Plastid dynamism integrates development and environment.

In land plants plastid type differentiation occurs concomitantly with cellular differentiation and the transition from one type to another is under developmental and environmental control. Plastid dynamism is based on a bilateral communication between plastids and nucleus through anterograde and retrograde signaling. Signaling occurs through the interaction with specific phytohormones (abscisic acid, strigolactones, jasmonates, gibberellins, brassinosteroids, ethylene, salicylic acid, cytokinin and auxin). The review is focused on the modulation of plastid capabilities at both transcriptional and post-translational levels at the crossroad between development and stress, with a particular attention to the chloroplast, because the most studied plastid type. The role of plastid-encoded and nuclear-encoded proteins for plastid development and stress responses, and the changes of plastid fate through the activity of stromules and plastoglobules, are discussed. Examples of plastid dynamism in response to soil stress agents (salinity, lead, cadmium, arsenic, and chromium) are described. Albinism and root greening are described based on the modulation activities of auxin and cytokinin. The physiological and functional responses of the sensory epidermal and vascular plastids to abiotic and biotic stresses along with their specific roles in stress sensing are described together with their potential modulation of retrograde signaling pathways. Future research perspectives include an in-depth study of sensory plastids to explore their potential for establishing a transgenerational memory to stress. Suggestions about anterograde and retrograde pathways acting at interspecific level and on the lipids of plastoglobules as a novel class of plastid morphogenic agents are provided.

Rice Phytoalexins: Half a Century of Amazing Discoveries; Part I: Distribution, Biosynthesis, Chemical Synthesis, and Biological Activities.

Cultivated rice is a staple food for more than half of the world's population, providing approximately 20% of the world's food energy needs. A broad spectrum of pathogenic microorganisms causes rice diseases leading to huge yield losses worldwide. Wild and cultivated rice species are known to possess a wide variety of antimicrobial secondary metabolites, known as phytoalexins, which are part of their active defense mechanisms. These compounds are biosynthesized transiently by rice in response to pathogens and certain abiotic stresses. Rice phytoalexins have been intensively studied for over half a century, both for their biological role and their potential application in agronomic and pharmaceutical fields. In recent decades, the growing interest of the research community, combined with advances in chemical, biological, and biomolecular investigation methods, has led to a notable acceleration in the growth of knowledge on rice phytoalexins. This review provides an overview of the knowledge gained in recent decades on the diversity, distribution, biosynthesis, chemical synthesis, and bioactivity of rice phytoalexins, with particular attention to the most recent advances in this research field.

New Paradigms in Brassinosteroids, Strigolactones, Sphingolipids, and Nitric Oxide Interaction in the Control of Lateral and Adventitious Root Formation.

The root system is formed by the primary root (PR), which forms lateral roots (LRs) and, in some cases, adventitious roots (ARs), which in turn may produce their own LRs. The formation of ARs is also essential for vegetative propagation in planta and in vitro and for breeding programs. Root formation and branching is coordinated by a complex developmental network, which maximizes the plant's ability to cope with abiotic stress. Rooting is also a response caused in a cutting by wounding and disconnection from the donor plant. Brassinosteroids (BRs) are steroid molecules perceived at the cell surface. They act as plant-growth-regulators (PGRs) and modulate plant development to provide stress tolerance. BRs and auxins control the formation of LRs and ARs. The auxin/BR interaction involves other PGRs and compounds, such as nitric oxide (NO), strigolactones (SLs), and sphingolipids (SPLs). The roles of these interactions in root formation and plasticity are still to be discovered. SLs are carotenoid derived PGRs. SLs enhance/reduce LR/AR formation depending on species and culture conditions. These PGRs possibly crosstalk with BRs. SPLs form domains with sterols within cellular membranes. Both SLs and SPLs participate in plant development and stress responses. SPLs are determinant for auxin cell-trafficking, which is essential for the formation of LRs/ARs in planta and in in vitro systems. Although little is known about the transport, trafficking, and signaling of SPLs, they seem to interact with BRs and SLs in regulating root-system growth. Here, we review the literature on BRs as modulators of LR and AR formation, as well as their crosstalk with SLs and SPLs through NO signaling. Knowledge on the control of rooting by these non-classical PGRs can help in improving crop productivity and enhancing AR-response from cuttings.

