Differential regulation of serine acetyltransferase is involved in nickel hyperaccumulation in Thlaspi goesingense

When growing in its native habitat, Thlaspi goesingense can hyperaccumulate 1.2% of its shoot dry weight as nickel. We reported previously that both constitutively elevated activity of serine acetyltransferase (SAT) and concentration of glutathione (GSH) are involved in the ability of T. goesingense to tolerate nickel. A feature of SAT is its feedback inhibition by L-cysteine. To understand the role of this regulation of SAT by Cys on GSH-mediated nickel tolerance in T. goesingense, we characterized the enzymatic properties of SATs from T. goesingense. We demonstrate that all three isoforms of SAT in T. goesingense are insensitive to inhibition by Cys. Further, two amino acids (proline and alanine) in the C-terminal region of the cytosolic SAT (SAT-c) from T. goesingense are responsible for converting the enzyme from a Cys-sensitive to a Cys-insensitive form. Furthermore, the Cys-insensitive isoform of SAT-c confers elevated resistance to nickel when expressed in Escherichia coli and Arabidopsis thaliana, supporting a role for altered regulation of SAT by Cys in nickel tolerance in T. goesingense.     

Loss-of-function of Constitutive Expresser of Pathogenesis Related Genes5 affects potassium homeostasis in Arabidopsis thaliana

Here, we demonstrate that the reduction in leaf K(+) observed in a mutant previously identified in an ionomic screen of fast neutron mutagenized Arabidopsis thaliana is caused by a loss-of-function allele of CPR5, which we name cpr5-3. This observation establishes low leaf K(+) as a new phenotype for loss-of-function alleles of CPR5. We investigate the factors affecting this low leaf K(+) in cpr5 using double mutants defective in salicylic acid (SA) and jasmonic acid (JA) signalling, and by gene expression analysis of various channels and transporters. Reciprocal grafting between cpr5 and Col-0 was used to determine the relative importance of the shoot and root in causing the low leaf K(+) phenotype of cpr5. Our data show that loss-of-function of CPR5 in shoots primarily determines the low leaf K(+) phenotype of cpr5, though the roots also contribute to a lesser degree. The low leaf K(+) phenotype of cpr5 is independent of the elevated SA and JA known to occur in cpr5. In cpr5 expression of genes encoding various Cyclic Nucleotide Gated Channels (CNGCs) are uniquely elevated in leaves. Further, expression of HAK5, encoding the high affinity K(+) uptake transporter, is reduced in roots of cpr5 grown with high or low K(+) supply. We suggest a model in which low leaf K(+) in cpr5 is driven primarily by enhanced shoot-to-root K(+) export caused by a constitutive activation of the expression of various CNGCs. This activation may enhance K(+) efflux, either indirectly via enhanced cytosolic Ca(2+) and/or directly by increased K(+) transport activity. Enhanced shoot-to-root K(+) export may also cause the reduced expression of HAK5 observed in roots of cpr5, leading to a reduction in uptake of K(+). All ionomic data presented is publically available at www.ionomicshub.org.     

Altered germination and subcellular localization patterns for PUB44/SAUL1 in response to stress and phytohormone treatments

Background

In plants, the ubiquitin-proteasome system is emerging as a significant regulatory system throughout the plant lifecycle. The ubiquitination of a target protein requires the sequential actions of the E1, E2 and E3 enzymes, with the latter E3 enzyme conferring target selection in this process. There are a large number of predicted E3 enzymes in plant genomes, and very little is known about the functions of many of these predicted genes. Here we report here an analysis of two closely-related members of the Arabidopsis Plant U-box (PUB) family of E3 ubiquitin ligases, PUB43 and PUB44.

