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.

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.

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.

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.

MtABCG20 is an ABA exporter influencing root morphology and seed germination of Medicago truncatula.

Abscisic acid (ABA) integrates internal and external signals to coordinate plant development, growth and architecture. It plays a central role in stomatal closure, and prevents germination of freshly produced seeds and germination of non-dormant seeds under unfavorable circumstances. Here, we describe a Medicago truncatula ATP-binding cassette (ABC) transporter, MtABCG20, as an ABA exporter present in roots and germinating seeds. In seeds, MtABCG20 was found in the hypocotyl-radicle transition zone of the embryonic axis. Seeds of mtabcg20 plants were more sensitive to ABA upon germination, due to the fact that ABA translocation within mtabcg20 embryos was impaired. Additionally, the mtabcg20 produced fewer lateral roots and formed more nodules compared with wild-type plants in conditions mimicking drought stress. Heterologous expression in Arabidopsis thaliana provided evidence that MtABCG20 is a plasma membrane protein that is likely to form homodimers. Moreover, export of ABA from Nicotiana tabacum BY2 cells expressing MtABCG20 was faster than in the BY2 without MtABCG20. Our results have implications both in legume crop research and determination of the fundamental molecular processes involved in drought response and germination.

ABA-Induced Stomatal Closure Involves ALMT4, a Phosphorylation-Dependent Vacuolar Anion Channel of Arabidopsis.

Stomatal pores are formed between a pair of guard cells and allow plant uptake of CO2 and water evaporation. Their aperture depends on changes in osmolyte concentration of guard cell vacuoles, specifically of K+ and Mal2- Efflux of Mal2- from the vacuole is required for stomatal closure; however, it is not clear how the anion is released. Here, we report the identification of ALMT4 (ALUMINUM ACTIVATED MALATE TRANSPORTER4) as an Arabidopsis thaliana ion channel that can mediate Mal2- release from the vacuole and is required for stomatal closure in response to abscisic acid (ABA). Knockout mutants showed impaired stomatal closure in response to the drought stress hormone ABA and increased whole-plant wilting in response to drought and ABA. Electrophysiological data show that ALMT4 can mediate Mal2- efflux and that the channel activity is dependent on a phosphorylatable C-terminal serine. Dephosphomimetic mutants of ALMT4 S382 showed increased channel activity and Mal2- efflux. Reconstituting the active channel in almt4 mutants impaired growth and stomatal opening. Phosphomimetic mutants were electrically inactive and phenocopied the almt4 mutants. Surprisingly, S382 can be phosphorylated by mitogen-activated protein kinases in vitro. In brief, ALMT4 likely mediates Mal2- efflux during ABA-induced stomatal closure and its activity depends on phosphorylation.

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.

Arabidopsis ABCG34 contributes to defense against necrotrophic pathogens by mediating the secretion of camalexin.

Plant pathogens cause huge yield losses. Plant defense often depends on toxic secondary metabolites that inhibit pathogen growth. Because most secondary metabolites are also toxic to the plant, specific transporters are needed to deliver them to the pathogens. To identify the transporters that function in plant defense, we screened Arabidopsis thaliana mutants of full-size ABCG transporters for hypersensitivity to sclareol, an antifungal compound. We found that atabcg34 mutants were hypersensitive to sclareol and to the necrotrophic fungi Alternaria brassicicola and Botrytis cinereaAtABCG34 expression was induced by Abrassicicola inoculation as well as by methyl-jasmonate, a defense-related phytohormone, and AtABCG34 was polarly localized at the external face of the plasma membrane of epidermal cells of leaves and roots. atabcg34 mutants secreted less camalexin, a major phytoalexin in Athaliana, whereas plants overexpressing AtABCG34 secreted more camalexin to the leaf surface and were more resistant to the pathogen. When treated with exogenous camalexin, atabcg34 mutants exhibited hypersensitivity, whereas BY2 cells expressing AtABCG34 exhibited improved resistance. Analyses of natural Arabidopsis accessions revealed that AtABCG34 contributes to the disease resistance in naturally occurring genetic variants, albeit to a small extent. Together, our data suggest that AtABCG34 mediates camalexin secretion to the leaf surface and thereby prevents Abrassicicola infection.

The wheat ABC transporter Lr34 modifies the lipid environment at the plasma membrane.

