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Auxin Response Factor 8 plays a key role in late stamen development: its splice variants ARF8.4 and ARF8.2 control stamen elongation and anther dehiscence. Here, we characterized the role of ARF8 isoforms in pollen fertility. By phenotypic and ultrastructural analysis of arf8-7 mutant stamens, we found defects in pollen germination and viability caused by alterations in exine structure and pollen coat deposition. Furthermore, tapetum degeneration, a prerequisite for proper pollen wall formation, is delayed in arf8-7 anthers. In agreement, the genes encoding the transcription factors TDF1, AMS, MS188, and MS1, required for exine and pollen coat formation, and tapetum development, are downregulated in arf8-7 stamens. Consistently, the sporopollenin content is decreased, and the expression of sporopollenin synthesis/transport and pollen coat protein biosynthetic genes, regulated by AMS and MS188, is reduced. Inducible expression of the full-length isoform ARF8.1 in arf8-7 inflorescences complements the pollen (and tapetum) phenotype and restores the expression of the above transcription factors. Chip-qPCR assay revealed that ARF8.1 directly targets the promoters of TDF1, AMS and MS188. In conclusion, the ARF8.1 isoform controls pollen and tapetum development acting directly on the expression of TDF1, AMS and MS188, which belong to the pollen/tapetum genetic pathway.

In Arabidopsis, stamen elongation, which ensures male fertility, is controlled by the auxin response factor ARF8, which regulates the expression of the auxin repressor IAA19. Here, we uncover a role for light in controlling stamen elongation. By an extensive genetic and molecular analysis we show that the repressor of light signaling COP1, through its targets HY5 and HYH, controls stamen elongation, and that HY5 - oppositely to ARF8 - directly represses the expression of IAA19 in stamens. In addition, we show that in closed flower buds, when light is shielded by sepals and petals, the blue light receptors CRY1/CRY2 repress stamen elongation. Coherently, at flower disclosure and in subsequent stages, stamen elongation is repressed by the red and far-red light receptors PHYA/PHYB. In conclusion, different light qualities - sequentially perceived by specific photoreceptors - and the downstream COP1-HY5/HYH module finely tune auxin-induced stamen elongation and thus male fertility.

In this work, arsenic (As) accumulation and distribution over time in Pteris vittata young fronds from adult plants and in whole plantlets, grown on a highly contaminated As-soil, was determined by μ-XRF. A linear increase in As content up to 60 days was found in young fronds at different times, and a progressive distribution from the apex to the base of the fronds was observed. In whole plantlets, As signal was detectable from 9 to 20 days in the apex of a few fronds and fiddleheads. Later, up to 60 days, As was localized in all fronds, in the rhizome and in basal part of the roots. The dynamics of expression of As-related genes revealed a good correlation between As content and the level of the As (III)-antiporter PvACR3 transcript in plantlets roots and fronds and in young fronds. Moreover, the transcription of As (V)-related gametophytic genes PvGAPC1, PvOCT4 increases over time during As accumulation while PvGSTF1 is expressed only in roots. Here, we demonstrate the suitability of the μ-XRF technique to monitor As accumulation, which allowed us to propose that As is initially directly transported to fiddleheads and apex of fronds, is later distributed to the whole fronds and simultaneously accumulated in the rhizome and roots. We also provide indications on the expression of candidate genes possibly involved in As (hyper)accumulation.

During 2017 an alert was raised in relation to a possible vaccination failure occurred in Friuli Venezia Giulia Region (Italy) between 2009 and 2015, exposing multiple cohorts of children and the entire community to vaccine preventable diseases outbreak risk. The Codroipo case resulted in 20,441 vaccine doses being in doubt, thus prompting the healthcare system to react in order to revaccinate 5444 children through planning and implementing network actions and multiple channels of communication.

This study evaluated the phytoextraction capacity of the fern Pteris vittata grown on a natural arsenic-rich soil of volcanic-origin from the Viterbo area in central Italy. This calcareous soil is characterized by an average arsenic concentration of 750 mg kg−1, of which 28% is bioavailable. By means of micro-energy dispersive X-ray fluorescence spectrometry (μ-XRF) we detected As in P. vittata fronds after just 10 days of growth, while a high As concentrations in fronds (5,000 mg kg−1), determined by Inductively coupled plasma-optical emission spectrometry (ICP-OES), was reached after 5.5 months. Sixteen arsenate-tolerant bacterial strains were isolated from the P. vittata rhizosphere, a majority of which belong to the Bacillus genus, and of this majority only two have been previously associated with As. Six bacterial isolates were highly As-resistant (> 100 mM) two of which, homologous to Paenarthrobacter ureafaciens and Beijerinckia fluminensis, produced a high amount of IAA and siderophores and have never been isolated from P. vittata roots. Furthermore, five isolates contained the arsenate reductase gene (arsC). We conclude that P. vittata can efficiently phytoextract As when grown on this natural As-rich soil and a consortium of bacteria, largely different from that usually found in As-polluted soils, has been found in P. vittata rhizosphere.

