Adela Olmedilla

A specific ultrastructural method to reveal DNA : the NAMA-Ur

We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.

Localization in Roots and Flowers of Pea Chloroplastic Thioredoxin f and Thioredoxin m Proteins Reveals New Roles in Nonphotosynthetic Organs

Plant thioredoxins (TRXs) are involved in redox regulation of a wide variety processes and usually exhibit organ specificity. We report strong evidence that chloroplastic TRXs are localized in heterotrophic tissues and suggest some ways in which they might participate in several metabolic and developmental processes. The promoter regions of the chloroplastic f and m1 TRX genes were isolated from a pea (Pisum sativum) plant genomic bank. Histochemical staining for β-glucuronidase (GUS) in transgenic homozygous Arabidopsis (Arabidopsis thaliana) plants showed preferential expression of the 444-bp PsTRXf1 promoter in early seedlings, stems, leaves, and roots, as well as in flowers, stigma, pollen grains, and filaments. GUS activity under the control of the 1,874-bp PsTRXm1 promoter was restricted to the leaves, roots, seeds, and flowers. To gain insight into the translational regulation of these genes, a series of deletions of 5′ elements in both TRX promoters were analyzed. The results revealed that a 126-bp construct of the PsTRXf2 promoter was unable to reproduce the expression pattern observed with the full promoter. The differences in expression and tissue specificity between PsTRXm1 and the deleted promoters PsTRXm2 and PsTRXm3 suggest the existence of upstream positive or negative regulatory regions that affect tissue specificity, sucrose metabolism, and light regulation. PsTRXm1 expression is finely regulated by light and possibly by other metabolic factors. In situ hybridization experiments confirmed new localizations of these chloroplastic TRX transcripts in vascular tissues and flowers, and therefore suggest possible new functions in heterotrophic tissues related to cell division, germination, and plant reproduction.

Peroxynitrite mediates programmed cell death both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.)

Programmed cell death (PCD) has been found to be induced after pollination both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.). Reactive oxygen species (ROS) and nitric oxide (NO) are known to be produced in the pistil and pollen during pollination but their contribution to PCD has so far remained elusive. The possible role of ROS and NO was investigated in olive pollen–pistil interaction during free and controlled pollination and it was found that bidirectional interaction appears to exist between the pollen and the stigma, which seems to regulate ROS and NO production. Biochemical evidence strongly suggesting that both O2˙− and NO are essential for triggering PCD in self-incompatibility processes was also obtained. It was observed for the first time that peroxynitrite, a powerful oxidizing and nitrating agent generated during a rapid reaction between O2˙− and NO, is produced during pollination and that this is related to an increase in protein nitration which, in turn, is strongly associated with PCD. It may be concluded that peroxynitrite mediates PCD during pollen–pistil interaction in Olea europaea L. both in self-incompatible pollen and papillar cells.

Programmed-cell-death hallmarks in incompatible pollen and papillar stigma cells of Olea europaea L. under free pollination

Programmed cell death (PCD) is a process that occurs both in animals and in plants and is an essential element in developmental processes. Pollination is a key factor in fruit production and self-incompatibility is one of the main limiting factors of this process. PCD has recently been put forward as a possible cause of pollen-growth arrest. As far as the olive is concerned, no data have been published concerning the mechanisms involved in hindering the growth of pollen tubes in incompatible pollen. Thus, we have studied olive pistils excised from freely pollinated flowers at different stages before and during the progamic phase using different cytochemical techniques, including trypan blue staining. To discover whether the elimination of incompatible pollen might be associated to PCD, we applied different tests to the excised pistils: (1) TUNEL assay; (2) DNA degradation analysis; (3) detection of caspase-3-like activity. Once we had determined that PCD was involved in pollen selection after free pollination, we conducted experiments after controlled pollination in pistils excised from flowers: (a) developing in the absence of pollen; (b) pollinated with sterile pollen that does not germinate; (c) self-pollinated; (d) pollinated with compatible pollen. Our results demonstrate that the growth of tubes in incompatible pollen is halted in the stylar area in a way that suggests the intervention of PCD. Furthermore, any pollen, even if sterile, seemed to accelerate PCD in papillar cells in the olive.Programmed cell death (PCD) is a process that occurs both in animals and in plants and is an essential element in developmental processes. Pollination is a key factor in fruit production and self-incompatibility is one of the main limiting factors of this process. PCD has recently been put forward as a possible cause of pollen-growth arrest. As far as the olive is concerned, no data have been published concerning the mechanisms involved in hindering the growth of pollen tubes in incompatible pollen. Thus, we have studied olive pistils excised from freely pollinated flowers at different stages before and during the progamic phase using different cytochemical techniques, including trypan blue staining. To discover whether the elimination of incompatible pollen might be associated to PCD, we applied different tests to the excised pistils: (1) TUNEL assay; (2) DNA degradation analysis; (3) detection of caspase-3-like activity. Once we had determined that PCD was involved in pollen selection after free pollination, we conducted experiments after controlled pollination in pistils excised from flowers: (a) developing in the absence of pollen; (b) pollinated with sterile pollen that does not germinate; (c) self-pollinated; (d) pollinated with compatible pollen. Our results demonstrate that the growth of tubes in incompatible pollen is halted in the stylar area in a way that suggests the intervention of PCD. Furthermore, any pollen, even if sterile, seemed to accelerate PCD in papillar cells in the olive.

