Correction: Anglana et al. Dittrichia viscosa Selection Strategy Based on Stress Produces Stable Clonal Lines for Phytoremediation Applications. Plants 2023, 12, 2499.

In the original publication [...].

Methanolic Extracts of D. viscosa Specifically Affect the Cytoskeleton and Exert an Antiproliferative Effect on Human Colorectal Cancer Cell Lines, According to Their Proliferation Rate.

Numerous studies have reported the pharmacological effects exhibited by Dittrichia viscosa, (D. viscosa) including antioxidant, cytotoxic, antiproliferative, and anticancer properties. In our research, our primary objective was to validate a prescreening methodology aimed at identifying the fraction that demonstrates the most potent antiproliferative and anticancer effects. Specifically, we investigated the impact of various extract fractions on the cytoskeleton using a screening method involving transgenic plants. Tumors are inherently heterogeneous, and the components of the cytoskeleton, particularly tubulin, are considered a strategic target for antitumor agents. To take heterogeneity into account, we used different lines of colorectal cancer, specifically one of the most common cancers regardless of gender. In patients with metastasis, the effectiveness of chemotherapy has been limited by severe side effects and by the development of resistance. Additional therapies and antiproliferative molecules are therefore needed. In our study, we used colon-like cell lines characterized by the expression of gastrointestinal differentiation markers (such as the HT-29 cell line) and undifferentiated cell lines showing the positive regulation of epithelial-mesenchymal transition and TGFß signatures (such as the DLD-1, SW480, and SW620 cell lines). We showed that all three of the D. viscosa extract fractions have an antiproliferative effect but the pre-screening on transgenic plants anticipated that the methanolic fraction may be the most promising, targeting the cytoskeleton specifically and possibly resulting in fewer side effects. Here, we show that the preliminary use of screening in transgenic plants expressing subcellular markers can significantly reduce costs and focus the advanced characterization only on the most promising therapeutic molecules.

Dittrichia viscosa Selection Strategy Based on Stress Produces Stable Clonal Lines for Phytoremediation Applications.

Dittrichia viscosa uptake and translocation of the metalloid As is not fully understood and some data are contradictory, but its adaptability to this pollutant is known and is dependent on its genetic variability. D. viscosa is not a hyperaccumulator plant, but it can grow in high-drought conditions while still producing large biomass, even tolerating significant concentrations of As3+ and As5+. In spite of these remarkable characteristics, adaptive modification of performances is not predictable in wild populations. In previous work, we established experimental clonal populations to perform a functional study on the aquaporin NIP1.1. Here, we propose a strategy to select a clonal population of D. viscosa with a defined phenotype related to As tolerance and to reduced NIP1.1 expression levels for phytoremediation applications. From the previous work, we selected four independent clones, two of them belonging to the weak population (W8 and W9) and the other two belonging to the strong population (S1 and S3). The weak and strong populations differ for a different expression ratio root/shoot of DvNip1;1 that brings a different tolerance to As presence. The stress response of the populations, revealed by the CAT enzymatic test, was statistically correlated to the clones, but not to As uptake. Performance of the selected plants on a second unrelated metallic pollutant, Cd, was evaluated, showing that Cd uptake is also independent from the tolerant phenotype. In vitro culture methods using solid media and temporary immersion bioreactors were compared to propose an optimized combined protocol. The procedure yielded propagation of genetically stable tolerant clonal lines with good uptake of As and Cd. The plants, mass-produced with the developed in vitro protocol, were able to maintain their acquired abilities and are potentially able be later applied in phytoremediation or contaminated areas' re-naturalization.

Dimethylmonothioarsenate Is Highly Toxic for Plants and Readily Translocated to Shoots.

