Cadmium exposure induced light/dark- and time-dependent redox changes at subcellular level in Arabidopsis plants.

Cadmium (Cd) is one of the most toxic heavy metals for plants and humans. Reactive oxygen species (ROS) are some of the primary signaling molecules produced after Cd treatment in plants but the contribution of different organelles and specific cell types, together with the impact of light is unknown. We used Arabidopsis lines expressing GRX1-roGFP2 (glutaredoxin1-roGFP) targeted to different cell compartments and analysed changes in redox state over 24 h light/dark cycle in Cd-treated leaf discs. We imaged redox state changes in peroxisomes and chloroplasts in leaf tissue. Chloroplasts and peroxisomes were the most affected organelles in the dark and blocking the photosynthetic electron transport chain (pETC) by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) promotes higher Cd-dependent oxidation in all organelles. Peroxisomes underwent the most rapid changes in redox state in response to Cd and DCMU and silencing chloroplastic NTRC (NADPH thioredoxin reductase C) considerably increases peroxisome oxidation. Total NAD(P)H and cytosolic NADH decreased during exposure to Cd, while Ca+2 content in chloroplasts and cytosol increased in the dark period. Our results demonstrate a Cd-, time- and light-dependent increase of oxidation of all organelles analysed, that could be in part triggered by disturbances in pETC and photorespiration, the decrease of NAD(P)H availability, and differential antioxidants expression at subcellular level.

The Role of Two Linear ß-Glucans Activated by c-di-GMP in Rhizobium etli CFN42.

Bacterial exopolysaccharides (EPS) have been implicated in a variety of functions that assist in bacterial survival, colonization, and host-microbe interactions. Among them, bacterial linear ß-glucans are polysaccharides formed by D-glucose units linked by ß-glycosidic bonds, which include curdlan, cellulose, and the new described Mixed Linkage ß-Glucan (MLG). Bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a universal bacterial second messenger that usually promote EPS production. Here, we report Rhizobium etli as the first bacterium capable of producing cellulose and MLG. Significant amounts of these two ß-glucans are not produced under free-living laboratory conditions, but their production is triggered upon elevation of intracellular c-di-GMP levels, both contributing to Congo red (CR+) and Calcofluor (CF+) phenotypes. Cellulose turned out to be more relevant for free-living phenotypes promoting flocculation and biofilm formation under high c-di-GMP conditions. None of these two EPS are essential for attachment to roots of Phaseolus vulgaris, neither for nodulation nor for symbiotic nitrogen fixation. However, both ß-glucans separately contribute to the fitness of interaction between R. etli and its host. Overproduction of these ß-glucans, particularly cellulose, appears detrimental for symbiosis. This indicates that their activation by c-di-GMP must be strictly regulated in time and space and should be controlled by different, yet unknown, regulatory pathways.

Characterization of the biochemical basis for copper homeostasis and tolerance in Biscutella auriculata L.

Biscutella auriculata L. is a plant that belongs to the Brassicaceae family and it has been found growing in a metal-contaminated area of the San Quíntín mine (Ciudad Real, Spain). The purpose of this work was to evaluate the mechanisms that allow this plant to tolerate high concentrations of copper. Seedlings were grown in a semi-hydroponic system for 15 days under 125 µM of Cu (NO3 )2 . Exposure to copper resulted in growth inhibition and reduction in the photosynthetic parameters. Copper was mainly accumulated in vascular tissue and vacuoles of the roots and only a minor proportion was transferred to the shoot. Biothiol analysis showed a greater enhancement of reduced glutathione in leaves and increases of phytochelatins (PC2 and PC3) in both leaves and roots. Copper treatment induced oxidative stress, which triggered a response of the enzymatic and non-enzymatic antioxidant mechanisms. The results show that B. auriculata is able to tolerate high metal levels through the activation of specific mechanisms to neutralize the oxidative stress produced and also by metal sequestration through phytochelatins. The preferential accumulation of copper in roots provides clues for further studies on the use of this plant for phytostabilization and environmental recovery purposes in Cu-contaminated areas.

Is Autophagy Involved in Pepper Fruit Ripening?