The role of reactive oxygen species and nitric oxide in programmed cell death associated with self-incompatibility

Successful sexual reproduction often relies on the ability of plants to recognize self- or genetically-related pollen and prevent pollen tube growth soon after germination in order to avoid self-fertilization. Angiosperms have developed different reproductive barriers, one of the most extended being self-incompatibility (SI). With SI, pistils are able to reject self or genetically-related pollen thus promoting genetic variability. There are basically two distinct systems of SI: gametophytic (GSI) and sporophytic (SSI) based on their different molecular and genetic control mechanisms. In both types of SI, programmed cell death (PCD) has been found to play an important role in the rejection of self-incompatible pollen. Although reactive oxygen species (ROS) were initially recognized as toxic metabolic products, in recent years, a new role for ROS has become apparent: the control and regulation of biological processes such as growth, development, response to biotic and abiotic environmental stimuli, and PCD. Together with ROS, nitric oxide (NO) has become recognized as a key regulator of PCD. PCD is an important mechanism for the controlled elimination of targeted cells in both animals and plants. The major focus of this review is to discuss how ROS and NO control male-female cross-talk during fertilization in order to trigger PCD in self-incompatible pollen, providing a highly effective way to prevent self-fertilization.

Programmed cell death during the transition from multicellular structures to globular embryos in barley androgenesis

Androgenesis represents one of the most fascinating examples of cell differentiation in plants. In barley, the conversion of stressed uninucleate microspores into embryo-like structures is highly efficient. One of the bottlenecks in this process is the successful release of embryo-like structures out of the exine wall of microspores. In the present work, morphological and biochemical studies were performed during the transition from multicellular structures to globular embryos. Exine wall rupture and subsequent globular embryo formation were observed only in microspores that divided asymmetrically. Independent divisions of the generative and the vegetative nuclei gave rise to heterogeneous multicellular structures, which were composed of two different cellular domains: small cells with condensed chromatin structure and large cells with normal chromatin structure. During exine wall rupture, the small cells died and their death marked the site of exine wall rupture. Cell death in the small cell domain showed typical features of plant programmed cell death. Chromatin condensation and DNA degradation preceded cell detachment and cytoplasm dismantling, a process that was characterized by the formation of vesicles and vacuoles that contained cytoplasmic material. This morphotype of programmed cell death was accompanied by an increase in the activity of caspase-3-like proteases. The orchestration of such a death program culminated in the elimination of the small generative domain, and further embryogenesis was carried out by the large vegetative domain. To date, this is the first report to show evidence that programmed cell death takes part in the development of microspore-derived embryos.