Principal Findings

Homozygous pub44/pub44 mutant seedlings were found displayed a seedling lethal phenotype and this corresponded with widespread cell death lesions throughout the cotyledons and roots. Interestingly, heterozygous PUB44/pub44 seedlings were wild-type in appearance yet displayed intermediate levels of cell death lesions in comparison to pub44/pub44 seedlings. In contrast, homozygous pub43/pub43 mutants were viable and did not show any signs of cell death despite the PUB43 gene being more highly expressed than PUB44. The PUB44 mutants are not classical lesion mimic mutants as they did not have increased resistance to plant pathogens. We also observed increased germination rates in mutant seeds for both PUB44 and PUB43 under inhibitory concentrations of abscisic acid. Finally, the subcellular localization of PUB44 was investigated with transient expression assays in BY-2 cells. Under varying conditions, PUB44 was observed to be localized to the cytoplasm, plasma membrane, or nucleus.

Conclusions

Based on mutant plant analyses, the Arabidopsis PUB43 and PUB44 genes are proposed to function during seed germination and early seedling growth. Given PUB44''s ability to shuttle from the nucleus to the plasma membrane, PUB44 may be active in different subcellular compartments as part of these biological functions.     

Regulation of Alr1 Mg transporter activity by intracellular magnesium

Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni(2+), Mn(2+), Zn(2+) and Co(2+). Using Ni(2+) uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms.     

In vitro studies of haemolysis by some staphylococci grown in chemically defined media

Staphylococcus aureus (five strains) and Staph. epidermidis (one strain) have been evaluated for comparative growth and haemolysin titre in both brain heart infusion (BHI) and in developed, nutritionally adequate, chemically defined media (CDMs) varying only in amino acid composition. The ability to show a particular haemolytic profile was strain-dependent and the haemolytic titre (HU₅₀/ml) was both strain- and medium-dependent. Highest titres of both alpha and beta type haemolysins were obtained in BHI. Maximum titres were in general detected in the late exponential phase in both CDMs and BHI. Titres declined during the stationary phase in CDMs. Staphylococcus epidermidis produced a delta-type haemolysis profile on BHI-based blood agars, but only rabbit blood was sensitive in agars based on a developed, chemically defined medium (CDM/A; 13 amino acids) in which all six staphylococci grew. The addition of yeast extract to CDM/A increased alpha haemolysin titre, but suppressed beta haemolysin formation; beta haemolysin was, however, detected in yeast extract/phosphate-buffered saline. Strain Wood 46 degraded haemoglobin, but only in (initially) whole blood; red blood cell-free haemoglobin-rich plates (BHI) were unaffected during growth. A novel haemolytic profile is described for Staph. aureus NCTC 8532 growing on blood agars based on CDM/A and may relate to the production of methaemoglobin during haemolysis.     

Slc39a1 to 3 (subfamily II) Zip genes in mice have unique cell-specific functions during adaptation to zinc deficiency

Subfamily II of the solute carrier (Slc)39a family contains three highly conserved members (ZIPs 1-3) that share a 12-amino acid signature sequence present in the putative fourth transmembrane domain and function as zinc transporters in transfected cells. The physiological significance of this genetic redundancy is unknown. Here we report that the complete elimination of all three of these Zip genes, by targeted mutagenesis and crossbreeding mice, causes no overt phenotypic effect. When mice were fed a zinc-adequate diet, several indicators of zinc status were indistinguishable between wild-type and triple-knockout mice, including embryonic morphogenesis and growth, alkaline phosphatase activity in the embryo, ZIP4 protein in the visceral yolk sac, and initial rates (30 min) of accumulation/retention of (67)Zn in liver and pancreas. When mice were fed a zinc-deficient diet, embryonic membrane-bound alkaline phosphatase activity was reduced to a much greater extent, and 80% of the embryos of the triple-knockout mice developed abnormally compared with 12% of the embryos of wild-type mice. During zinc deficiency, the accumulation/retention (3 h) of (67)Zn in the liver and pancreas of weanlings was significantly impaired in the triple-knockout mice compared with wild-type mice. Thus none of these three mammalian Zip genes apparently plays a critical role in zinc homeostasis when zinc is replete, but they play important, noncompensatory roles when this metal is deficient.