Phospholipids (PLs) are emerging as important factors that initiate signal transduction cascades at the plasma membrane. Their distribution within biological membranes is tightly regulated, e.g. by ATP-binding cassette (ABC) transporters, which preferably translocate PLs from the cytoplasmic to the exoplasmic membrane leaflet and are therefore called PL-floppases. Here, we demonstrate that a plant ABC transporter, Lr34 from wheat (Triticum aestivum), is involved in plasma membrane remodeling characterized by an intracellular accumulation of phosphatidic acid and enhanced outward translocation of phosphatidylserine. In addition, the content of phosphatidylinositol 4,5-bisphosphate in the cytoplasmic leaflet of the plasma membrane was reduced in the presence of the ABC transporter. When heterologously expressed in Saccharomyces cerevisiae, Lr34 promoted oil body formation in a mutant defective in PL-transfer in the secretory pathway. Our results suggest that PL redistribution by Lr34 potentially affects the membrane-bound proteome and contributes to the previously reported stimuli-independent activation of biotic and abiotic stress responses and neutral lipid accumulation in transgenic Lr34-expressing barley plants.

Purification and functional characterization of the vacuolar malate transporter tDT from Arabidopsis.

The exact transport characteristics of the vacuolar dicarboxylate transporter tDT from Arabidopsis are elusive. To overcome this limitation, we combined a range of experimental approaches comprising generation/analysis of tDT overexpressors, 13CO2 feeding and quantification of 13C enrichment, functional characterization of tDT in proteoliposomes, and electrophysiological studies on vacuoles. tdt knockout plants showed decreased malate and increased citrate concentrations in leaves during the diurnal light-dark rhythm and after onset of drought, when compared with wildtypes. Interestingly, under the latter two conditions, tDT overexpressors exhibited malate and citrate levels opposite to tdt knockout plants. Highly purified tDT protein transports malate and citrate in a 1:1 antiport mode. The apparent affinity for malate decreased with decreasing pH, whereas citrate affinity increased. This observation indicates that tDT exhibits a preference for dianion substrates, which is supported by electrophysiological analysis on intact vacuoles. tDT also accepts fumarate and succinate as substrates, but not α-ketoglutarate, gluconate, sulfate, or phosphate. Taking tDT as an example, we demonstrated that it is possible to reconstitute a vacuolar metabolite transporter functionally in proteoliposomes. The displayed, so far unknown counterexchange properties of tDT now explain the frequently observed reciprocal concentration changes of malate and citrate in leaves from various plant species. tDT from Arabidopsis is the first member of the well-known and widely present SLC13 group of carrier proteins, exhibiting an antiport mode of transport.

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.

Vacuolar Transporters - Companions on a Longtime Journey.

Biochemical and electrophysiological studies on plant vacuolar transporters became feasible in the late 1970s and early 1980s, when methods to isolate large quantities of intact vacuoles and purified vacuolar membrane vesicles were established. However, with the exception of the H+-ATPase and H+-PPase, which could be followed due to their hydrolytic activities, attempts to purify tonoplast transporters were for a long time not successful. Heterologous complementation, T-DNA insertion mutants, and later proteomic studies allowed the next steps, starting from the 1990s. Nowadays, our knowledge about vacuolar transporters has increased greatly. Nevertheless, there are several transporters of central importance that have still to be identified at the molecular level or have even not been characterized biochemically. Furthermore, our knowledge about regulation of the vacuolar transporters is very limited, and much work is needed to get a holistic view about the interplay of the vacuolar transportome. The huge amount of information generated during the last 35 years cannot be summarized in such a review. Therefore, I decided to concentrate on some aspects where we were involved during my research on vacuolar transporters, for some our laboratories contributed more, while others contributed less.

The Vacuolar Transportome of Plant Specialized Metabolites.

The plant vacuole is a cellular compartment that is essential to plant development and growth. Often plant vacuoles accumulate specialized metabolites, also called secondary metabolites, which constitute functionally and chemically diverse compounds that exert in planta many essential functions and improve the plant's fitness. These metabolites provide, for example, chemical defense against herbivorous and pathogens or chemical attractants (color and fragrance) to attract pollinators. The chemical composition of the vacuole is dynamic, and is altered during development and as a response to environmental changes. To some extent these alterations rely on vacuolar transporters, which import and export compounds into and out of the vacuole, respectively. During the past decade, significant progress was made in the identification and functional characterization of the transporters implicated in many aspects of plant specialized metabolism. Still, deciphering the molecular players underlying such processes remains a challenge for the future. In this review, we present a comprehensive summary of the most recent achievements in this field.

Vacuolar Transporters for Cadmium and Arsenic in Plants and their Applications in Phytoremediation and Crop Development.