The reuse of Pteris vittata plants for multiple phytofiltration cycles is a main issue to allow an efficient phytoremediation of arsenic (As)-contaminated groundwater. Here, we assessed the capacity of phytofiltration of P. vittata plants grown for two cycles on naturally As-contaminated drinking water (collected in Central Italy), spaced by a growth cycle on non-contaminated water (N cycle). P. vittata young plants, with extensive frond and root development, were suspended individually in 15 L of water with initial As of 59 µg/L, without any additional treatment or water refilling. During cycle 1, in 45 days P. vittata plants reduced As concentration below 10 µg/L, the allowed EU limits for drinking water. During the subsequent 30 day-N cycle on non-contaminated water, no leaching of As from the roots was observed, while the water pH increased 0.9 Units, but is within the allowed limits. During cycle 2, under the same conditions as cycle 1, As concentration decreased below 10 µg/L in less than seven days. These results show that P. vittata young plants, previously used for the phytofiltration of As, do not extrude As and, when reused, remove As much more rapidly. No additional treatments were required during phytofiltration and thus this represents a sustainable, efficient, and scalable strategy.

An attempt to link phytomanagement, art and social involvement is presented. The “Remediation” Project started up from the search of sustainable solutions for the management of a riparian area in t...

An innovative approach, based on hyperspectral imaging (HSI) coupled with chemometrics, allowing the detection of arsenic (As) in the hyper-accumulator fern Pteris Vittata L., is presented in this study. The aim of this work was to investigate the possibility of monitoring by HSI the As sequestration capacity of plants grown on As-contaminated soils, in order to perform soil remediation. The proposed approach is based on the acquisition by HSI in the SWIR range (1000-2500 nm) of fern leaves, followed by the implementation of a classification model based on Partial Least Square Discriminant Analysis (PLS-DA). Following this procedure, false color maps, representative of the chemical elements distribution on the leaves were obtained, where As is clearly detected without performing any chemical analysis. The proposed approach is not invasive and not destructive. Comparative evaluations were carried out analyzing Pteris Vittata L. leaves collected from plants grown on natural soils containing different As concentrations. To evaluate reliability, robustness and analytical correctness of the proposed HSI approach, micro X-ray fluorescence (μXRF) analyses were carried out on the same samples in order to quantitatively and topologically assess As presence in the leaves of the plants. The achieved results are very promising for monitoring the phytoremediation process by detecting and controlling the uptake of As plants growing on contaminated soils.

Here, we present a new scheme of analysis combining micro-energy dispersive X-ray fluorescence spectrometry (μ-XRF) with a multivariate approach that allows to establish the inter-correlation of multiple elements and their elemental map in plants. The main advantage of this procedure is that XRF spectral profiles can be analysed directly, by means of principal component analysis (PCA), allowing a quick interpretation of the results. Furthermore, this analysis requires small amounts of plant material and can be performed in whole individual seedlings in the hydrated state without chemical extraction. With this technology, we determined the distribution of arsenic (As) and the variation and spatial distribution of multiple elements in whole tobacco seedlings grown in the presence of different arsenate concentrations. We observed that As is detectable mainly in roots, primarily in the basal part, but also in the root apex in seedlings grown in the highest arsenate concentration. The low rate of As translocation from roots to shoots and the significant increase in S are consistent with previous evidence showing that As is retained in roots by forming complexes with thiol peptides. We also found a significant non-linear increase in P, as arsenate is taken up by phosphate transporters and induces the expression of genes encoding them. A decrease in Mn, Fe and Zn proportional to the accumulation of As and in the same tissues, suggests a competition of these elements with As for cellular transporters.

In addition to the full-length transcript ARF8.1, a splice variant (ARF8.2) of the auxin response factor gene ARF8 has been reported. Here, we identified an intron-retaining variant of ARF8.2, ARF8.4, whose translated product is imported into the nucleus and has tissue-specific localization in Arabidopsis thaliana. By inducibly expressing each variant in arf8-7 flowers, we show that ARF8.4 fully complements the short-stamen phenotype of the mutant and restores the expression of AUX/IAA19, encoding a key regulator of stamen elongation. By contrast, the expression of ARF8.2 and ARF8.1 had minor or no effects on arf8-7 stamen elongation and AUX/IAA19 expression. Coexpression of ARF8.2 and ARF8.4 in both the wild type and arf8-7 caused premature anther dehiscence: We show that ARF8.2 is responsible for increased expression of the jasmonic acid biosynthetic gene DAD1 and that ARF8.4 is responsible for premature endothecium lignification due to precocious expression of transcription factor gene MYB26. Finally, we show that ARF8.4 binds to specific auxin-related sequences in both the AUX/ IAA19 and MYB26 promoters and activates their transcription more efficiently than ARF8.2. Our data suggest that ARF8.4 is a tissue-specific functional splice variant that controls filament elongation and endothecium lignification by directly regulating key genes involved in these processes.

