Surface wax esters contribute to drought tolerance in Arabidopsis

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.     

Inhibition by light of CO2 evolution from dark respiration: Comparison of two gas exchange methods

Two approaches to determine the fraction (μ) of mitochondrial respiration sustained during illumination by measuring CO₂ gas exchange are compared. In single leaves, the respiration rate in the light (`day respiration' rate R<sub>d</sub>) is determined as the ordinate of the intersection point of A–c<sub>i</sub> curves at various photon flux densities and compared with the CO<sub>2</sub> evolution rate in darkness (`night respiration' rate R_n). Alternatively, using leaves with varying values of CO₂ compensation concentration (Γ), intracellular resistance (r_i) and R_n, an average number for μ can be derived from the linear regression between Γ and the product r_iċR_n. Both methods also result in a number c* for that intercellular CO₂ concentration at which net CO₂ uptake rate is equal to –R_d. c* is an approximate value of the photocompensation point Γ* (Γ in the absence of mitochondrial respiration), which is related to the CO₂/O₂ specificity factor of Rubisco S_c/o. The presuppositions and limitations for application of both approaches are discussed. In leaves of Nicotiana tabacum, at 22 °C, single leaf measurements resulted in mean values of μ = 0.71 and c_* = 34 μmol mol⁻¹. At the photosynthetically active photon flux density of 960 μmol quanta m⁻² s⁻¹, nearly the same numbers were derived from the linear relationship between Γ and r_iċR_n. c* and R_d determined by single leaf measurements varied between 31 and 41 μmol mol⁻¹ and between 0.37 and 1.22 μmol m⁻² s⁻¹, respectively. A highly significant negative correlation between c* and R_d was found. From the regression equation we obtained estimates for Γ* (39 μmol mol⁻¹), S_c/o (96.5 mol mol⁻¹) and the mesophyll CO₂ transfer resistance (7.0 mol⁻¹ m² s). This revised version was published online in June 2006 with corrections to the Cover Date.     

Functional characterization of the atypical Hsp70 subunit of yeast ribosome-associated complex

Eukaryotic ribosomes carry a stable chaperone complex termed ribosome-associated complex consisting of the J-domain protein Zuo1 and the Hsp70 Ssz1. Zuo1 and Ssz1 together with the Hsp70 homolog Ssb1/2 form a functional triad involved in translation and early polypeptide folding processes. Strains lacking one of these components display slow growth, cold sensitivity, and defects in translational fidelity. Ssz1 diverges from canonical Hsp70s insofar that neither the ability to hydrolyze ATP nor binding to peptide substrates is essential in vivo. The exact role within the chaperone triad and whether or not Ssz1 can hydrolyze ATP has remained unclear. We now find that Ssz1 is not an ATPase in vitro, and even its ability to bind ATP is dispensable in vivo. Furthermore, Ssz1 function was independent of ribosome-associated complex formation, indicating that Ssz1 is not merely a structural scaffold for Zuo1. Finally, Ssz1 function in vivo was inactivated when both nucleotide binding and Zuo1 interaction via the C-terminal domain were disrupted in the same mutant. The two domains of this protein thus cooperate in a way that allows for severe interference in either but not in both of them.     

Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 Å crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G–ribosome complex.     

Isolation and characterization of <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> and <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis> phytochelatin synthases

The synthesis of phytochelatins (PC) represents a major metal and metalloid detoxification mechanism in various species. PC most likely play a role in the distribution and accumulation of Cd and possibly other metals. However, to date, no studies have investigated the phytochelatin synthase (PCS) genes and their expression in the Cd-hyperaccumulating species. We used functional screens in two yeast species to identify genes expressed by two Cd hyperaccumulators (Arabidopsis halleri and Thlaspi caerulescens) and involved in cellular Cd tolerance. As a result of these screens, PCS genes were identified for both species. PCS1 was in each case the dominating cDNA isolated. The deduced sequences of AhPCS1 and TcPCS1 are very similar to AtPCS1 and their identity is particularly high in the proposed catalytic N-terminal domain. We also identified in A. halleri and T. caerulescens orthologues of AtPCS2 that encode functional PCS. As compared to A. halleri and A. thaliana, T. caerulescens showed the lowest PCS expression. Furthermore, concentrations of PC in Cd-treated roots were the highest in A. thaliana, intermediate in A. halleri and the lowest in T. caerulescens. This mirrors the known capacity of these species to translocate Cd to the shoot, with T. caerulescens being the best translocator. Very low or undetectable concentrations of PC were measured in A. halleri and T. caerulescens shoots, contrary to A. thaliana. These results suggest that extremely efficient alternative Cd sequestration pathways in leaves of Cd hyperaccumulators prevent activation of PC synthase by Cd²⁺ ions.     