Convergence between Development and Stress: Ectopic Xylem Formation in Arabidopsis Hypocotyl in Response to 24-Epibrassinolide and Cadmium.

Ectopic xylary element (EXE) formation in planta is a poorly investigated process, and it is unknown if it occurs as a response to the soil pollutant Cadmium (Cd). The pericycle cells of Arabidopsis thaliana hypocotyl give rise to EXEs under specific hormonal inputs. Cadmium triggers pericycle responses, but its role in EXE formation is unknown. Brassinosteroids (BRs) affect numerous developmental events, including xylogenesis in vitro, and their exogenous application by 24-epibrassinolide (eBL) helps to alleviate Cd-stress by increasing lateral/adventitious rooting. Epibrassinolide's effects on EXEs in planta are unknown, as well as its relationship with Cd in the control of the process. The research aims to establish an eBL role in pericycle EXE formation, a Cd role in the same process, and the possible interaction between the two. Results show that 1 nM eBL causes an identity reversal between the metaxylem and protoxylem within the stele, and its combination with Cd reduces the event. All eBL concentrations increase EXEs, also affecting xylary identity by changing from protoxylem to metaxylem in a concentration-dependent manner. Cadmium does not affect EXE identity but increases EXEs when combined with eBL. The results suggest that eBL produces EXEs to form a mechanical barrier against the pollutant.

Brassinosteroids Mitigate Cadmium Effects in Arabidopsis Root System without Any Cooperation with Nitric Oxide.

The heavy metal cadmium (Cd) affects root system development and quiescent center (QC)-definition in Arabidopsis root-apices. The brassinosteroids-(BRs)-mediated tolerance to heavy metals has been reported to occur by a modulation of nitric oxide (NO) and root auxin-localization. However, how BRs counteract Cd-action in different root types is unknown. This research aimed to find correlations between BRs and NO in response to Cd in Arabidopsis's root system, monitoring their effects on QC-definition and auxin localization in root-apices. To this aim, root system developmental changes induced by low levels of 24-epibrassinolide (eBL) or by the BR-biosynthesis inhibitor brassinazole (Brz), combined or not with CdSO4, and/or with the NO-donor nitroprusside (SNP), were investigated using morpho-anatomical and NO-epifluorescence analyses, and monitoring auxin-localization by the DR5::GUS system. Results show that eBL, alone or combined with Cd, enhances lateral (LR) and adventitious (AR) root formation and counteracts QC-disruption and auxin-delocalization caused by Cd in primary root/LR/AR apices. Exogenous NO enhances LR and AR formation in Cd-presence, without synergism with eBL. The NO-signal is positively affected by eBL, but not in Cd-presence, and BR-biosynthesis inhibition does not change the low NO-signal caused by Cd. Collectively, results show that BRs ameliorate Cd-effects on all root types acting independently from NO.

Jasmonates, Ethylene and Brassinosteroids Control Adventitious and Lateral Rooting as Stress Avoidance Responses to Heavy Metals and Metalloids.

Developmental and environmental signaling networks often converge during plant growth in response to changing conditions. Stress-induced hormones, such as jasmonates (JAs), can influence growth by crosstalk with other signals like brassinosteroids (BRs) and ethylene (ET). Nevertheless, it is unclear how avoidance of an abiotic stress triggers local changes in development as a response. It is known that stress hormones like JAs/ET and BRs can regulate the division rate of cells from the first asymmetric cell divisions (ACDs) in meristems, suggesting that stem cell activation may take part in developmental changes as a stress-avoidance-induced response. The root system is a prime responder to stress conditions in soil. Together with the primary root and lateral roots (LRs), adventitious roots (ARs) are necessary for survival in numerous plant species. AR and LR formation is affected by soil pollution, causing substantial root architecture changes by either depressing or enhancing rooting as a stress avoidance/survival response. Here, a detailed overview of the crosstalk between JAs, ET, BRs, and the stress mediator nitric oxide (NO) in auxin-induced AR and LR formation, with/without cadmium and arsenic, is presented. Interactions essential in achieving a balance between growth and adaptation to Cd and As soil pollution to ensure survival are reviewed here in the model species Arabidopsis and rice.