Arsenic is one of the most relevant environmental pollutants and human health threats. Several arsenic species occur in soil pore waters. Recently, it was discovered that these include inorganic and organic thioarsenates. Among the latter, dimethylmonothioarsenate (DMMTA) is of particular concern because in mammalian cells, its toxicity was found to exceed even that of arsenite. We investigated DMMTA toxicity for plants in experiments with Arabidopsis thaliana and indeed observed stronger growth inhibition than with arsenite. DMMTA caused a specific, localized deformation of root epidermal cells. Toxicity mechanisms apparently differ from those of arsenite since no accumulation of reactive oxygen species was observed in DMMTA-exposed root tips. Also, there was no contribution of the phytochelatin pathway to the DMMTA detoxification as indicated by exposure experiments with respective mutants and thiol profiling. RNA-seq analysis found strong transcriptome changes dominated by stress-responsive genes. DMMTA was taken up more efficiently than the methylated oxyarsenate dimethylarsenate and highly mobile within plants as revealed by speciation analysis. Shoots showed clear indications of DMMTA toxicity such as anthocyanin accumulation and a decrease in chlorophyll and carotenoid levels. The toxicity and efficient translocation of DMMTA within plants raise important food safety issues.

Actin and Microtubules Differently Contribute to Vacuolar Targeting Specificity during the Export from the ER.

Plants rely on both actin and microtubule cytoskeletons to fine-tune sorting and spatial targeting of membranes during cell growth and stress adaptation. Considerable advances have been made in recent years in the comprehension of the relationship between the trans-Golgi network/early endosome (TGN/EE) and cytoskeletons, but studies have mainly focused on the transport to and from the plasma membrane. We address here the relationship of the cytoskeleton with different endoplasmic reticulum (ER) export mechanisms toward vacuoles. These emergent features of the plant endomembrane traffic are explored with an in vivo approach, providing clues on the traffic regulation at different levels beyond known proteins' functions and interactions. We show how traffic of vacuolar markers, characterized by different vacuolar sorting determinants, diverges at the export from the ER, clearly involving different components of the cytoskeleton.

Aquatic Mosses as Adaptable Bio-Filters for Heavy Metal Removal from Contaminated Water.

Heavy metals (HMs) are released into the environment by many human activities and persist in water even after remediation. The efficient filtration of solubilized HMs is extremely difficult. Phytoremediation appears a convenient tool to remove HMs from polluted water, but it is limited by the choice of plants able to adapt to filtration of polluted water in terms of space and physiological needs. Biomasses are often preferred. Aquatic moss biomasses, thanks to gametophyte characteristics, can act as live filtering material. The potential for phytoremediation of Hypnales aquatic mosses has been poorly investigated compared to aquatic macrophytes. Their potential is usually indicated as a tool for bioindication and environmental monitoring more than for pollutant removal. When phytoremediation has been considered, insufficient attention has been paid to the adaptability of biomasses to different needs. In this study the heavy metal uptake of moss Taxiphyllum barbieri grown in two different light conditions, was tested with high concentrations of elements such as Pb, Cd, Zn, Cu, As, and Cr. This moss produces dense mats with few culture needs. The experimental design confirmed the capacity of the moss to accumulate HMs accordingly to their physiology and then demonstrated that a significant proportion of HMs was accumulated within a few hours. In addition to the biosorption effect, an evident contribution of the active simplistic mass can be evidenced. These reports of HM accumulation within short time intervals, show how this moss is particularly suitable as an adaptable bio-filter, representing a new opportunity for water eco-sustainable remediation.

AQUA1 is a mercury sensitive poplar aquaporin regulated at transcriptional and post-translational levels by Zn stress.