Structural organization and cytochemical features of the pistil in Olive (Olea europaea L.) cv. Picual at anthesis

Pistil structure and composition are critical in recognizing and permitting the germination of suitable pollen grains. We have studied the structure of the different component tissues of the pistil, their organization and cytochemical features of olive flowers, Olea europaea L., at anthesis, an essential first step for understanding the processes of pollen-pistil interaction and fertilization. The pistil from olive cv. Picual trees is characterized by a wet bilobed stigma, a solid style and a bilocular ovary containing four ovules. The stigma is composed of external multicellular papillae and a non-papillate inner region of secretory cells. An exudate is observed on the surface of the papillae at anthesis, the moment when the flowers (first) open, but the anthers are not yet dehiscent. The inner secretory cells of the stigma and those of the stylar transmitting tissue are continuous, constituting a funnel-shaped zone which extends from within the stigma to the style base. The outer surface of the ovary and style epidermis is surrounded by a cuticle layer, while internally, the locule wall, formed by the innermost cells of the endocarp, consists of two layers of periclinally oriented cells with thicker cell walls. Starch granules are distributed differentially, concentrated most densely in the style (adjacent to the vascular bundles), in the distil region of the ovary, and in the micropylar ends of the ovules. Well-developed vascular bundles are present in the lower part of the stigma, the style and in the pericarp of the ovary. The histochemical identification of sugars and lipid substances within and around the vascular bundles suggests that they are involved in the transport of these materials. Ultrastructural observations confirm the presence of exudates on the papillar surface and confirm the secretory characteristics of the inner stigmatic cells. They also demonstrate marked differences in size, form, and vacuolar and cytoplasmic contents among the cells of the various style and upper ovary tissues. We provide the first detailed cytological description at anthesis of all the olive pistil tissues, indicating the structural and cytochemical basis for the pistil behavior which will transpire during the progamic phase.

Plant flavonoids target Pseudomonas syringae pv. tomato DC3000 flagella and type III secretion system

Flavonoids are among the most abundant plant secondary metabolites involved in plant protection against pathogens, but micro-organisms have developed resistance mechanisms to those compounds. We previously demonstrated that the MexAB-OprM efflux pump mediates resistance of Pseudomonas syringae pv. tomato (Pto) DC3000 to flavonoids, facilitating its survival and the colonization of the host. Here, we have shown that tomato plants respond to Pto infection producing flavonoids and other phenolic compounds. The effects of flavonoids on key traits of this model plant-pathogen bacterium have also been investigated observing that they reduce Pto swimming and swarming because of the loss of flagella, and also inhibited the expression and assembly of a functional type III secretion system. Those effects were more severe in a mutant lacking the MexAB-OprM pump. Our results suggest that flavonoids inhibit the function of the GacS/GacA two-component system, causing a depletion of rsmY RNA, therefore affecting the synthesis of two important virulence factors in Pto DC3000, flagella and the type III secretion system. These data provide new insights into the flavonoid role in the molecular dialog between host and pathogen.

Spatial patterns of histone mRNA expression during grain development and germination in rice

The pattern of distribution of histone H3 mRNA during the development of the rice grain and its germination was monitored by in situ hybridization and confirmed by Northern blot analysis. In ovaries sampled before and after fertilization, a 3H-labeled histone RNA probe was localized in the cells of the pericarp, outer integument and nucellus but binding of the probe decreased as these tissues senesced. In the developing embryo, the histone message was first detected in the scutellum; later, all parts of the embryo except the shoot apex, newly formed leaf primordia and the quiescent center of the root, revealed the presence of transcripts. Considerable binding of the probe also occurred in the endosperm as its cells began to accumulate starch. Cells of the embryo and endosperm of mature grains displayed very little or no histone mRNA, although during germination, these transcripts reappeared in the cells of both embryo and endosperm. There was a good correlation between the presence or absence of binding to the 32P-labeled histone gene DNA by RNA extracted from grains of different stages of development and germination, as revealed by Northern blot analysis and by spatial localization following in situ hybridization.

Methylation pattern of nuclear ribosomal RNA genes from rice (Oryza sativa)

Abstract A restriction map of rice ( Oryza sativa ) rDNA was established for Eco RI, Bam HI and Bg 1II. The methylation pattern of these genes was studied using Hpa II and Msp I. Surprisingly, rice rDNA was found much more sensitive to these enzymes than the rDNA of the other plant species studied so far. No difference was observed between the methylation pattern of rDNA prepared from embryos grown in aerobic or anaerobic conditions although there is a marked difference between the rates of rRNA synthesis in these two situations.