VOZ1 and VOZ2 transcription factors regulate arsenic tolerance and distribution in rice and Arabidopsis.

Rice is the major source of arsenic (As) intake in humans, as this staple crop readily accumulates As in the grain. Identifying the genes and molecular mechanisms underlying As accumulation and tolerance is a crucial step toward developing rice with reduced As levels. We identified 25 rice genes that improve As tolerance in yeast cells by expressing a complementary DNA (cDNA) library generated from As-treated rice roots. Among them, a zinc finger-type transcription factor VASCULAR PLANT ONE- ZINC FINGER 1 (OsVOZ1) (OsVOZ1) conferred the most pronounced As tolerance. OsVOZ1 inhibits As accumulation in yeast via activation of As efflux transporter Acr3p by post-transcriptional modification in yeast. The Arabidopsis voz1 voz2 double-knockout mutant exhibited As hypersensitivity, altered As concentrations in various tissues, and reduced As transport activity via the phloem. Arabidopsis and rice VOZs were highly expressed in phloem cells in various tissues, which are critical for As distribution in plant tissues. The double-knockdown and single-knockout plants of OsVOZ1 and OsVOZ2 reduced As accumulation in their seeds. These findings suggest that rice and Arabidopsis VOZs regulate the translocation of As into tissues by regulating the phloem loading of this element.

Role of ubiquitination in arsenic tolerance in plants.

Arsenic (As) is harmful to all living organisms, including humans and plants. To limit As uptake and avoid its toxicity, plants employ systems that regulate the uptake of As from the soil and its translocation from roots to grains. Ubiquitination, a highly conserved post-translational modification (PTM) in all eukaryotes, plays crucial roles in modulating As detoxification mechanisms in budding yeast (Saccharomyces cerevisiae), but little is known about its roles in As tolerance and transport in plants. In this opinion article we review recent findings and suggest that ubiquitination plays a crucial role in regulating As transport in plants. We also propose ideas for future research to explore the importance of ubiquitination for enhancing As tolerance in crops.

Arabidopsis ABCG27 plays an essential role in flower and leaf development by modulating abscisic acid content.

Abscisic acid (ABA) is a phytohormone that mediates stress responses and regulates plant development. Several ATP-binding cassette (ABC) transporters in the G subfamily of ABC (ABCG) proteins have been reported to transport ABA. We investigated whether there are any other ABCG proteins that mediate plant developmental processes regulated by ABA in Arabidopsis (Arabidopsis thaliana). The ABCG27 gene was upregulated in response to exogenous ABA treatment. The abcg27 knockout mutant exhibited two developmental defects: epinastic leaves and abnormally long pistils, which reduced fertility and silique length. ABCG27 expression was induced threefold when flower buds were exposed to exogenous ABA, and the promoter of ABCG27 had two ABA-responsive elements. ABA content in the pistil and true leaves were increased in the abcg27 knockout mutant. Detached abcg27 pistils exposed to exogenous ABA grew longer than those of the wild-type control. ABCG27 fused to GFP localized to the plasma membrane when expressed in Arabidopsis mesophyll protoplasts. A transcriptome analysis of the pistils and true leaves of the wild type and abcg27 knockout mutant revealed that the expression of organ development-related genes changed in the knockout mutant. In particular, the expression of trans-acting small interference (ta-si) RNA processing enzyme genes, which regulate flower and leaf development, was low in the knockout mutant. Together, these results suggest that ABCG27 most likely function as an ABA transporter at the plasma membrane, modulating ABA levels and thereby regulating the development of the pistils and leaves under normal, non-stressed conditions.

Correction: Characterization of Brassica rapa metallothionein and phytochelatin synthase genes potentially involved in heavy metal detoxification.

[This corrects the article DOI: 10.1371/journal.pone.0252899.].

Identification of an Isoflavonoid Transporter Required for the Nodule Establishment of the Rhizobium-Fabaceae Symbiotic Interaction.

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by, for example, forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

An evergreen mind and a heart for the colors of fall.

With the finest biochemical and molecular approaches, convincing explorative strategies, and long-term vision, Stefan Hörtensteiner succeeded in elucidating the biochemical pathway responsible for chlorophyll degradation. After having contributed to the identification of key chlorophyll degradation products in the course of the past 25 years, he gradually identified and characterized most of the crucial players in the PAO/phyllobilin degradation pathway of chlorophyll. He was one of the brightest plant biochemists of his generation, and his work opened doors to a better understanding of plant senescence, tetrapyrrole homeostasis, and their complex regulation. He sadly passed away on 5 December 2020, aged 57.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

The Full-Size ABCG Transporter of Medicago truncatula Is Involved in Strigolactone Secretion, Affecting Arbuscular Mycorrhiza.