Aquaporins are water channel proteins that regulate plant development, growth, and response to environmental stresses. Populus trichocarpa is one of the plants with the highest number of aquaporins in its genome, but only few of them have been characterized at the whole plant functional level. Here we analyzed a putative aquaporin gene, aqua1, a gene that encodes for a protein of 257 amino acid with the typical NPA (Asp-Pro-Ala) signature motif of the aquaporin gene family. aqua1 was down-regulated of ∼10 fold under excess Zn in both leaves and roots, and conferred Zn tolerance when expressed in yeast Zn hypersensitive strain. In vivo localization of AQUA1-GFP in Arabidopsis protoplast showed a heterogeneous distribution of this protein on different membranes destined to form aggregates related to autophagic multivesicular bodies. Zn-dependent AQUA1-GFP re-localization was perturbed by phosphatases' and kinases' inhibitors that could affect both intracellular trafficking and aquaporins' activity. Exposed to high concentration of Zn, AQUA1 also co-localized with AtTIP1;1, a well-known Arabidopsis vacuolar marker, probably in pro-vacuolar multivesicular bodies. These findings suggest that high concentration of Zn down-regulates aqua1 and causes its re-localization in new forming pro-vacuoles. This Zn-dependent re-localization appears to be mediated by mechanisms regulating intracellular trafficking and aquaporins' post-translational modifications. This functional characterization of a poplar aquaporin in response to excess Zn will be a useful reference for understanding aquaporins' roles and regulation in response to high concentration of Zn in poplar.

A LHCB9-dependent photosystem I megacomplex induced under low light in Physcomitrella patens.

Photosystem I of the moss Physcomitrella patens has special properties, including the capacity to undergo non-photochemical fluorescence quenching. We studied the organization of photosystem I under different light and carbon supply conditions in wild-type moss and in moss with the lhcb9 (light-harvesting complex) knockout genotype, which lacks an antenna protein endowed with red-shifted absorption forms. Wild-type moss, when grown on sugars and in low light, accumulated LHCB9 proteins and a large form of the photosystem I supercomplex, which, besides the canonical four LHCI subunits, included a LHCII trimer and four additional LHC monomers. The lhcb9 knockout produced an angiosperm-like photosystem I supercomplex with four LHCI subunits irrespective of the growth conditions. Growth in the presence of sublethal concentrations of electron transport inhibitors that caused oxidation or reduction of the plastoquinone pool prevented or promoted, respectively, the accumulation of LHCB9 and the formation of the photosystem I megacomplex. We suggest that LHCB9 is a key subunit regulating the antenna size of photosystem I and the ability to avoid the over-reduction of plastoquinone: this condition is potentially dangerous in the shaded and sunfleck-rich environment typical of mosses, whose plastoquinone pool is reduced by both photosystem II and the oxidation of sugar substrates.

A Newly Identified Flower-Specific Splice Variant of AUXIN RESPONSE FACTOR8 Regulates Stamen Elongation and Endothecium Lignification in Arabidopsis.

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.

Variation in Membrane Trafficking Linked to SNARE AtSYP51 Interaction With Aquaporin NIP1;1.

SYP51 and 52 are the two members of the SYP5 Qc-SNARE gene family in Arabidopsis thaliana. These two proteins, besides their high level of sequence identity (85%), have shown to have differential functional specificity and possess a different interactome. Here we describe a unique and specific interaction of SYP51 with an ER aquaporin, AtNIP1;1 (also known as NLM1) indicated to be able to transport arsenite [As(III)] and previously localized on PM. In the present work we investigate in detail such localization in vivo and characterize the interaction with SYP51. We suggest that this interaction may reveal a new mechanism regulating tonoplast invagination and recycling. We propose this interaction to be part of a regulatory mechanism associated with direct membrane transport from ER to tonoplast and Golgi mediated vesicle trafficking. We also demonstrate that NIP1;1 is important for plant tolerance to arsenite but does not alter its uptake or translocation. To explain such phenomenon the hypothesis that SYP51/NIP1;1 interaction modifies ER and vacuole ability to accumulate arsenite is discussed.

Trafficking routes to the plant vacuole: connecting alternative and classical pathways.

Due to the numerous roles plant vacuoles play in cell homeostasis, detoxification, and protein storage, the trafficking pathways to this organelle have been extensively studied. Recent evidence, however, suggests that our vision of transport to the vacuole is not as simple as previously imagined. Alternative routes have been identified and are being characterized. Intricate interconnections between routes seem to occur in various cases, complicating the interpretation of data. In this review, we aim to summarize the published evidence and link the emerging data with previous findings. We discuss the current state of information on alternative and classical trafficking routes to the plant vacuole.

