Cation diffusion facilitator proteins of Beta vulgaris reveal diversity of metal handling in dicotyledons.

Manganese (Mn), iron (Fe), and zinc (Zn) are essential for diverse processes in plants, but their availability is often limiting or excessive. Cation diffusion facilitator (CDF) proteins have been implicated in the allocation of those metals in plants, whereby most of our mechanistic understanding has been obtained in Arabidopsis. It is unclear to what extent this can be generalized to other dicots. We characterized all CDFs/metal tolerance proteins of sugar beet (Beta vulgaris spp. vulgaris), which is phylogenetically distant from Arabidopsis. Analysis of subcellular localization, substrate selectivities, and transcriptional regulation upon exposure to metal deficiencies and toxicities revealed unexpected deviations from their Arabidopsis counterparts. Localization and selectivity of some members were modulated by alternative splicing. Notably, unlike in Arabidopsis, Mn- and Zn-sequestrating members were not induced in Fe-deficient roots, pointing to differences in the Fe acquisition machinery. This was supported by low Zn and Mn accumulation under Fe deficiency and a strikingly increased Fe accumulation under Mn and Zn excess, coinciding with an induction of BvIRT1. High Zn load caused a massive upregulation of Zn-BvMTPs. The results suggest that the employment of the CDF toolbox is highly diverse amongst dicots, which questions the general applicability of metal homeostasis models derived from Arabidopsis.

AtDTX25, a member of the multidrug and toxic compound extrusion family, is a vacuolar ascorbate transporter that controls intracellular iron cycling in Arabidopsis.

Iron (Fe) is an essential element, its transport is regulated by the cell redox balance. In seeds, Fe enters the embryo as Fe2+ and is stored in vacuoles as Fe3+ . Through its ferric reduction activity, ascorbate plays a major role in Fe redox state and therefore Fe transport within the seed. We searched for ascorbate membrane transporters responsible for controlling Fe reduction by screening the yeast ferric reductase-deficient fre1 strain and isolated AtDTX25, a member of the Multidrug And Toxic compound Extrusion (MATE) family. AtDTX25 was shown to mediate ascorbate efflux when expressed in yeast and Xenopus oocytes, in a pH-dependent manner. In planta, AtDTX25 is highly expressed during germination and encodes a vacuolar membrane protein. Isolated vacuoles from AtDTX25-1 knockout mutant contained less ascorbate and more Fe than wild-type (WT), and mutant seedlings were highly sensitive to Fe deficiency. Iron imaging further showed that the remobilisation of Fe from vacuoles was highly impaired in mutant seedlings. Taken together, our results established AtDTX25 as a vacuolar ascorbate transporter, required during germination to promote the reduction of the pool of stored Fe3+ and its remobilisation to feed the developing seedling.

Developing a Sufficient Protocol for the Enhancement of α-Glucosidase Inhibitory Activity by Urena lobata L. Aeroponic Hairy Roots Using Exogenous Factors, a Precursor, and an Elicitor.

Aeroponics is considered as a potential method for the culture of herbal plants due to the high growth rate, quantity and quality enhancement of secondary metabolites, and substantial environmental progress associated with this method. The aim of this study was to develop a sufficient protocol for successful Urena lobata hairy root induction by Agrobacterium rhizogenes ATCC 15834, using a precursor and elicitor to enhance α-glucosidase inhibitory activity (GIA) of aeroponic hairy roots (AHRs) in greenhouse conditions. In this study, we found that the optimized procedure (10 min, Woody plant medium (WPM), 1/25 salt strength) had an outstanding effect with a reduction in the rooting time (RT), promotion of the rooting rate (RR), and increase in the fresh weight (FW) and dry weight (DW) compared with the original procedure (30 min, Murashige and Skoog (MS) medium, 1/25 salt strength) after 30 days of culture. The highest DW, GIA, flavonoid (FLA) and phenolic (PHEL) contents were observed for individual addition of 10 mM phenylalanine (PA) or 50 mM chitosan (CS) in the late exponential phase (eighth week) with 15 days of elicitation compared to the control AHRs. However, individual treatment was less effective than the combination of the two. Positive correlations among the GIA, FLA and PHEL indicate that AHRs accumulated phenolic compounds, leading to an increase in the GIA by a synergistic effect. In conclusion, the culture of Urena lobata AHRs with PA and CS is an efficient procedure to produce GIA material in greenhouse conditions.

Pseudomonas PS01 Isolated from Maize Rhizosphere Alters Root System Architecture and Promotes Plant Growth.

