyl groups, and flavonoids. CONCLUSIONS: Our findings support the hypothesis that the lysigenous appearance of the oil glands is a technical artifact. These tissue distortions must be considered when interpreting the development of this type of secretory structure. Moreover, the basic analyses of chemical constituents show for the first time that the glands of neotropical Myrtaceae are potential reservoirs of some compounds such as flavonoids previously reported as novelties for a few other myrtaceous species. Because some of them are non-lipid compounds, the idea that the glands are just oil repositories is no longer applicable.

Evolutionary features of microspore and pollen grain development in Cyperaceae.

KEY MESSAGE: Asymmetric meiosis leading to the release of pollen grains as pseudomonads is a synapomorphy in Cyperaceae, but differences in microspore development are relevant in the family's evolutionary history. Cyperaceae members present atypical microsporogenesis, in which one meiotic product is functional while the other three degenerate, culminating in pseudomonad pollen formation. Differences during development, such as pseudomonad shape and degenerative microspore positioning, are seen throughout the family, but no phylogenetic interpretation has been made regarding these variances thus far. In this study, we analyzed the early- and late-diverging sedge genera Hypolytrum and Eleocharis, respectively, while comparing them to data available in the literature and conducting an ancestral character reconstruction for pseudomonad traits. Light microscopy results show that pseudomonad development in Hypolytrum is homologous to several other sedge genera, presenting apical degenerative microspores. However, pseudomonads are round and centrally arranged in the anther locule in this case, which consists of a pleisiomorphic trait for the family. The basal positioning of degenerative microspores is restricted to Rhynchospora, consisting of an apomorphic trait for this genus. Despite these differences, ultrastructural analysis of Eleocharis pseudomonad revealed shared features with other genera studied, which include variations in chromatin condensation and cytoplasmic turnover in functional cells. These common features seem related to the different cellular fates seen during microspore development and further corroborate the synapomorphic status of pseudomonads in sedges.

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.

Nuclear variations and tapetum polyploidy related to pollen grain development in Passiflora L. (Passifloraceae).

Tapetal cells comprise an anther tissue fundamental to pollen grain development. They are associated with endoreduplication events, which culminate in polyploid and multinucleated cells, high metabolic activity, and different organelle arrangements to support all the development of the pollen grains. Passiflora species present a secretory tapetum, with diversity in the number and size of nuclei. Tapetal cells undergo numerous changes in a short period of development when compared to the plant's life span. To improve our knowledge of tapetum development, tests assessing ploidy levels, anatomy, cytochemistry, transmission electron microscopy, flow cytometry, as well as conventional and molecular cytogenetics were used in Passiflora actinia and P. elegans. The current data show striking differences in nuclear organisation during tapetal cell development, including mono to quadrinucleate cells, and ploidy levels from 2n to 32n. One of the most peculiar features was the atypical behaviour of the endoplasmic reticulum (ER), which accumulated in the cell border, similar to a 'cER', as well as large dictyosomes. This endomembrane configuration may be related to the tapetum nutritional network and secretion of compounds at the end of meiosis. Another atypical feature of the ER was the formation of an invagination to establish 'binucleated' polyploid cells. This membrane projection appears when the nuclei form two lobes, as well as when it organises a nucleoplasmic reticulum. These data demonstrate that there are important ultrastructural changes in tapetal cells, including organelle arrangements, ploidy levels, and nuclear activity, common to P. actinia and P. elegans, but different from the plant model A. thaliana.

Tightly controlled expression of OsbHLH35 is critical for anther development in rice.

