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.

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.

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.

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.

The Arabidopsis BET bromodomain factor GTE4 regulates the mitotic cell cycle.

Proteins containing bromodomains are capable of binding to acetylated histone tails and have a role in recognizing and deciphering acetylated chromatin. Plant BET proteins contain one bromodomain. Twelve BET-encoding genes have been identified in the Arabidopsis genome. Two of these genes have been functionally characterized, one shows a role in seed germination, the other is involved in the establishment of leaf shape. Recently, we characterized a third AtBET gene, named GTE4. We demonstrated that GTE4 is involved in the activation and maintenance of cell division in the meristems and by this controls cell numbers in differentiated organs. Moreover, the quiescent center (QC) identity is partially lost in the apex of the primary root of gte4 mutant, and there is a premature switch from mitosis to endocycling. Genes involved in the retinoblastoma (RB)-E2F pathway, which is important for coupling cell division and cell differentiation in plants and animals, were either up- or down-regulated in the gte4 mutant. In this report we also show that the defect in germination observed in gte4 mutant seeds is not rescued by the action of GA3. Further the root pole of the mutant embryo shows irregular cytokinesis in the procambial stem cells, and the QC of the lateral root shows a partial, but not transient, loss of QC identity. These additional results reinforce the importance of GTE4 in the control of cell proliferation.

Adventitious rooting is enhanced by methyl jasmonate in tobacco thin cell layers.

Adventitious roots (ARs) are induced by auxins. Jasmonic acid (JA) and methyl jasmonate (MeJA) are also plant growth regulators with many effects on development, but their role on ARs needs investigation. To this aim, we analyzed AR formation in tobacco thin cell layers (TCLs) cultured with 0.01-10 microM MeJA, either under root-inductive conditions, i.e., on medium containing 10 microM indole-3-butyric acid (IBA) and 0.1 microM kinetin, or without hormones. The explants were excised from the cultivars Samsun, Xanthii and Petite Havana, and from genotypes with altered AR-forming ability in response to auxin, namely the non-rooting rac mutant and the over-rooting Agrobacterium rhizogenes rolB transgenic line. Results show that NtRNR1 (G1/S) and Ntcyc29 (G2/M) gene activity, cell proliferation and meristemoid formation were stimulated in hormone-cultured TCLs by submicromolar MeJA concentrations. The meristemoids developed either into ARs and xylogenic nodules, or into xylogenic nodules only (rac TCLs). MeJA-induced meristemoid over-production characterized rolB TCLs. No rooting or xylogenesis occurred under hormone-free conditions, independently of MeJA and genotype. Endogenous JA progressively (days 1-4) increased in hormone-cultured TCLs in the absence of MeJA. JA levels were enhanced by 0.1 microM MeJA, on both days 1 and 4. Endogenous IBA was the only auxin detected, both in the free form and as IBA-glucose. Free IBA increased up to day 2, remaining constant thereafter (day 4). Its level was enhanced by 0.1 microM MeJA only on day 1, while IBA conjugation was not affected by MeJA. Taken together, these results show that an interplay between jasmonates and auxins regulates AR formation and xylogenesis in tobacco TCLs.