Autophagy is a universal self-degradation process involved in the removal and recycling of cellular constituents and organelles; however, little is known about its possible role in fruit ripening, in which the oxidation of lipids and proteins and changes in the metabolism of different cellular organelles occur. In this work, we analyzed several markers of autophagy in two critical maturation stages of pepper (Capsicum annuum L.) fruits where variations due to ripening become clearly visible. Using two commercial varieties that ripen to yellow and red fruits respectively, we studied changes in the gene expression and protein content of several autophagy (ATG) components, ATG4 activity, as well as the autophagy receptor NBR1 and the proteases LON1 and LON2. Additionally, the presence of intravacuolar vesicles was analyzed by electron microscopy. Altogether, our data reveal that autophagy plays a role in the metabolic changes which occur during ripening in the two studied varieties, suggesting that this process may be critical to acquiring final optimal quality of pepper fruits.

Selective Autophagy of Peroxisomes in Plants: From Housekeeping to Development and Stress Responses.

Peroxisomes are dynamic organelles involved in multiple functions, including oxygen and nitrogen reactive species metabolism. In plants, these organelles have a close relationship with chloroplasts and mitochondria, characterized by intense metabolic activity and signal transduction. Peroxisomes undergo rapid changes in size, morphology, and abundance depending on the plant development stage and environmental conditions. As peroxisomes are essential not only for redox homeostasis but also for sensing stress, signaling transduction, and cell survival, their formation and degradation need to be rigorously regulated. In this review, new insights into the regulation of plant peroxisomes are briefly described, with a particular emphasis on pexophagy components and their regulation.

Cadmium induces reactive oxygen species-dependent pexophagy in Arabidopsis leaves.

Cadmium treatment induces transient peroxisome proliferation in Arabidopsis leaves. To determine whether this process is regulated by pexophagy and to identify the mechanisms involved, we analysed time course-dependent changes in ATG8, an autophagy marker, and the accumulation of peroxisomal marker PEX14a. After 3 hr of Cd exposure, the transcript levels of ATG8h, ATG8c, a, and i were slightly up-regulated and then returned to normal. ATG8 protein levels also increased after 3 hr of Cd treatment, although an opposite pattern was observed in PEX14. Arabidopsis lines expressing GFP-ATG8a and CFP-SKL enabled us to demonstrate the presence of pexophagic processes in leaves. The Cd-dependent induction of pexophagy was demonstrated by the accumulation of peroxisomes in autophagy gene (ATG)-related Arabidopsis knockout mutants atg5 and atg7. We show that ATG8a colocalizes with catalase and NBR1 in the electron-dense peroxisomal core, thus suggesting that NBR1 may be an autophagic receptor for peroxisomes, with catalase being possibly involved in targeting pexophagy. Protein carbonylation and peroxisomal redox state suggest that protein oxidation may trigger pexophagy. Cathepsine B, legumain, and caspase 6 may also be involved in the regulation of pexophagy. Our results suggest that pexophagy could be an important step in rapid cell responses to cadmium.

Visualization and characterization of Pseudomonas syringae pv. tomato DC3000 pellicles.

Cellulose, whose production is controlled by c-di-GMP, is a commonly found exopolysaccharide in bacterial biofilms. Pseudomonas syringae pv. tomato (Pto) DC3000, a model organism for molecular studies of plant-pathogen interactions, carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose. The high intracellular levels of the second messenger c-di-GMP induced by the overexpression of the heterologous diguanylate cyclase PleD stimulate cellulose production and enhance air-liquid biofilm (pellicle) formation. To characterize the mechanisms involved in Pto DC3000 pellicle formation, we studied this process using mutants lacking flagella, biosurfactant or different extracellular matrix components, and compared the pellicles produced in the absence and in the presence of PleD. We have discovered that neither alginate nor the biosurfactant syringafactin are needed for their formation, whereas cellulose and flagella are important but not essential. We have also observed that the high c-di-GMP levels conferred more cohesion to Pto cells within the pellicle and induced the formation of intracellular inclusion bodies and extracellular fibres and vesicles. Since the pellicles were very labile and this greatly hindered their handling and processing for microscopy, we have also developed new methods to collect and process them for scanning and transmission electron microscopy. These techniques open up new perspectives for the analysis of fragile biofilms in other bacterial strains.