Glutathione S-transferase related detoxification processes are correlated with receptor-mediated vacuolar sorting mechanisms.

KEY MESSAGE: Triticum durum Glutathione S-transferase Z1 is specifically responsive to glyphosate. Its expression influences the receptor-mediated vacuolar sorting mechanisms involved in tolerance mechanisms. A zeta subfamily glutathione S-transferase gene from Triticum durum (cv Cappelli) (TdGSTZ1) was characterized as part of a complex detoxification mechanism. The effect of different abiotic stresses on TdGSTZ1 revealed that the gene is unexpectedly responsive to glyphosate (GLY) herbicide despite it should not be part of tolerance mechanisms. Its role in the non-target-site mechanism of GLY resistance was then investigated. To analyze the GLY and the TdGSTZ1 overexpression effects on vacuolar sorting mechanisms, we performed transient transformation experiments in Nicotiana tabacum protoplasts using two vacuolar markers, AleuGFPgl133 and GFPgl133Chi, labeling the Sar1 dependent or independent sorting, respectively. We observed that the adaptive reaction of tobacco protoplasts vacuolar system to the treatment with GLY could be partially mimicked by the overexpression of TdGSTZ1 gene. To confirm the influence of GLY on the two vacuolar markers accumulation and the potential involvement of the secretion pathway activity in detoxification events, Arabidopsis thaliana transgenic plants overexpressing the non-glycosylated versions of the two markers were analyzed. The results suggested that GLY treatment specifically altered different vacuolar sorting characteristics, suggesting an involvement of the receptor-mediated AleuGFP sorting mechanism in GLY resistance. Finally, the expression analysis of selected genes confirmed that the non-target-site GLY resistance mechanisms are related to vacuolar sorting.

Cisplatin, Oxaliplatin, and Kiteplatin Subcellular Effects Compared in a Plant Model.

The immediate visual comparison of platinum chemotherapeutics' effects in eukaryotic cells using accessible plant models of transgenic Arabidopsis thaliana is reported. The leading anticancer drug cisplatin, a third generation drug used for colon cancer, oxaliplatin and kiteplatin, promising Pt-based anticancer drugs effective against resistant lines, were administered to transgenic A. thaliana plants monitoring their effects on cells from different tissues. The transgenic plants' cell cytoskeletons were labelled by the green fluorescent protein (GFP)-tagged microtubule-protein TUA6 (TUA6-GFP), while the vacuolar organization was evidenced by two soluble chimerical GFPs (GFPChi and AleuGFP) and one transmembrane GFP-tagged tonoplast intrinsic protein 1-1 (TIP1.1-GFP). The three drugs showed easily recognizable effects on plant subcellular organization, thereby providing evidence for a differentiated drug targeting. Genetically modified A. thaliana are confirmed as a possible rapid and low-cost screening tool for better understanding the mechanism of action of human anticancer drugs.

New insights on plant cell elongation: a role for acetylcholine.

We investigated the effect of auxin and acetylcholine on the expression of the tomato expansin gene LeEXPA2, a specific expansin gene expressed in elongating tomato hypocotyl segments. Since auxin interferes with clathrin-mediated endocytosis, in order to regulate cellular and developmental responses we produced protoplasts from tomato elongating hypocotyls and followed the endocytotic marker, FM4-64, internalization in response to treatments. Tomato protoplasts were observed during auxin and acetylcholine treatments after transient expression of chimerical markers of volume-control related compartments such as vacuoles. Here we describe the contribution of auxin and acetylcholine to LeEXPA2 expression regulation and we support the hypothesis that a possible subcellular target of acetylcholine signal is the vesicular transport, shedding some light on the characterization of this small molecule as local mediator in the plant physiological response.