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.

Here, we investigated the role of auxin distribution in controlling Arabidopsis thaliana late stamen development. We analysed auxin distribution in anthers by monitoring DR5 activity: at different flower developmental stages; inhibiting auxin transport; in the rpk2-3 and ems1 mutants devoid of middle layer (ML) or tapetum, respectively; and in the auxin biosynthesis yuc6 and perception afb1-3 mutants. We ran a phenotypic, DR5::GUS and gene expression analysis of yuc6rpk2 and afb1rpk2 double mutants, and of 1-N-naphthylphthalamic acid (NPA)-treated flower buds. We show that an auxin maximum, caused by transport from the tapetum, is established in the ML at the inception of late stamen development. rpk2-3 mutant stamens lacking the ML have an altered auxin distribution with excessive accumulation in adjacent tissues, causing non-functional pollen grains, indehiscent anthers and reduced filament length; the expression of genes controlling stamen development is also altered in rpk2-3 as well as in NPA-treated flower buds. By decreasing auxin biosynthesis or perception in the rpk2-3 background, we eliminated these developmental and gene expression anomalies. We propose that the auxin maximum in the ML plays a key role in late stamen development, as it ensures correct and coordinated pollen maturation, anther dehiscence and filament elongation.

Highlight AtABCC3 detoxifies cadmium by transporting phytochelatin–cadmium complexes into the vacuoles, and it can functionally complement abcc1 abcc2 mutants., The heavy metal cadmium (Cd) is a widespread environmental contaminant with harmful effects on living cells. In plants, phytochelatin (PC)-dependent Cd detoxification requires that PC–Cd complexes are transported into vacuoles. Here, it is shown that Arabidopsis thaliana seedlings defective in the ABCC transporter AtABCC3 (abcc3) have an increased sensitivity to different Cd concentrations, and that seedlings overexpressing AtABCC3 (AtABCC3ox) have an increased Cd tolerance. The cellular distribution of Cd was analysed in protoplasts from abcc3 mutants and AtABCC3 overexpressors grown in the presence of Cd, by means of the Cd-specific fluorochromes 5-nitrobenzothiazole coumarin (BTC-5N) and Leadmium™ Green AM dye. This analysis revealed that Cd is mostly localized in the cytosol of abcc3 mutant protoplasts whereas there is an increase in vacuolar Cd in protoplasts from AtABCC3ox plants. Overexpression of AtABCC3 in cad1-3 mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that AtABCC3 acts via PCs. In addition, overexpression of AtABCC3 in atabcc1 atabcc2 mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of AtABCC3 transcript in wild type seedlings was lower than that of AtABCC1 and AtABCC2 in the absence of Cd but higher after Cd exposure, and even higher in atabcc1 atabcc2 mutants. The results point to AtABCC3 as a transporter of PC–Cd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2.

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.

It has been suggested that, in Arabidopsis, auxin controls the timing of anther dehiscence, possibly by preventing premature endothecium lignification. We show here that auxin content in anthers peaks before the beginning of dehiscence and decreases when endothecium lignification occurs. We show that, in the auxin-perception mutants afb1-3 and tir1 afb2 afb3, endothecium lignification and anther dehiscence occur earlier than wild-type, and the gene encoding the transcription factor MYB26, which is required for endothecium lignification, is over-expressed specifically at early stages; in agreement, MYB26 expression is reduced in naphthalene acetic acid-treated anthers, and afb1 myb26 double mutants show no endothecial lignification, suggesting that auxin acts through MYB26. As jasmonic acid (JA) controls anther dehiscence, we analysed how auxin and JA interact. In the JA-defective opr3 mutant, indehiscent anthers show normal timing of endothecium lignification, suggesting that JA does not control this event. We show that expression of the OPR3 and DAD1 JA biosynthetic genes is enhanced in afb1-3 and tir1 afb2 afb3 flower buds, but is reduced in naphthalene acetic acid-treated flower buds, suggesting that auxin negatively regulates JA biosynthesis. The double mutant afb1 opr3 shows premature endothecium lignification, as in afb1-3, and indehiscent anthers due to lack of JA, which is required for stomium opening. By treating afb1 opr3 and opr3 inflorescences with JA, we show that a high JA content and precocious endothecium lignification both contribute to induction of early anther dehiscence. We propose that auxin controls anther dehiscence timing by negatively regulating two key events: endothecium lignification via MYB26, and stomium opening via the control of JA biosynthesis.