Nitric Oxide Cooperates With Auxin to Mitigate the Alterations in the Root System Caused by Cadmium and Arsenic.

Oryza sativa L. is a worldwide food-crop frequently growing in cadmium (Cd)/arsenic (As) polluted soils, with its root-system as the first target of the pollutants. Root-system development involves the establishment of optimal indole-3-acetic acid (IAA) levels, also requiring the conversion of the IAA natural precursor indole-3-butyric acid (IBA) into IAA, causing nitric oxide (NO) formation. Nitric oxide is a stress-signaling molecule. In rice, a negative interaction of Cd or As with endogenous auxin has been demonstrated, as some NO protective effects. However, a synergism between the natural auxins (IAA and/or IBA) and NO was not yet determined and might be important for ameliorating rice metal(oid)-tolerance. With this aim, the stress caused by Cd/As toxicity in the root cells and the possible recovery by either NO or auxins (IAA/IBA) were evaluated after Cd or As (arsenate) exposure, combined or not with the NO-donor compound sodium-nitroprusside (SNP). Root fresh weight, membrane electrolyte leakage, and H2O2 production were also measured. Moreover, endogenous IAA/IBA contents, transcription-levels of OsYUCCA1 and OsASA2 IAA-biosynthetic-genes, and expression of the IAA-influx-carrier OsAUX1 and the IAA-responsive DR5::GUS construct were analyzed, and NO-epifluorescence levels were measured. Results showed that membrane injury by enhanced electrolyte leakage occurred under both pollutants and was reduced by the treatment with SNP only in Cd-presence. By contrast, no membrane injury was caused by either exogenous NO or IAA or IBA. Cd- and As-toxicity also resulted into a decreased root fresh weight, mitigated by the combination of each pollutant with either IAA or IBA. Cd and As decreased the endogenous NO-content, increased H2O2 formation, and altered auxin biosynthesis, levels and distribution in both adventitious (ARs) and mainly lateral roots (LRs). The SNP-formed NO counteracted the pollutants' effects on auxin distribution/levels, reduced H2O2 formation in Cd-presence, and enhanced AUX1-expression, mainly in As-presence. Each exogenous auxin, but mainly IBA, combined with Cd or As at 10 µM, mitigated the pollutants' effects by increasing LR-production and by increasing NO-content in the case of Cd. Altogether, results demonstrate that NO and auxin(s) work together in the rice root system to counteract the specific toxic-effects of each pollutant.

Jasmonic Acid Methyl Ester Induces Xylogenesis and Modulates Auxin-Induced Xylary Cell Identity with NO Involvement.

In Arabidopsis basal hypocotyls of dark-grown seedlings, xylary cells may form from the pericycle as an alternative to adventitious roots. Several hormones may induce xylogenesis, as Jasmonic acid (JA), as well as indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) auxins, which also affect xylary identity. Studies with the ethylene (ET)-perception mutant ein3eil1 and the ET-precursor 1-aminocyclopropane-1-carboxylic acid (ACC), also demonstrate ET involvement in IBA-induced ectopic metaxylem. Moreover, nitric oxide (NO), produced after IBA/IAA-treatments, may affect JA signalling and interact positively/negatively with ET. To date, NO-involvement in ET/JA-mediated xylogenesis has never been investigated. To study this, and unravel JA-effects on xylary identity, xylogenesis was investigated in hypocotyls of seedlings treated with JA methyl-ester (JAMe) with/without ACC, IBA, IAA. Wild-type (wt) and ein3eil1 responses to hormonal treatments were compared, and the NO signal was quantified and its role evaluated by using NO-donors/scavengers. Ectopic-protoxylem increased in the wt only after treatment with JAMe(10 µM), whereas in ein3eil1 with any JAMe concentration. NO was detected in cells leading to either xylogenesis or adventitious rooting, and increased after treatment with JAMe(10 µM) combined or not with IBA(10 µM). Xylary identity changed when JAMe was applied with each auxin. Altogether, the results show that xylogenesis is induced by JA and NO positively regulates this process. In addition, NO also negatively interacts with ET-signalling and modulates auxin-induced xylary identity.