Transgenic tobacco plants overexpressing chloroplastic ferredoxin-NADP(H) reductase display normal rates of photosynthesis and increased tolerance to oxidative stress

Ferredoxin-NADP(H) reductase (FNR) catalyzes the last step of photosynthetic electron transport in chloroplasts, driving electrons from reduced ferredoxin to NADP+. This reaction is rate limiting for photosynthesis under a wide range of illumination conditions, as revealed by analysis of plants transformed with an antisense version of the FNR gene. To investigate whether accumulation of this flavoprotein over wild-type levels could improve photosynthetic efficiency and growth, we generated transgenic tobacco (Nicotiana tabacum) plants expressing a pea (Pisum sativum) FNR targeted to chloroplasts. The alien product distributed between the thylakoid membranes and the chloroplast stroma. Transformants grown at 150 or 700 micromol quanta m(-2) s(-1) displayed wild-type phenotypes regardless of FNR content. Thylakoids isolated from plants with a 5-fold FNR increase over the wild type displayed only moderate stimulation (approximately 20%) in the rates of electron transport from water to NADP+. In contrast, when donors of photosystem I were used to drive NADP+ photoreduction, the activity was 3- to 4-fold higher than the wild-type controls. Plants expressing various levels of FNR (from 1- to 3.6-fold over the wild type) failed to show significant differences in CO2 assimilation rates when assayed over a range of light intensities and CO2 concentrations. Transgenic lines exhibited enhanced tolerance to photooxidative damage and redox-cycling herbicides that propagate reactive oxygen species. The results suggest that photosynthetic electron transport has several rate-limiting steps, with FNR catalyzing just one of them.     

Variability of photosynthetic gas exchange parameters, dark respiration, and stomatal numbers in species of Polygonum

Within the genus Polygonum a large variation was found between species with regard to stomatal number, gas phase resistance, intracellular resistance and dark respiration. Interspecific variation in CO₂ compensation concentration and intercellular CO₂ concentration at constant external concentration were comparatively small. Correlations were found between stomatal number and gas phase resistance, stomatal number and Γ, and Γ and the product of dark respiration rate and intracellular resistance. The influence of dark respiration and stomatal number on photosynthetic gas exchange is discussed. It was concluded that dark respiration in light was enhanced by 22% as a mean value in 9 Polygonum species and by 62% in Polygonum lapathifolium .     

Phytochelatin Synthesis Is Essential for the Detoxification of Excess Zinc and Contributes Significantly to the Accumulation of Zinc