We previously described the expression of a tumour-targeting antibody (mAb H10) in Nicotiana benthamiana by vacuum-agro-infiltration and the remarkable yields of highly pure protein achieved. The objective of the present work was to investigate different strategies for transient overexpression of the mAb H10 in which glycan configuration was modulated and assess how these strategies affect the accumulation yield and stability of the antibody. To this aim, three procedures have been assayed: (1) Site-directed mutagenesis to abolish the glycosylation site; (2) endoplasmic reticulum retention (C-terminal SEKDEL fusion) to ensure predominantly high-mannose type glycans; and (3) expression in a N. benthamiana RNAi down-regulated line in which β1,2-xylosyltransferase and α1,3-fucosyltransferase gene expression is silenced. The three antibody variants (H10-Mut) (H10-SEKDEL) (H10(XylT/FucT)) were transiently expressed, purified and characterised for their glycosylation profile, expression/purification yield and antibody degradation pattern. Glycosylation analysis of H10(XylT/FucT) demonstrated the absence of plant complex-type sugars, while H10-SEKDEL, although substantially retained in the ER, revealed the presence of β1,2-xylose and α1,3-fucose residues, indicating a partial escape from the ER retrieval system. Antibody accumulation and purification yields were not enhanced by ER retention. All H10 antibody glyco-forms revealed greater degradation compared to the original, resulting mostly in the formation of Fab fragments. In the case of aglycosylated H10-Mut, more than 95% of the heavy chain was cleaved, confirming the pivotal role of the sugar moiety in protein stability. Identification of possible 'fragile' sites in the H10 antibody hinge region could be of general interest for the development of new strategies to reduce antibody degradation and increase the yield of intact IgGs in plants.

Previous studies demonstrated that expression of the Arabidopsis phytochelatin (PC) biosynthetic gene AtPCS1 in Nicotiana tabacum plants increases the Cd tolerance in the presence of exogenous glutathione (GSH). In this paper, the Cd tolerance of Arabidopsis plants over-expressing AtPCS1 (AtPCSox lines) has been analysed and the differences between Arabidopsis and tobacco are shown. Based on the analysis of seedling fresh weight, primary root length, and alterations in root anatomy, evidence is provided that, at relatively low Cd concentrations, the Cd tolerance of AtPCSox lines is lower than the wild type, while AtPCS1 over-expressing tobacco is more tolerant to Cd than the wild type. At higher Cd concentrations, Arabidopsis AtPCSox seedlings are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Exogenous GSH, in contrast to what was observed in tobacco, did not increase the Cd tolerance of AtPCSox lines. The PC content in wild-type Arabidopsis at low Cd concentrations is more than three times higher than in tobacco and substantial differences were also found in the PC chain lengths. These data indicate that the differences in Cd tolerance and in its dependence on exogenous GSH between Arabidopsis and tobacco are due to species-specific differences in the endogenous content of PCs and GSH and may be in the relative abundance of PCs of different length.

The aims of this work were to obtain a human antibody against the tumour-associated antigen tenascin-C (TNC) and to compare the yield and quality of plant-produced antibody in either stable transgenics or using a transient expression system. To this end, the characterization of a full-sized human immunoglobulin G (IgG) [monoclonal antibody H10 (mAb H10)], derived from a selected single-chain variable fragment (scFv) and produced in plants, is presented. The human mAb gene was engineered for plant expression, and Nicotiana tabacum transgenic lines expressing both heavy (HC) and light (LC) chain were obtained and evaluated for antibody expression levels, in vivo assembly and functionality. Affinity-purified H10 from transgenics (yield, 0.6-1.1 mg/kg fresh weight) revealed that more than 90% of HC was specifically degraded, leading to the formation of functional antigen-binding fragments (Fab). Consequently, H10 was transiently expressed in Nicotiana benthamiana plants through an Agrobacterium-mediated gene-transfer system. Moreover, the use of the p19 silencing suppressor gene from artichoke mottled crinkle virus raised antibody expression levels by an order of magnitude (yields of purified H10, 50-100 mg/kg fresh weight). Approximately 75% of purified protein consisted of full-sized antibody functionally binding to TNC (K(D) = 14 nm), and immunohistochemical analysis on tumour tissues revealed specific accumulation around tumour blood vessels. The data indicate that the purification yields of mAb H10, using a transient expression system boosted by the p19 silencing suppressor, are exceptionally high when compared with the results reported previously, providing a technique for the over-expression of anticancer mAbs by a rapid, cost-effective, molecular farming approach.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00425-015-2428-8) contains supplementary material, which is available to authorized users.