Anther development is a complex process regulated by a myriad of transcription factors belonging to distinct protein families. In this study, we focus on the functional characterization of OsbHLH35, a basic Helix-Loop-Helix (bHLH) TF that regulates anther development in rice. Plants overexpressing OsbHLH35 presented small and curved anthers, leading to a reduction of 72 % on seed production. Rice transgenic plants expressing GUS reporter gene under the control of OsbHLH35 promoter (pOsbHLH35::GUS) showed that this TF specifically accumulates in anthers at the meiosis stage and in other spikelet tissues. Yeast one-hybrid screening identified three members of the Growth-Regulating Factor (GRF) family, OsGRF3, OsGRF4, and OsGRF11, as transcriptional regulators of OsbHLH35. Transactivation assay showed that OsGRF11 negatively regulates OsbHLH35 expression in Arabidopsis protoplasts. This regulation was also observed in planta through the analysis of transgenic plants overexpressing OsGRF11 (OsGRF11OE), confirming that OsGRF11 is a negative regulator of OsbHLH35 in rice. Our data suggest that OsbHLH35 plays an essential role in anther development in rice and the fine control of its expression is crucial to ensure proper seed production.

Are unusual ultrastructural features occurring in the pollen endomembrane system of Cyperaceae and other angiosperms?

Cyperaceae representatives present peculiar microsporogenesis and microgametogenesis, which raises the question of how regular the sedge pollen is. In order to answer this question, preanthesis pollen grains of Cyperaceae individuals were analyzed under different tools such as light and transmission electron microscopy, which included cytochemistry and immunogold procedures. The results showed that maturing pollen in Cyperaceae presents some unusual endomembrane behaviors. Endoplasmic reticulum and dictyosomes are concerned in classic secretion pathways in vegetative cells, and possibly the late breakdown of degenerative microspores. However, cortical and concentric endoplasmic reticulum are also present and are possibly related to other functions aside the biosynthetic pathway. Unconventional secretion of large membrane-bound bodies containing cell wall precursors was also observed and confirmed by immunogold. However, since these bodies most likely receive material from dictyosomes, as observed in silver nitrate reaction, the "unconventional" status of this secretion is debatable. Reports of the literature show that these unusual endomembrane formations are not exclusive of the sedge pollen, but little attention have been given to them so far. This could represent an opportunity to re-examine our understanding on the endomembrane system in pollen cells in general.

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.

Heat transfer in the tank-inflorescence of Nidularium innocentii (Bromeliaceae): Experimental and finite element analysis based on X-ray microtomography.

In Bromeliaceae, various traits have evolved for the uptake and storage of water; however, their roles in bromeliad inflorescences remain unresolved. This study investigates the role of water in the flowers and inflorescences of Nidularium innocentii, and describes water as a protection mechanism. Individuals were divided into groups with and without water provision in inflorescences. Both groups were maintained with water in soil and leaves under the same environmental conditions. During anthesis, individuals were collected, and inflorescences were measured. Another specimen was prepared and scanned using X-ray microtomography (µCT), generating a high-resolution 3D model that was converted into a discretized geometry. Heat transfer finite element analysis (FEA) of the µCT-based geometry was then performed to simulate external temperature dissipation with the presence and absence of water in 3D. Flower size in the control group was significantly larger, and many injuries were observed in the drought group. FEA data indicated that the water environment led to lower temperature variation when compared to the air environment by significantly alleviating thermal amplitude. Water acted as a temperature stabilizer for the inflorescence, while its absence initiated physiological stress responses.

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.

Manipulation of VviAGL11 expression changes the seed content in grapevine (Vitis vinifera L.).

Seedlessness in grapes is a desirable trait, especially for in natura consumption. Previously, we showed that VviAGL11 is the main responsible gene for seed morphogenesis in grapevine. Here we tested the function of this gene in grapevine with the use of plant plasmids. VviAGL11 was cloned into silencing and overexpression versions of p28iIR plasmid. Reproductive grapevine bunches from different seeded and seedless cultivars were separately treated with VviAGL11-harboring plasmids, along with controls. Plasmids were detected in leaves after a month of treatment, and berries, leaves, stems and seeds were analyzed for ectopic gene expression by RT-qPCR after 90 days of plasmid injection. Fruits from the seedless 'Linda' treated with the VviAGL11-overexpression plasmid showed high expression levels of VviAGL11 and exhibited small seeds that were not found in the untreated control samples. Mature grapes from seeded 'Italia' and 'Ruby' bunches treated with the VviAGL11-silencing plasmid showed decreased VviAGL11 expression, reduced number of seeds and increased number of seed traces. The present study confirms that VviAGL11 is a key master regulator of seed morphogenesis in grapevine and corroborates with the applicability of plant plasmids as promising biotechnological tools to functionally test genes in perennial plants in a rapid and confident way.

