Cytokinin Determines Thiol-Mediated Arsenic Tolerance and Accumulation.

The presence of arsenic in soil and water is a constant threat to plant growth in many regions of the world. Phytohormones act in the integration of growth control and stress response, but their role in plant responses to arsenic remains to be elucidated. Here, we show that arsenate [As(V)], the most prevalent arsenic chemical species in nature, causes severe depletion of endogenous cytokinins (CKs) in the model plant Arabidopsis (Arabidopsis thaliana). We found that CK signaling mutants and transgenic plants with reduced endogenous CK levels showed an As(V)-tolerant phenotype. Our data indicate that in CK-depleted plants exposed to As(V), transcript levels of As(V)/phosphate-transporters were similar or even higher than in wild-type plants. In contrast, CK depletion provoked the coordinated activation of As(V) tolerance mechanisms, leading to the accumulation of thiol compounds such as phytochelatins and glutathione, which are essential for arsenic sequestration. Transgenic CK-deficient Arabidopsis and tobacco lines show a marked increase in arsenic accumulation. Our findings indicate that CK is an important regulatory factor in plant adaptation to arsenic stress.

Assessment of homozygosity in transgenic plants using selectable markers.

Production of homozygous transgenic plants is a prerequisite for the phenotypic analysis and/or for the commercial release of transgenic plants for cultivation. Here we present a simple protocol for the selection of homozygous transgenics using antibiotics as a selectable marker. The protocol has been used to select homozygous rice transgenic plants using hygromycin antibiotic. However, the described protocol can be used for selction of homozygous in any transgenic plants using a appropriate selectable marker. For complete details on the use and execution of this protocol, please refer to Passricha et al. (2016).1.

An exopolysaccharide-producing novel Agrobacterium pusense strain JAS1 isolated from snake plant enhances plant growth and soil water retention.

A peculiar bacterial growth was very often noticed in leaf-initiated tissue cultures of Sansevieria trifasciata, a succulent belonging to the Asparagaceae family. The isolate left trails of some highly viscous material on the walls of the suspension vessels or developed a thick overlay on semisolid media without adversities in plant growth. FTIR identified this substance to be an extracellular polysaccharide. Various morphological, biochemical tests, and molecular analyses using 16S rRNA, atpD, and recA genes characterized this isolate JAS1 as a novel strain of Agrobacterium pusense. Its mucoidal growth over Murashige and Skoog media yielded enormous exopolysaccharide (7252 mg l-1), while in nutrient agar it only developed fast-growing swarms. As a qualifying plant growth-promoting bacteria, it produces significant indole-3-acetic acid (86.95 mg l-1), gibberellic acid (172.98 mg l-1), ammonia (42.66 µmol ml-1). Besides, it produces siderophores, 1-aminocyclopropane-1-carboxylic acid deaminase, fixes nitrogen, forms biofilms, and productively solubilizes soil inorganic phosphates, and zinc. Under various treatments with JAS1, wheat and chickpea resulted in significantly enhanced shoot and root growth parameters. PGP effects of JAS1 positively enhanced plants' physiological growth parameters reflecting significant increments in overall chlorophyll, carotenoids, proline, phenols, flavonoids, and sugar contents. In addition, the isolated strain maintained both plant and soil health under an intermittent soil drying regime, probably by both its PGP and EPS production attributes, respectively.

Transfer of stabilising mutations between different secondary active transporter families.

Integral membrane transporters play essential roles in the movement of substrates across biological membranes. One approach to produce transporters suitable for structural studies is to introduce mutations that reduce conformational flexibility and increase stability. However, it can be difficult to predict which mutations will result in a more stable protein. Previously, we stabilised the uric acid-xanthine transporter, UapA, a member of the SLC23 family, through introduction of a single-point mutation, G411V, trapping the protein in the inward-facing conformation. Here, we attempted to stabilise the structurally related BOR1 transporter from Arabidopsis thaliana, a member of the SLC4 family, by introducing the equivalent substitution. We identified possible residues, P362 and M363, in AtBOR1, likely to be equivalent to the G411 of UapA, and generated four mutants, P362V or L and M363F or Y. Stability analysis using heated Fluorescent Size Exclusion Chromatography indicated that the M363F/Y mutants were more stable than the WT AtBOR1 and P362V/L mutants. Furthermore, functional complementation analysis revealed that the M363F/Y mutants exhibited reduced transport activity compared to the P362V/L and WT proteins. Purification and crystallisation of the M363F/Y proteins yielded crystals that diffracted better than WT (5.5 vs 7 Å). We hypothesise that the increased bulk of the F and Y substitutions limits the ability of the protein to undergo the conformational rearrangements associated with transport. These proteins represent a basis for future studies on AtBOR1.

Structural and functional insights into the mechanism of action of plant borate transporters.

Boron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are homologues of the human SLC4 family of transporters, which includes well studied mammalian transporters such as the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) variants based (i) on known disease causing mutations of hAE1 (S466R, A500R) and (ii) a loss of function mutation (D311A) identified in the yeast BOR protein, ScBOR1p. The AtBOR1 variants express in yeast and localise to the plasma membrane, although both S466R and A500R exhibit lower expression than the WT AtBOR1 and D311A. The D311A, S466R and A500R mutations result in a loss of borate efflux activity in a yeast bor1p knockout strain. A. thaliana plants containing these three individual mutations exhibit substantially decreased growth phenotypes in soil under conditions of low boron. These data confirm an important role for D311 in the function of the protein and show that mutations equivalent to disease-causing mutations in hAE1 have major effects in AtBOR1. We also obtained a low resolution cryo-EM structure of a BOR protein from Oryza sativa, OsBOR3, lacking the 30 C-terminal amino acid residues. This structure confirms the gate and core domain organisation previously observed for related proteins, and is strongly suggestive of an inward facing conformation.

Arsenite provides a selective signal that coordinates arsenate uptake and detoxification through the regulation of PHR1 stability in Arabidopsis.

In nature, plants acquire nutrients from soils to sustain growth, and at the same time, they need to avoid the uptake of toxic compounds and/or possess tolerance systems to cope with them. This is particularly challenging when the toxic compound and the nutrient are chemically similar, as in the case of phosphate and arsenate. In this study, we demonstrated that regulatory elements of the phosphate starvation response (PSR) coordinate the arsenate detoxification machinery in the cell. We showed that arsenate repression of the phosphate transporter PHT1;1 is associated with the degradation of the PSR master regulator PHR1. Once arsenic is sequestered into the vacuole, PHR1 stability is restored and PHT1;1 expression is recovered. Furthermore, we identified an arsenite responsive SKP1-like protein and a PHR1 interactor F-box (PHIF1) as constituents of the SCF complex responsible for PHR1 degradation.We found that arsenite, the form to which arsenate is reduced for compartmentalization in vacuoles, represses PHT1;1 expression, providing a highly selective signal versus phosphate to control PHT1;1 expression in response to arsenate. Collectively, our results provide molecular insights into a sensing mechanism that regulates arsenate/phosphate uptake depending on the plant's detoxification capacity.

Determination of Boron Content Using a Simple and Rapid Miniaturized Curcumin Assay.

To determine boron quantity in soil, water and biological samples, several protocols are available. Colorimetric assays are the simplest and cheapest methods which can be used to determine boron concentration. However, published protocols do not give straightforward guidance for beginners to adopt these protocols for routine use in the laboratory. Based on a previously published available procedure, we present a detailed and modified version of a curcumin based colorimetric protocol to determine boron concentration extracted from any sample. Our modified protocol is able to determine up to 0.2 nmole of Boron in a sample volume of 300 µl.