Jasmonate promotes auxin-induced adventitious rooting in dark-grown Arabidopsis thaliana seedlings and stem thin cell layers by a cross-talk with ethylene signalling and a modulation of xylogenesis.

BACKGROUND: Adventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA + Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA + Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored. RESULTS: MeJA, at 0.01 µM, enhances AR-formation, when combined with IBA + Kin, and the response of the early-JA-biosynthesis mutant dde2-2 and the JA-signalling mutant coi1-16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression. CONCLUSIONS: The JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species.

From Robinia pseudoacacia L. nectar to Acacia monofloral honey: biochemical changes and variation of biological properties.

BACKGROUND: Robinia pseudoacacia L. nectar and its derivative monofloral honey were systematically compared in this study, to understand how much the starting solution reflected the final product, after re-elaboration by Apis mellifera ligustica Spinola. RESULTS: Subjected to dehydration in the hive, nectar changed in its water and sugar content when transformed into honey, as physicochemical and gas chromatographic-mass spectrometric analyses revealed. Spectrophotometric measurements and characterization by high-performance liquid chromatography-diode array detection of 18 plant molecules demonstrated honey to be richer than nectar in secondary metabolites. For the first time, the hypothesis of the existence of a nectar redox cycle in R. pseudoacacia was reported, as previously described for Nicotiana sp., based on 1D-protein profiles, western blot analysis and detection of H2 O2 and ascorbate. The bioactivity of both matrices was also investigated. Antiradical in vitro tests showed that Acacia honey was more antioxidant than nectar, which was even able to induce oxidative stress directly in a eukaryotic cell system. Antimicrobial assays demonstrated that nectar was bacteriostatic, due to H2 O2 activity, whereas honey was even bactericidal. CONCLUSION: All these data support the ecological role of nectar and honey in nature: protection of the gynoecium from pathogens and preservation from degradative processes, respectively. © 2018 Society of Chemical Industry.

Indole-3-Butyric Acid Induces Ectopic Formation of Metaxylem in the Hypocotyl of Arabidopsis thaliana without Conversion into Indole-3-Acetic Acid and with a Positive Interaction with Ethylene.

The role of the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) and of the auxin-interacting phytohormone ethylene, on the ectopic formation of primary xylem (xylogenesis in planta) is still little known. In particular, auxin/ethylene-target tissue(s), modality of the xylary process (trans-differentiation vs. de novo formation), and the kind of ectopic elements formed (metaxylem vs. protoxylem) are currently unknown. It is also unclear whether IBA may act on the process independently of conversion into IAA. To investigate these topics, histological analyses were carried out in the hypocotyls of Arabidopsis wild type seedlings and ech2ibr10 and ein3eil1 mutants, which are blocked in IBA-to-IAA conversion and ethylene signalling, respectively. The seedlings were grown under darkness with either IAA or IBA, combined or not with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Adventitious root formation was also investigated because this process may compete with xylogenesis. Our results show that ectopic formation of protoxylem and metaxylem occurred as an indirect process starting from the pericycle periclinal derivatives of the hypocotyl basal part. IAA favoured protoxylem formation, whereas IBA induced ectopic metaxylem with ethylene cooperation through the EIN3EIL1 network. Ectopic metaxylem differentiation occurred independently of IBA-to-IAA conversion as mediated by ECH2 and IBR10, and in the place of IBA-induced adventitious root formation.

The quiescent center and the stem cell niche in the adventitious roots of Arabidopsis thaliana.

Adventitious rooting is essential for the survival of numerous species from vascular cryptogams to monocots, and is required for successful micropropagation. The tissues involved in AR initiation may differ in planta and in in vitro systems. For example, in Arabidopsis thaliana, ARs originate from the hypocotyl pericycle in planta and the stem endodermis in in vitro cultured thin cell layers. The formation of adventitious roots (ARs) depends on numerous factors, among which the hormones, auxin, in particular. In both primary and lateral roots, growth depends on a functional stem cell niche in the apex, maintained by an active quiescent center (QC), and involving the expression of genes controlled by auxin and cytokinin. This review summarizes current knowledge about auxin and cytokinin control on genes involved in the definition and maintenance of QC, and stem cell niche, in the apex of Arabidopsis ARs in planta and in longitudinal thin cell layers.