Comparative adventitious root development in pre-etiolated and flooded Arabidopsis hypocotyls exposed to different auxins.

Adventitious roots (ARs) emerge from stems, leaves or hypocotyls, being strategic for clonal propagation. ARs may develop spontaneously, upon environmental stress or hormonal treatment. Auxins strongly influence AR development (ARD), depending on concentration and kind. However, the role of different types of auxin is rarely compared at the molecular level. Rooting triggered by light exposure and flooding was examined in intact etiolated Arabidopsis thaliana hypocotyls treated with distinct auxin types. Morphological aspects, rooting-related gene expression profiles, and IAA immunolocalization were recorded. NAA and 2,4-D effects were highly dose-dependent; at higher concentrations NAA inhibited root growth and 2,4-D promoted callus formation. NAA yielded the highest number of roots, but inhibited elongation. IAA increased the number of roots with less interference in elongation, yielding the best overall rooting response. IAA was localized close to the tissues of root origin. Auxin stimulated ARD was marked by increased expression of PIN1 and GH3.3. NAA treatment induced expression of CYCB1, GH3.6 and ARF8. These NAA-specific responses may be associated with the development of numerous shorter roots. In contrast, expression of the auxin action inhibitor IAA28 was induced by IAA. Increased PIN1 expression indicated the relevance of auxin efflux transport for focusing in target cells, whereas GH3.3 suggested tight control of auxin homeostasis. IAA28 increased expression during IAA-induced ARD differs from what was previously reported for lateral root development, pointing to yet another possible difference in the molecular programs of these two developmental processes.

The MADS-box gene Agamous-like 11 is essential for seed morphogenesis in grapevine.

Despite the wide appreciation of seedless grapes, little is known about the molecular mechanisms that drive the stenospermocarpic seedless-type phenotype in grapevine. In order to address the molecular mechanisms that control seedlessness in grapevine, our study aimed to characterize VviAGL11, a class D MADS-box transcription factor gene that has been proposed as the major candidate gene involved in Vitis vinifera seed morphogenesis. VviAGL11 allelic variations in seeded and seedless grapevine cultivars were determined, and its correlations with allele-specific steady-state mRNA levels were investigated. VviAGL11 relative expression was significantly higher in seeds at 2, 4, and 6 weeks after fruit set, whereas in the seedless grape its transcript levels were extremely low in all stages analyzed. In situ hybridization revealed transcript accumulation specifically in the dual endotesta layer of the seeds, which is responsible for elongation and an increase of cell number, a necessary step to determine the lignification and the final seed size. No hybridization signals were visible in the seedless grapevine tissues, and a morphoanatomical analysis showed an apparent loss of identity of the endotesta layer of the seed traces. Ectopic expression of VviAGL11 in the Arabidopsis SEEDSTICK mutant background restored the wild-type phenotype and confirmed the direct role of VviAGL11 in seed morphogenesis, suggesting that depletion of its expression is responsible for the erroneous development of a highly essential seed layer, therefore culminating in the typical apirenic phenotype.

Comparative transcriptional analysis provides new insights into the molecular basis of adventitious rooting recalcitrance in Eucalyptus.