The objectives of this study were to evaluate the plant growth promoting effects on Arabidopsis by Pseudomonas sp. strains associated with rhizosphere of crop plants grown in Mekong Delta, Vietnam. Out of all the screened isolates, Pseudomonas PS01 isolated from maize rhizosphere showed the most prominent plant growth promoting effects on Arabidopsis and maize (Zea mays). We also found that PS01 altered root system architecture (RSA). The full genome of PS01 was resolved using high-throughput sequencing. Phylogenetic analysis identified PS01 as a member of the Pseudomonas putida subclade, which is closely related to Pseudomonas taiwanensis.. PS01 genome size is 5.3 Mb, assembled in 71 scaffolds comprising of 4820 putative coding sequence. PS01 encodes genes for the indole-3-acetic acid (IAA), acetoin and 2,3-butanediol biosynthesis pathways. PS01 promoted the growth of Arabidopsis and altered the root system architecture by inhibiting primary root elongation and promoting lateral root and root hair formation. By employing gene expression analysis, genetic screening and pharmacological approaches, we suggested that the plant-growth promoting effects of PS01 and the alteration of RSA might be independent of bacterial auxin and could be caused by a combination of different diffusible compounds and volatile organic compounds (VOCs). Taken together, our results suggest that PS01 is a potential candidate to be used as bio-fertilizer agent for enhancing plant growth.

Plant growth-promoting rhizobacterium Pseudomonas PS01 induces salt tolerance in Arabidopsis thaliana.

OBJECTIVES: Plant growth-promoting rhizobacteria (PGPR) may contribute to sustainable crop production by improving plant growth and/or plant tolerance to abiotic stresses. Soil salinity, which limits the productivity of crop plants, is one of the major concerns of modern agriculture, especially in countries heavily affected by climate change as Vietnam. Currently, only a few reports have studied local PGPR isolated in Vietnam, particular Pseudomonas. Therefore, our study aimed to isolate and identify a region-specific Pseudomonas strain and evaluate the effects of this strain on germination, growth promotion and gene expression of Arabidopsis thaliana under salt stress. RESULTS: The Pseudomonas named PS01 was isolated from maize rhizosphere collected in Ben Tre province, Vietnam. This strain was identified as a member of the Pseudomonas putida subclade. Pseudomonas PS01 could improve the germination rate of Arabidopsis seeds in 150 mM NaCl. A. thaliana plants inoculated with Pseudomonas PS01 survived under salt stress conditions up to 225 mM NaCl, while all non-inoculated plants were dead above 200 mM NaCl. The transcriptional levels of genes related to stress tolerance showed that only LOX2 was up-regulated, while APX2 and GLYI7 were down-regulated in inoculated plants in comparison to the non-inoculated controls. In turn, RD29A and RD29B did not show any significant changes in their expression profiles.

Chloroplast-localized BICAT proteins shape stromal calcium signals and are required for efficient photosynthesis.

The photosynthetic machinery of plants must be regulated to maximize the efficiency of light reactions and CO2 fixation. Changes in free Ca2+ in the stroma of chloroplasts have been observed at the transition between light and darkness, and also in response to stress stimuli. Such Ca2+ dynamics have been proposed to regulate photosynthetic capacity. However, the molecular mechanisms of Ca2+ fluxes in the chloroplasts have been unknown. By employing a Ca2+ reporter-based approach, we identified two chloroplast-localized Ca2+ transporters in Arabidopsis thaliana, BICAT1 and BICAT2, that determine the amplitude of the darkness-induced Ca2+ signal in the chloroplast stroma. BICAT2 mediated Ca2+ uptake across the chloroplast envelope, and its knockout mutation strongly dampened the dark-induced [Ca2+ ]stroma signal. Conversely, this Ca2+ transient was increased in knockout mutants of BICAT1, which transports Ca2+ into the thylakoid lumen. Knockout mutation of BICAT2 caused severe defects in chloroplast morphology, pigmentation and photosynthetic light reactions, rendering bicat2 mutants barely viable under autotrophic growth conditions, while bicat1 mutants were less affected. These results show that BICAT transporters play a role in chloroplast Ca2+ homeostasis. They are also involved in the regulation of photosynthesis and plant productivity. Further work will be required to reveal whether the effect on photosynthesis is a direct result of their role as Ca2+ transporters.

Ascorbate efflux as a new strategy for iron reduction and transport in plants.

Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)3Cit2Mal2, Fe(III)3Cit3Mal1, Fe(III)Cit2). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled (55)Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.