Strigolactones (SLs) are plant-derived signaling molecules that stimulate the hyphal branching of arbuscular mycorrhizal fungi (AMF), and consequently promote symbiotic interaction between the fungus and the plant. Currently, our knowledge on the molecular mechanism of SL transport is restricted to the Solanaceae family. In the Solanaceae family, SL translocation toward the rhizosphere occurs through the exodermis via hypodermal passage cells and involves a member of the G subfamily, of the ATP-binding cassette (ABC) membrane transporters. Most Fabaceae species, including those that are agriculturally important, have a different root anatomy compared to most angiosperm plants (i.e., lacking an exodermis). Thus, we have investigated how SL transport occurs in the model legume Medicago truncatula. Here, we show that overexpression of a SL transporter from petunia (PaPDR1) enhances AMF colonization rates in M. truncatula. This result demonstrates the importance of ABCG proteins for the translocation of orobanchol-type molecules to facilitate arbuscular mycorrhiza, regardless of root anatomy and phylogenetic relationships. Moreover, our research has led to the identification of Medicago ABCG59, a close homologue of Petunia PDR1, that exhibits root specific expression and is up-regulated by phosphate starvation as well as in the presence of rac-GR24, a synthetic SL. Its promoter is active in cortical cells, root tips, and the meristematic zone of nodules. The mtabcg59 loss-of-function mutant displayed a reduced level of mycorrhization compared to the WT plants but had no impact on the number of nodules after Sinorhizobium meliloti inoculation. The reduced mycorrhization indicates that less SLs are secreted by the mutant plants, which is in line with the observation that mtabcg59 exudates exhibit a reduced stimulatory effect on the germination of the parasitic plant Phelipanche ramosa compared to the corresponding wild type.

Non-intrinsic ATP-binding cassette proteins ABCI19, ABCI20 and ABCI21 modulate cytokinin response at the endoplasmic reticulum in Arabidopsis thaliana.

KEY MESSAGE: The non-intrinsic ABC proteins ABCI20 and ABCI21 are induced by light under HY5 regulation, localize to the ER, and ameliorate cytokinin-driven growth inhibition in young Arabidopsis thaliana seedlings. The plant ATP-binding cassette (ABC) I subfamily (ABCIs) comprises heterogeneous proteins containing any of the domains found in other ABC proteins. Some ABCIs are known to function in basic metabolism and stress responses, but many remain functionally uncharacterized. ABCI19, ABCI20, and ABCI21 of Arabidopsis thaliana cluster together in a phylogenetic tree, and are suggested to be targets of the transcription factor ELONGATED HYPOCOTYL 5 (HY5). Here, we reveal that these three ABCIs are involved in modulating cytokinin responses during early seedling development. The ABCI19, ABCI20 and ABCI21 promoters harbor HY5-binding motifs, and ABCI20 and ABCI21 expression was induced by light in a HY5-dependent manner. abci19 abci20 abci21 triple and abci20 abci21 double knockout mutants were hypersensitive to cytokinin in seedling growth retardation assays, but did not show phenotypic differences from the wild type in either control medium or auxin-, ABA-, GA-, ACC- or BR-containing media. ABCI19, ABCI20, and ABCI21 were expressed in young seedlings and the three proteins interacted with each other, forming a large protein complex at the endoplasmic reticulum (ER) membrane. These results suggest that ABCI19, ABCI20, and ABCI21 fine-tune the cytokinin response at the ER under the control of HY5 at the young seedling stage.

Transportomics for the Characterization of Plant Apocarotenoid Transmembrane Transporters.

Apocarotenoids are carotenoid derivatives produced by the nonenzymatic or enzymatic cleavage of carotenoids, followed by different enzymatic modifications. In plants, apocarotenoids play different roles, such as attraction of pollinators and seeds dispersal, defense against pathogens and herbivores, protection against photo-oxidative stresses, stimulation and inhibition of plant growth and regulation of biological processes in the case of phytohormones abscisic acid and strigolactones. While carotenoids are in general plastid-localized metabolites, apocarotenoids can reach different final destinations inside or outside the cell. The mechanisms of apocarotenoid transport through biological membranes have been poorly studied. This chapter describes a method to characterize transmembrane transporters involved in the transport of polar and amphipathic apocarotenoids. This protocol was successfully used to in vitro characterize the transport activity of ATP-binding cassette (ABC) and multidrug and toxic extrusion (MATE) in microsomes isolated from Saccharomyces cerevisiae expressing these plant transporters.

ABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron Stigmas.

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.

ABCG36/PEN3/PDR8 Is an Exporter of the Auxin Precursor, Indole-3-Butyric Acid, and Involved in Auxin-Controlled Development.

The PDR-type ABCG transporter, ABCG36/PDR8/PEN3, is thought to be implicated in the export of a few structurally unrelated substrates, including the auxin precursor, indole-3-butyric acid (IBA), although a clear-cut proof of transport is lacking. An outward facing, lateral root (LR) location for ABCG36 fuelled speculations that it might secrete IBA into the rhizosphere. Here, we provide strong evidence that ABCG36 catalyzes the export of IBA - but not of indole-3-acetic acid - through the plasma membrane. ABCG36 seems to function redundantly with the closely related isoform ABCG37/PDR9/PIS1 in a negative control of rootward IBA transport in roots, which might be dampened by concerted, lateral IBA export. Analyses of single and double mutant phenotypes suggest that both ABCG36 and ABCG37 function cooperatively in auxin-controlled plant development. Both seem to possess a dual function in the control of auxin homeostasis in the root tip and long-range transport in the mature root correlating with non-polar and polar expression profiles in the LR cap and epidermis, respectively.