Adventitious rooting (AR) is essential in clonal propagation. Eucalyptus globulus is relevant for the cellulose industry due to its low lignin content. However, several useful clones are recalcitrant to AR, often requiring exogenous auxin, adding cost to clonal garden operations. In contrast, E. grandis is an easy-to-root species widely used in clonal forestry. Aiming at contributing to the elucidation of recalcitrance causes in E. globulus, we conducted a comparative analysis with these two species differing in rooting competence, combining gene expression and anatomical techniques. Recalcitrance in E. globulus is reversed by exposure to exogenous indole-3-acetic acid (IAA), which promotes important gene expression modifications in both species. The endogenous content of IAA was significantly higher in E. grandis than in E. globulus. The cambium zone was identified as an active area during AR, concentrating the first cell divisions. Immunolocalization assay showed auxin accumulation in cambium cells, further indicating the importance of this region for rooting. We then performed a cambium zone-specific gene expression analysis during AR using laser microdissection. The results indicated that the auxin-related genes TOPLESS and IAA12/BODENLOS and the cytokinin-related gene ARR1may act as negative regulators of AR, possibly contributing to the hard-to-root phenotype of E. globulus.

Cold tolerance in rice germinating seeds revealed by deep RNAseq analysis of contrasting indica genotypes.

Rice productivity is largely affected by low temperature, which can be harmful throughout plant development, from germination to grain filling. Germination of indica rice cultivars under cold is slow and not uniform, resulting in irregular emergence and small plant population. To identify and characterize novel genes involved in cold tolerance during the germination stage, two indica rice genotypes (sister lines previously identified as cold-tolerant and cold-sensitive) were used in parallel transcriptomic analysis (RNAseq) under cold treatment (seeds germinating at 13 °C for 7 days). We detected 1,361 differentially expressed transcripts. Differences in gene expression found by RNAseq were confirmed for 11 selected genes using RT-qPCR. Biological processes enhanced in the cold-tolerant seedlings include: cell division and expansion (confirmed by anatomical sections of germinating seeds), cell wall integrity and extensibility, water uptake and membrane transport capacity, sucrose synthesis, generation of simple sugars, unsaturation of membrane fatty acids, wax biosynthesis, antioxidant capacity (confirmed by histochemical staining of H2O2), and hormone and Ca(2+)-signaling. The cold-sensitive seedlings respond to low temperature stress increasing synthesis of HSPs and dehydrins, along with enhanced ubiquitin/proteasome protein degradation pathway and polyamine biosynthesis. Our findings can be useful in future biotechnological approaches aiming to cold tolerance in indica rice.

Initial development of the endocarp in Lithraea brasiliensis Marchand (Anacardiaceae): with taxonomic notes.

Investigation into the initial developmental stages of a given structure is fundamental for precise characterization as well as for comparative analysis in relation to other taxa when homologies are established. For the Anacardiaceae family, investigations of the initial development of the pericarp or its basic histological sites, the epicarp, mesocarp and endocarp, are relevant since these regions are of taxonomic and phylogenetic importance. The initial stages of endocarp development in Lithraea brasiliensis were studied using light microscopy. In L. brasiliensis, the fruits are of the drupe type. The endocarp originates exclusively in the epidermis of the locular cavity and is composed of only three strata. The crystalliferous layer, typical in fruits of the Anacardiaceae family, originates in the carpelar mesophyll and runs adjacent to the outermost layer of the endocarp. The endocarp in Lithraea brasiliensis is of the Anacardium type. The results of the present study are important for the identification of the Lithraea species that occur in the state of Rio Grande do Sul. In L. molleoides, the crystalliferous layer was shown to originate from the locular epidermis and this developmental aspect enables it to be differentiated from L. brasiliensis and demonstrates that there is variability of endocarp development in Lithraea.

New insights into aluminum tolerance in rice: the ASR5 protein binds the STAR1 promoter and other aluminum-responsive genes.