Multiple CsrA Proteins Control Key Virulence Traits in Pseudomonas syringae pv. tomato DC3000.

The phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 has a complex Gac-rsm global regulatory pathway that controls virulence, motility, production of secondary metabolites, carbon metabolism, and quorum sensing. However, despite the fact that components of this pathway are known, their physiological roles have not yet been established. Regarding the CsrA/RsmA type proteins, five paralogs, three of which are well conserved within the Pseudomonas genus (csrA1, csrA2, and csrA3), have been found in the DC3000 genome. To decipher their function, mutants lacking the three most conserved CsrA proteins have been constructed and their physiological outcomes examined. We show that they exert nonredundant functions and demonstrate that CsrA3 and, to a lesser extent, CsrA2 but not CsrA1 alter the expression of genes involved in a variety of pathways and systems important for motility, exopolysaccharide synthesis, growth, and virulence. Particularly, alginate synthesis, syringafactin production, and virulence are considerably de-repressed in a csrA3 mutant, whereas growth in planta is impaired. We propose that the linkage of growth and symptom development is under the control of CsrA3, which functions as a pivotal regulator of the DC3000 life cycle, repressing virulence traits and promoting cell division in response to environmental cues.

FleQ coordinates flagellum-dependent and -independent motilities in Pseudomonas syringae pv. tomato DC3000.

Motility plays an essential role in bacterial fitness and colonization in the plant environment, since it favors nutrient acquisition and avoidance of toxic substances, successful competition with other microorganisms, the ability to locate the preferred hosts, access to optimal sites within them, and dispersal in the environment during the course of transmission. In this work, we have observed that the mutation of the flagellar master regulatory gene, fleQ, alters bacterial surface motility and biosurfactant production, uncovering a new type of motility for Pseudomonas syringae pv. tomato DC3000 on semisolid surfaces. We present evidence that P. syringae pv. tomato DC3000 moves over semisolid surfaces by using at least two different types of motility, namely, swarming, which depends on the presence of flagella and syringafactin, a biosurfactant produced by this strain, and a flagellum-independent surface spreading or sliding, which also requires syringafactin. We also show that FleQ activates flagellum synthesis and negatively regulates syringafactin production in P. syringae pv. tomato DC3000. Finally, it was surprising to observe that mutants lacking flagella or syringafactin were as virulent as the wild type, and only the simultaneous loss of both flagella and syringafactin impairs the ability of P. syringae pv. tomato DC3000 to colonize tomato host plants and cause disease.

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.

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.

Histochemical location of key enzyme activities involved in receptivity and self-incompatibility in the olive tree (Olea europaea L.).

Stigma-surface and style enzymes are important for pollen reception, selection and germination. This report deals with the histochemical location of the activity of four basic types of enzyme involved in these processes in the olive (Olea europaea L.). The detection of peroxidase, esterase and acid-phosphatase activities at the surface of the stigma provided evidence of early receptivity in olive pistils. The stigma maintained its receptivity until the arrival of pollen. Acid-phosphatase activity appeared in the style at the moment of anthesis and continued until the fertilization of the ovule. RNase activity was detected in the extracellular matrix of the styles of flowers just before pollination and became especially evident in pistils after self-pollination. This activity gradually decreased until it practically disappeared in more advanced stages. RNase activity was also detected in pollen tubes growing in pollinated pistils and appeared after in vitro germination in the presence of self-incompatible pistils. These findings suggest that RNases may well be involved in intraspecific pollen rejection in olive flowers. To the best of our knowledge this is the first time that evidence of enzyme activity in stigma receptivity and pollen selection has been described in this species.

Role of peroxynitrite in programmed cell death induced in self-incompatible pollen.

Reactive oxygen species and NO are involved in the signaling pathway of programmed cell death (PCD). Information concerning the role of these molecules in self-incompatible pollination is scarce especially in non-model species studied in vivo. We recently reported that in the olive tree, compatible and self-incompatible pollen have different levels of reactive oxygen and nitrogen species and that PCD is induced in self-incompatible pollen. Levels of O 2 (.-) and NO are higher in pollen after self-incompatible pollination than after compatible pollination. The presence of these reactive species was concomitant with the presence of peroxynitrite. Similar results were obtained on pollen-germination experiments both in vivo and in vitro. These data, together with observations made after treating pollinated flowers with scavengers, suggest that peroxynitrite plays a role in PCD induced after self-incompatible pollination and we propose here a model to describe the way in which it might work.