Soil contamination by heavy metals and metalloids such as cadmium (Cd) and arsenic (As) poses a major threat to the environment and to human health. Vacuolar sequestration is one of the main mechanisms by which plants control toxic materials including Cd and As. Understanding the mechanisms of heavy metal tolerance and accumulation can be useful for both phytoremediation and safe crop development. In this review, we summarize recent advances in deciphering the molecular mechanisms underlying vacuolar sequestration of Cd and As, and discuss potential biotechnological applications of this knowledge and efforts towards attaining these goals.

Engineering rice with lower grain arsenic.

Arsenic (As) is a poisonous element that causes severe skin lesions and cancer in humans. Rice (Oryza sativa L.) is a major dietary source of As in humans who consume this cereal as a staple food. We hypothesized that increasing As vacuolar sequestration would inhibit its translocation into the grain and reduce the amount of As entering the food chain. We developed transgenic rice plants expressing two different vacuolar As sequestration genes, ScYCF1 and OsABCC1, under the control of the RCc3 promoter in the root cortical and internode phloem cells, along with a bacterial γ-glutamylcysteine synthetase driven by the maize UBI promoter. The transgenic rice plants exhibited reduced root-to-shoot and internode-to-grain As translocation, resulting in a 70% reduction in As accumulation in the brown rice without jeopardizing agronomic traits. This technology could be used to reduce As intake, particularly in populations of South East Asia suffering from As toxicity and thereby improve human health.

Changes in the allocation of endogenous strigolactone improve plant biomass production on phosphate-poor soils.

Strigolactones (SLs) are carotenoid-derived phytohormones shaping plant architecture and inducing the symbiosis with endomycorrhizal fungi. In Petunia hybrida, SL transport within the plant and towards the rhizosphere is driven by the ABCG-class protein PDR1. PDR1 expression is regulated by phytohormones and by the soil phosphate abundance, and thus SL transport integrates plant development with nutrient conditions. We overexpressed PDR1 (PDR1 OE) to investigate whether increased endogenous SL transport is sufficient to improve plant nutrition and productivity. Phosphorus quantification and nondestructive X-ray computed tomography were applied. Morphological and gene expression changes were quantified at cellular and whole tissue levels via time-lapse microscopy and quantitative PCR. PDR1 OE significantly enhanced phosphate uptake and plant biomass production on phosphate-poor soils. PDR1 OE plants showed increased lateral root formation, extended root hair elongation, faster mycorrhization and reduced leaf senescence. PDR1 overexpression allowed considerable SL biosynthesis by releasing SL biosynthetic genes from an SL-dependent negative feedback. The increased endogenous SL transport/biosynthesis in PDR1 OE plants is a powerful tool to improve plant growth on phosphate-poor soils. We propose PDR1 as an as yet unexplored trait to be investigated for crop production. The overexpression of PDR1 is a valuable strategy to investigate SL functions and transport routes.

A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching.

Strigolactones were originally identified as stimulators of the germination of root-parasitic weeds that pose a serious threat to resource-limited agriculture. They are mostly exuded from roots and function as signalling compounds in the initiation of arbuscular mycorrhizae, which are plant-fungus symbionts with a global effect on carbon and phosphate cycling. Recently, strigolactones were established to be phytohormones that regulate plant shoot architecture by inhibiting the outgrowth of axillary buds. Despite their importance, it is not known how strigolactones are transported. ATP-binding cassette (ABC) transporters, however, are known to have functions in phytohormone translocation. Here we show that the Petunia hybrida ABC transporter PDR1 has a key role in regulating the development of arbuscular mycorrhizae and axillary branches, by functioning as a cellular strigolactone exporter. P. hybrida pdr1 mutants are defective in strigolactone exudation from their roots, resulting in reduced symbiotic interactions. Above ground, pdr1 mutants have an enhanced branching phenotype, which is indicative of impaired strigolactone allocation. Overexpression of Petunia axillaris PDR1 in Arabidopsis thaliana results in increased tolerance to high concentrations of a synthetic strigolactone, consistent with increased export of strigolactones from the roots. PDR1 is the first known component in strigolactone transport, providing new opportunities for investigating and manipulating strigolactone-dependent processes.

Plant hormone transporters: what we know and what we would like to know.

Hormone transporters are crucial for plant hormone action, which is underlined by severe developmental and physiological impacts caused by their loss-of-function mutations. Here, we summarize recent knowledge on the individual roles of plant hormone transporters in local and long-distance transport. Our inventory reveals that many hormones are transported by members of distinct transporter classes, with an apparent dominance of the ATP-binding cassette (ABC) family and of the Nitrate transport1/Peptide transporter family (NPF). The current need to explore further hormone transporter regulation, their functional interaction, transport directionalities, and substrate specificities is briefly reviewed.