Aluminum (Al) toxicity in plants is one of the primary constraints in crop production. Al³âº, the most toxic form of Al, is released into soil under acidic conditions and causes extensive damage to plants, especially in the roots. In rice, Al tolerance requires the ASR5 gene, but the molecular function of ASR5 has remained unknown. Here, we perform genome-wide analyses to identify ASR5-dependent Al-responsive genes in rice. Based on ASR5_RNAi silencing in plants, a global transcriptome analysis identified a total of 961 genes that were responsive to Al treatment in wild-type rice roots. Of these genes, 909 did not respond to Al in the ASR5_RNAi plants, indicating a central role for ASR5 in Al-responsive gene expression. Under normal conditions, without Al treatment, the ASR5_RNAi plants expressed 1.756 genes differentially compared to the wild-type plants, and 446 of these genes responded to Al treatment in the wild-type plants. Chromatin immunoprecipitation followed by deep sequencing identified 104 putative target genes that were directly regulated by ASR5 binding to their promoters, including the STAR1 gene, which encodes an ABC transporter required for Al tolerance. Motif analysis of the binding peak sequences revealed the binding motif for ASR5, which was confirmed via in vitro DNA-binding assays using the STAR1 promoter. These results demonstrate that ASR5 acts as a key transcription factor that is essential for Al-responsive gene expression and Al tolerance in rice.

The mitochondrial glutathione peroxidase GPX3 is essential for H2O2 homeostasis and root and shoot development in rice.

Glutathione (GSH) peroxidases (GPXs: EC 1.11.1.9 and EC1.11.1.12) are non-heme thiol peroxidases that catalyze the reduction of H2O2 or organic hydroperoxides to water, and they have been identified in almost all kingdoms of life. The rice glutathione peroxidase (OsGPX) gene family is comprised of 5 members spread throughout a range of sub cellular compartments. The OsGPX gene family is induced in response to exogenous H2O2 and cold stress. In contrast, they are down regulated in response to drought and UV-B light treatments. Transgenic rice plants have been generated that lack mitochondrial OsGPX3. These GPX3s plants showed shorter roots and shoots compared to non-transformed (NT) plants, and higher amounts of H2O2 mitochondrial release were observed in the roots of these plants cultivated under normal conditions. This accumulation of H2O2 is positively associated with shorter root length in GPX3s plants compared to NT ones. Moreover, GPX3 promoter analysis indicated that it is mainly expressed in root tissue. These results suggest that silencing the mitochondrial OsGPX3 gene impairs normal plant development and leads to a stress-induced morphogenic response via H2O2 accumulation.

Heterogeneity of silica and glycan-epitope distribution in epidermal idioblast cell walls in Adiantum raddianum laminae.

Laminae of Adiantum raddianum Presl., a fern belonging to the family Pteridaceae, are characterised by the presence of epidermal fibre-like cells under the vascular bundles. These cells were thought to contain silica bodies, but their thickened walls leave no space for intracellular silica suggesting it may actually be deposited within their walls. Using advanced electron microscopy in conjunction with energy dispersive X-ray microanalysis we showed the presence of silica in the cell walls of the fibre-like idioblasts. However, it was specifically localised to the outer layers of the periclinal wall facing the leaf surface, with the thick secondary wall being devoid of silica. Immunocytochemical experiments were performed to ascertain the respective localisation of silica deposition and glycan polymers. Epitopes characteristic for pectic homogalacturonan and the hemicelluloses xyloglucan and mannan were detected in most epidermal walls, including the silica-rich cell wall layers. The monoclonal antibody, LM6, raised against pectic arabinan, labelled the silica-rich primary wall of the epidermal fibre-like cells and the guard cell walls, which were also shown to contain silica. We hypothesise that the silicified outer wall layers of the epidermal fibre-like cells support the lamina during cell expansion prior to secondary wall formation. This implies that silicification does not impede cell elongation. Although our results suggest that pectic arabinan may be implicated in silica deposition, further detailed analyses are needed to confirm this. The combinatorial approach presented here, which allows correlative screening and in situ localisation of silicon and cell wall polysaccharide distribution, shows great potential for future studies.