OeFAD8, OeLIP and OeOSM expression and activity in cold-acclimation of Olea europaea, a perennial dicot without winter-dormancy.

MAIN CONCLUSION: Cold-acclimation genes in woody dicots without winter-dormancy, e.g., olive-tree, need investigation. Positive relationships between OeFAD8, OeOSM , and OeLIP19 and olive-tree cold-acclimation exist, and couple with increased lipid unsaturation and cutinisation. Olive-tree is a woody species with no winter-dormancy and low frost-tolerance. However, cold-tolerant genotypes were empirically selected, highlighting that cold-acclimation might be acquired. Proteins needed for olive-tree cold-acclimation are unknown, even if roles for osmotin (OeOSM) as leaf cryoprotectant, and seed lipid-transfer protein for endosperm cutinisation under cold, were demonstrated. In other species, FAD8, coding a desaturase producing α-linolenic acid, is activated by temperature-lowering, concomitantly with bZIP-LIP19 genes. The research was focussed on finding OeLIP19 gene(s) in olive-tree genome, and analyze it/their expression, and that of OeFAD8 and OeOSM, in drupes and leaves under different cold-conditions/developmental stages/genotypes, in comparison with changes in unsaturated lipids and cell wall cutinisation. Cold-induced cytosolic calcium transients always occurred in leaves/drupes of some genotypes, e.g., Moraiolo, but ceased in others, e.g., Canino, at specific drupe stages/cold-treatments, suggesting cold-acclimation acquisition only in the latter genotypes. Canino and Moraiolo were selected for further analyses. Cold-acclimation in Canino was confirmed by an electrolyte leakage from leaf/drupe membranes highly reduced in comparison with Moraiolo. Strong increases in fruit-epicarp/leaf-epidermis cutinisation characterized cold-acclimated Canino, and positively coupled with OeOSM expression, and immunolocalization of the coded protein. OeFAD8 expression increased with cold-acclimation, as the production of α-linolenic acid, and related compounds. An OeLIP19 gene was isolated. Its levels changed with a trend similar to OeFAD8. All together, results sustain a positive relationship between OeFAD8, OeOSM and OeLIP19 expression in olive-tree cold-acclimation. The parallel changes in unsaturated lipids and cutinisation concur to suggest orchestrated roles of the coded proteins in the process.

Overexpression of AtPCS1 in tobacco increases arsenic and arsenic plus cadmium accumulation and detoxification.

MAIN CONCLUSION: The heterologous expression of AtPCS1 in tobacco plants exposed to arsenic plus cadmium enhances phytochelatin levels, root As/Cd accumulation and pollutants detoxification, but does not prevent root cyto-histological damages. High phytochelatin (PC) levels may be involved in accumulation and detoxification of both cadmium (Cd) and arsenic (As) in numerous plants. Although polluted environments are frequently characterized by As and Cd coexistence, how increased PC levels affect the adaptation of the entire plant and the response of its cells/tissues to a combined contamination by As and Cd needs investigation. Consequently, we analyzed tobacco seedlings overexpressing Arabidopsis phytochelatin synthase1 gene (AtPCS1) exposed to As and/or Cd, to evaluate the levels of PCs and As/Cd, the cyto-histological modifications of the roots and the Cd/As leaf extrusion ability. When exposed to As and/or Cd the plants overexpressing AtPCS1 showed higher PC levels, As plus Cd root accumulation, and detoxification ability than the non-overexpressing plants, but a blocked Cd-extrusion from the leaf trichomes. In all genotypes, As, and Cd in particular, damaged lateral root apices, enhancing cell-vacuolization, causing thinning and stretching of endodermis initial cells. Alterations also occurred in the primary structure region of the lateral roots, i.e., cell wall lignification in the external cortex, cell hypertrophy in the inner cortex, crushing of endodermis and stele, and nuclear hypertrophy. Altogether, As and/or Cd caused damage to the lateral roots (and not to the primary one), with such damage not counteracted by AtPCS1 overexpression. The latter, however, positively affected accumulation and detoxification to both pollutants, highlighting that Cd/As accumulation and detoxification due to PCS1 activity do not reduce the cyto-histological damage.