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 O(2)(-) 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 O(2)(-) 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.

Using proteomics to study sexual reproduction in angiosperms.

While a relative latecomer to the postgenomics era of functional biology, the application of mass spectrometry-based proteomic analysis has increased exponentially over the past 10 years. Some of this increase is the result of transition of chemists, physicists, and mathematicians to the study of biology, and some is due to improved methods, increased instrument sensitivity, and better techniques of bioinformatics-based data analysis. Proteomic Biological processes are typically studied in isolation, and seldom are efforts made to coordinate results obtained using structural, biochemical, and molecular-genetic strategies. Mass spectrometry-based proteomic analysis can serve as a platform to bridge these disparate results and to additionally incorporate both temporal and anatomical considerations. Recently, proteomic analyses have transcended their initial purely descriptive applications and are being employed extensively in studies of posttranslational protein modifications, protein interactions, and control of metabolic networks. Herein, we provide a brief introduction to sample preparation, comparison of gel-based versus gel-free methods, and explanation of data analysis emphasizing plant reproductive applications. We critically review the results from the relatively small number of extant proteomics-based analyses of angiosperm reproduction, from flowers to seedlings, and speculate on the utility of this strategy for future developments and directions.

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.

Histological comparison between wheat embryos developing in vitro from isolated zygotes and those developing in vivo.

There is currently great interest shown in understanding the process of embryogenesis and, due to the relative inaccessibility of these structures in planta, extended studies are carried out in various in vitro systems. The culture of isolated zygotes in particular provides an excellent platform to study the process of in planta embryogenesis. However, very few comparisons have been made between zygotic embryos grown entirely in cultures and those grown in vivo. The present study analyses the differences and similarities between the in vitro and in vivo development of wheat zygotic embryos at the level of morphology and histology. The study was possible thanks to an efficient culture system and an appropriate method of preparing isolated wheat zygotes for microscopy. The in vitro embryos were fixed, embedded and sectioned in the two-celled, globular, club-shaped and fully differentiated stages. Embryos developing in vitro closely followed the morphology of their in planta counterparts and their cell types and tissues were also similar, demonstrating the applicability of the present culture system for studying the process of zygotic embryogenesis. However, some important differences were also detected in the case of in vitro development: the disturbance of or lack of initial polarity led to changes in the division symmetry of the zygotes and subsequently to the formation of uniform cells in the globular structures. Presumably, differences between the in vitro and in planta environments resulted in a lower level of differentiation and maturation in in vitro embryos and in abundant starch and protein accumulation in the scutellum.

HvPG1 and ECA1: two genes activated transcriptionally in the transition of barley microspores from the gametophytic to the embryogenic pathway.

Microspores genetically programmed to produce male gametes can be switched to the embryogenic pathway to give rise to haploid embryos. Microspore embryogenesis is usually induced in barley by stress pre-treatment applied to vacuolated microspores. We studied the expression of two genes during the early stages of microspore embryogenesis to gain further insight into the microspore transition from the gametophytic to the embryogenic pathway. RT-PCR together with in situ hybridization on sections (ISH) and whole-mount in situ hybridization (WISH) were used to analyse the expression of the early-culture abundant gene (ECA1), which is expressed in barley during microspore embryogenesis, and a polygalacturonase gene (HvPG1), a late pollen gene expressed during gametogenesis only after microspore division. Both ECA1 and HvPG1 genes were transcriptionally active after stress pre-treatment in the same populations of microspore-derived structures, representing the sporophytically induced ones. ECA1 transcripts were also detected after 3 days' culture. Our results point to the possibility of using ECA1 gene expression as a marker for the induction of microspore embryogenesis and the earliest stages of this process. Finally, we demonstrate that WISH is a suitable technique for studying gene expression in embryogenic microspore populations and, because different structures can be examined individually, is an appropriate complement to transcriptomic profile analyses in the study of early microspore embryogenesis.

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 beta-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.

Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development.

Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time hordein expression during microspore embryogenesis. Surprisingly, these hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.