Strigolactones Play an Important Role in Shaping Exodermal Morphology via a KAI2-Dependent Pathway.

The majority of land plants have two suberized root barriers: the endodermis and the hypodermis (exodermis). Both barriers bear non-suberized passage cells that are thought to regulate water and nutrient exchange between the root and the soil. We learned a lot about endodermal passage cells, whereas our knowledge on hypodermal passage cells (HPCs) is still very scarce. Here we report on factors regulating the HPC number in Petunia roots. Strigolactones exhibit a positive effect, whereas supply of abscisic acid (ABA), ethylene, and auxin result in a strong reduction of the HPC number. Unexpectedly the strigolactone signaling mutant d14/dad2 showed significantly higher HPC numbers than the wild-type. In contrast, its mutant counterpart max2 of the heterodimeric receptor DAD2/MAX2 displayed a significant decrease in HPC number. A mutation in the Petunia karrikin sensor KAI2 exhibits drastically decreased HPC amounts, supporting the hypothesis that the dimeric KAI2/MAX2 receptor is central in determining the HPC number.

Arabidopsis ABCG28 is required for the apical accumulation of reactive oxygen species in growing pollen tubes.

Tip-focused accumulation of reactive oxygen species (ROS) is tightly associated with pollen tube growth and is thus critical for fertilization. However, it is unclear how tip-growing cells establish such specific ROS localization. Polyamines have been proposed to function in tip growth as precursors of the ROS, hydrogen peroxide. The ABC transporter AtABCG28 may regulate ROS status, as it contains multiple cysteine residues, a characteristic of proteins involved in ROS homeostasis. In this study, we found that AtABCG28 was specifically expressed in the mature pollen grains and pollen tubes. AtABCG28 was localized to secretory vesicles inside the pollen tube that moved toward and fused with the plasma membrane of the pollen tube tip. Knocking out AtABCG28 resulted in defective pollen tube growth, failure to localize polyamine and ROS to the growing pollen tube tip, and complete male sterility, whereas ectopic expression of this gene in root hair could recover ROS accumulation at the tip and improved the growth under high-pH conditions, which normally prevent ROS accumulation and tip growth. Together, these data suggest that AtABCG28 is critical for localizing polyamine and ROS at the growing tip. In addition, this function of AtABCG28 is likely to protect the pollen tube from the cytotoxicity of polyamine and contribute to the delivery of polyamine to the growing tip for incorporation into the expanding cell wall.

Ideal Cereals With Lower Arsenic and Cadmium by Accurately Enhancing Vacuolar Sequestration Capacity.

Cereals are a staple food for many people around the world; however, they are also a major dietary source of toxic metal(loid)s. Many agricultural regions throughout the world are contaminated with toxic metal(loid)s, which can accumulate to high levels in the grains of cereals cultivated in these regions, posing serious health risks to consumers. Arsenic (As) and cadmium (Cd) are efficiently accumulated in cereals through metal transport pathways. Therefore, there is an urgent need to develop crops that contain greatly reduced levels of toxic metal(loid)s. Vacuolar sequestration of toxic metal(loid)s is a primary strategy for reducing toxic metal(loid)s in grains. However, until recently, detailed strategies and mechanisms for reducing toxic metal(loid)s in grain were limited by the lack of experimental data. New strategies to reduce As and Cd in grain by enhancing vacuolar sequestration in specific tissues are critical to develop crops that lower the daily intake of As and Cd, potentially improving human health. This review provides insights and strategies for developing crops with strongly reduced amounts of toxic metal(loid)s without jeopardizing agronomic traits.

Abscisic acid is a substrate of the ABC transporter encoded by the durable wheat disease resistance gene Lr34.

The wheat Lr34res allele, coding for an ATP-binding cassette transporter, confers durable resistance against multiple fungal pathogens. The Lr34sus allele, differing from Lr34res by two critical nucleotide polymorphisms, is found in susceptible wheat cultivars. Lr34res is functionally transferrable as a transgene into all major cereals, including rice, barley, maize, and sorghum. Here, we used transcriptomics, physiology, genetics, and in vitro and in vivo transport assays to study the molecular function of Lr34. We report that Lr34res results in a constitutive induction of transcripts reminiscent of an abscisic acid (ABA)-regulated response in transgenic rice. Lr34-expressing rice was altered in biological processes that are controlled by this phytohormone, including dehydration tolerance, transpiration and seedling growth. In planta seedling and in vitro yeast accumulation assays revealed that both LR34res and LR34sus act as ABA transporters. However, whereas the LR34res protein was detected in planta the LR34sus version was not, suggesting a post-transcriptional regulatory mechanism. Our results identify ABA as a substrate of the LR34 ABC transporter. We conclude that LR34res-mediated ABA redistribution has a major effect on the transcriptional response and physiology of Lr34res-expressing plants and that ABA is a candidate molecule that contributes to Lr34res-mediated disease resistance.