Recent Advances on Genetic and Physiological Bases of In Vitro Somatic Embryo Formation.

Somatic embryogenesis involves a broad repertoire of genes, and complex expression patterns controlled by a concerted gene regulatory network. The present work describes this regulatory network focusing on the main aspects involved, with the aim of providing a deeper insight into understanding the total reprogramming of cells into a new organism through a somatic way. To the aim, the chromatin remodeling necessary to totipotent stem cell establishment is described, as the activity of numerous transcription factors necessary to cellular totipotency reprogramming. The eliciting effects of various plant growth regulators on the induction of somatic embryogenesis is also described and put in relation with the activity of specific transcription factors. The role of programmed cell death in the process, and the related function of specific hemoglobins as anti-stress and anti-death compounds is also described. The tools for biotechnology coming from this information is highlighted in the concluding remarks.

Spermine either delays or promotes cell death in Nicotiana tabacum L. corolla depending on the floral developmental stage and affects the distribution of transglutaminase.

The role of spermine (SM) was studied to verify if SM supplied to Nicotiana tabacum flower can modulate programmed cell death (PCD) of the corolla. SM has strong effects on the development and senescence of excised flowers despite its low physiological levels. The timing and duration of SM treatment is a key factor; SM counteracts PCD (verified by morphological observations, pigment contents and DNA laddering) only in the narrow developmental window of corolla expansion. Before and after, SM promotes PCD. SM exerts its pro-survival role by delaying fresh weight loss, by inhibiting reduction of pigments and finally by preventing DNA degradation. Moreover, SM deeply alters the distribution of the PA-conjugating enzyme transglutaminase (TGase). TGase is present in the epidermis during development, but it sprays also in the cell walls of inner parenchyma at senescence. After SM treatment, parenchyma cells accumulate TGase, increase in size and their cell walls do not undergo stiffening contrarily to control cells. The subcellular localization of TGase has been validated by biolistic-transformation of onion epidermal cells. Results indicated that SM is a critical factor in the senescence of N. tabacum corolla by controlling biochemical and morphological parameters; the lasts are probably interconnected with the action of TGase.

The symbiosis between Nicotiana tabacum and the endomycorrhizal fungus Funneliformis mosseae increases the plant glutathione level and decreases leaf cadmium and root arsenic contents.

Over time, anthropogenic activities have led to severe cadmium (Cd) and arsenic (As) pollution in several environments. Plants inhabiting metal(loid)-contaminated areas should be able to sequester and detoxify these toxic elements as soon as they enter roots and leaves. We postulated here that an important role in protecting plants from excessive metal(loid) accumulation and toxicity might be played by arbuscular mycorrhizal (AM) fungi. In fact, human exploitation of plant material derived from Cd- and As-polluted environments may lead to a noxious intake of these toxic elements; in particular, a possible source of Cd and As for humans is given by cigarette and cigar smoke. We investigated the role of AM fungus Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler in protecting Nicotiana tabacum L. (cv. Petit Havana) from the above-mentioned metal(loid) stress. Our findings proved that the AM symbiosis is effective in increasing the plant tissue content of the antioxidant glutathione (GSH), in influencing the amount of metal(loid)-induced chelators as phytochelatins, and in reducing the Cd and As content in leaves and roots of adult tobacco plants. These results might also prove useful in improving the quality of commercial tobacco, thus reducing the risks to human health due to inhalation of toxic elements contained in smoking products.

ABCB1 and ABCB19 auxin transporters have synergistic effects on early and late Arabidopsis anther development.

Arabidopsis abcb1 abcb19 double mutants defective in the auxin transporters ABCB1/PGP1 and ABCB19/PGP19 are altered in stamen elongation, anther dehiscence and pollen maturation. To assess the contribution of these transporters to stamen development we performed phenotypic, histological analyses, and in situ hybridizations on abcb1 and abcb19 single mutant flowers. We found that pollen maturation and anther dehiscence are precocious in the abcb1 but not in the abcb19 mutant. Accordingly, endothecium lignification is altered only in abcb1 anthers. Both abcb1 and abcb1 abcb19 stamens also show altered early development, with asynchronous anther locules and a multilayer tapetum. DAPI staining showed that the timing of meiosis is asynchronous in abcb1 abcb19 anther locules, while only a small percentage of pollen grains are non-viable according to Alexander's staining. In agreement, TAM (TARDY ASYNCHRONOUS MEIOSIS), as well as BAM2 (BARELY ANY MERISTEM)-involved in tapetal cell development-are overexpressed in abcb1 abcb19 young flower buds. Correspondingly, ABCB1 and ABCB19 mRNA localization supports the observed phenotypes of abcb1 and abcb1 abcb19 mutant anthers. In conclusion, we provide evidence that auxin transport plays a significant role both in early and late stamen development: ABCB1 plays a major role during anther development, while ABCB19 has a synergistic role.

A Cd/Fe/Zn-responsive phytochelatin synthase is constitutively present in the ancient liverwort Lunularia cruciata (L.) dumort.

Lunularia cruciata occupies a very basal position in the phylogenetic tree of liverworts, which in turn have been recognized as a very early clade of land plants. It would therefore seem appropriate to take L. cruciata as the startingpoint for investigating character evolution in plants' metal(loid) response. One of the strongest evolutionary pressures for land colonization by plants has come from potential access to much greater amounts of nutritive ions from surface rocks, compared to water. This might have resulted in the need to precisely regulate trace element homeostasis and to minimize the risk of exposure to toxic concentrations of certain metals, prompting the evolution of a number of response mechanisms, such as synthesis of phytochelatins, metal(loid)-binding thiol-peptides. Accordingly, if the ability to synthesize phytochelatins and the occurrence of an active phytochelatin synthase are traits present in a basal liverwort species, and have been even reinforced in 'modern' tracheophytes, e.g. Arabidopsis thaliana, then such traits would presumably have played an essential role in plant fitness over time. Hence, we demonstrated here that: (i) L. cruciata compartmentalizes cadmium in the vacuoles of the phototosynthetic parenchyma by means of a phytochelatin-mediated detoxification strategy, and possesses a phytochelatin synthase that is activated by cadmium and homeostatic concentrations of iron(II) and zinc; and (ii) A. thaliana phytochelatin synthase displays a higher and broader response to several metal(loid)s [namely: cadmium, iron(II), zinc, copper, mercury, lead, arsenic(III)] than L. cruciata phytochelatin synthase.

Tapetum and middle layer control male fertility in Actinidia deliciosa.

BACKGROUND AND AIMS: Dioecism characterizes many crop species of economic value, including kiwifruit (Actinidia deliciosa). Kiwifruit male sterility occurs at the microspore stage. The cell walls of the microspores and the pollen of the male-sterile and male-fertile flowers, respectively, differ in glucose and galactose levels. In numerous plants, pollen formation involves normal functioning and degeneration timing of the tapetum, with calcium and carbohydrates provided by the tapetum essential for male fertility. The aim of this study was to determine whether the anther wall controls male fertility in kiwifruit, providing calcium and carbohydrates to the microspores. METHODS: The events occurring in the anther wall and microspores of male-fertile and male-sterile anthers were investigated by analyses of light microscopy, epifluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL assay) and transmission electron microscopy coupled with electron spectroscopy. The possibility that male sterility was related to anther tissue malfunctioning with regard to calcium/glucose/galactose provision to the microspores was also investigated by in vitro anther culture. KEY RESULTS: Both tapetum and the middle layer showed secretory activity and both degenerated by programmed cell death (PCD), but PCD was later in male-sterile than in male-fertile anthers. Calcium accumulated in cell walls of the middle layer and tapetum and in the exine of microspores and pollen, reaching higher levels in anther wall tissues and dead microspores of male-sterile anthers. A specific supply of glucose and calcium induced normal pollen formation in in vitro-cultured anthers of the male-sterile genotype. CONCLUSIONS: The results show that male sterility in kiwifruit is induced by anther wall tissues through prolonged secretory activity caused by a delay in PCD, in the middle layer in particular. In vitro culture results support the sporophytic control of male fertility in kiwifruit and open the way to applications to overcome dioecism and optimize kiwifruit production.