The synthesis of phytochelatins (PCs) is essential for the detoxification of nonessential metals and metalloids such as cadmium and arsenic in plants and a variety of other organisms. To our knowledge, no direct evidence for a role of PCs in essential metal homeostasis has been reported to date. Prompted by observations in Schizosaccharomyces pombe and Saccharomyces cerevisiae indicating a contribution of PC synthase expression to Zn²⁺ sequestration, we investigated a known PC-deficient Arabidopsis (Arabidopsis thaliana) mutant, cad1-3, and a newly isolated second strong allele, cad1-6, with respect to zinc (Zn) homeostasis. We found that in a medium with low cation content PC-deficient mutants show pronounced Zn²⁺ hypersensitivity. This phenotype is of comparable strength to the well-documented Cd²⁺ hypersensitivity of cad1 mutants. PC deficiency also results in significant reduction in root Zn accumulation. To be able to sensitively measure PC accumulation, we established an assay using capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry of derivatized extracts. Plants grown under control conditions consistently showed PC2 accumulation. Analysis of plants treated with same-effect concentrations revealed that Zn²⁺-elicited PC2 accumulation in roots reached about 30% of the level of Cd²⁺-elicited PC2 accumulation. We conclude from these data that PC formation is essential for Zn²⁺ tolerance and provides driving force for the accumulation of Zn. This function might also help explain the mysterious occurrence of PC synthase genes throughout the plant kingdom and in a wide range of other organisms.Both essential and nonessential metal ions can be toxic when present in excess. Zinc (Zn) ions, for instance, are used in biological systems as catalytic or structural components in a myriad of proteins (Frausto da Silva and Williams, 2001). In humans, about 10% of genes encode Zn-dependent proteins (Andreini et al., 2006) and it is reasonable to postulate similar numbers for plants. When the Zn-buffering capacity of a cell is exceeded, however, aberrant binding of Zn ions to thiols or other functional groups can occur, which disrupts the function of proteins. Also, Zn ions can displace other essential metal ions from their binding sites (Krämer and Clemens, 2005). Toxicity thresholds for Zn were found to range between 100 and 300 μg g⁻¹ dry weight depending on plant species and physiological state (Marschner, 1995). Ions of the nonessential metal cadmium (Cd) have a high affinity for various functional groups in biological molecules, in particular thiols. When taken up into a cell they can inactivate proteins by uncontrolled binding or cause oxidative stress by depleting glutathione pools (Clemens, 2006a).Because of the potential toxicity of metal ions, all living systems possess mechanisms to tightly regulate the distribution of metal ions and to minimize damage under conditions of excess metal supply (Eide, 2006; Grotz and Guerinot, 2006). Principally, detoxification of excess metal ions is assumed to be achieved by efflux, sequestration, and chelation. For instance, in the past few years evidence has been reported for a contribution of AtMTP1 (At2g46800) to vacuolar sequestration of Zn²⁺ (Kobae et al., 2004; Desbrosses-Fonrouge et al., 2005) and of AtHMA4 (At2g19110) to effluxing Zn²⁺ (Mills et al., 2005). Furthermore, the Arabidopsis halleri ortholog of HMA4 is essential for Zn and Cd hypertolerance (Hanikenne et al., 2008). Loss of ZIF1, a transporter of the major facilitator superfamily, results in Zn²⁺ hypersensitivity in Arabidopsis (Arabidopsis thaliana; Haydon and Cobbett, 2007).The synthesis of phytochelatins (PCs), glutathione-derived metal-binding peptides, represents a major detoxification mechanism for Cd and arsenic (As) ions in various species. More recently, PCs have also been implicated in long-distance transport of Cd in the phloem (Mendoza-Cozatl et al., 2008). Formation of PCs is catalyzed by PC synthases (PCS) and genes encoding this enzyme have been cloned from plants, fungi, and nematodes (Clemens et al., 1999, 2001; Ha et al., 1999; Vatamaniuk et al., 1999, 2001). Mutant lines of Arabidopsis, Schizosaccharomyces pombe, or Caenorhabditis elegans that are deficient in PC synthesis show a severe loss of Cd and As tolerance (Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al., 2001). For other metal ions only minor effects have been reported (Cobbett and Goldsbrough, 2002). Arabidopsis cad1-3 mutant plants, which are defective in AtPCS1, showed about a 2-fold increase in copper (Cu) and mercury sensitivity and no significant increase in Zn sensitivity (Ha et al., 1999). S. pombe PCS-deficient mutants are slightly more Cu²⁺ sensitive than wild-type cells (Clemens et al., 1999). PC-metal complexes have been detected in plant cells only with Cd, silver, Cu, and As (Maitani et al., 1996; Schmöger et al., 2000) even though synthesis of PCs is activated by a wide range of metal ions both in vivo and in vitro (Grill et al., 1987; Vatamaniuk et al., 2000; Oven et al., 2002). Thus, the role of PC synthesis in metal detoxification has so far been seen as being confined to Cd and As (Cobbett and Goldsbrough, 2002). This, however, leaves the question as to why PCS genes are so widespread and why the enzyme is expressed constitutively throughout the plant (Rea et al., 2004). It is not clear how the sporadic need to sequester excess Cd or As ions could have provided the selective pressure to maintain PCS expression throughout the plant kingdom and beyond (Clemens, 2006b). One explanation could be the second enzymatic function of PCS, i.e. breakdown of glutathione conjugates (GS conjugates) to the corresponding β-glutamyl-Cys conjugates (Blum et al., 2007). The catalytic activity of bacterial PCS-like proteins is similar (Harada et al., 2004; Tsuji et al., 2004; Vivares et al., 2005). Another possibility is involvement of PC synthesis in essential metal homeostasis, which has been discussed occasionally (Steffens et al., 1986; Rauser, 1990; Steffens, 1990). As early as 1986, Steffens et al. suggested this based on the observation that small amounts of PCs were detectable in tomato (Solanum lycopersicum) cell cultures in the absence of excess metals. Similarly, Grill et al. reported the induction of PC synthesis by Cu and Zn ions after transfer of cultured cells into fresh medium. The amount of detectable PCs was correlated linearly with the Zn²⁺ concentration in the culture medium and an involvement of PC synthesis in metal homeostasis was proposed (Grill et al., 1987, 1988). However, no genetic evidence for such a role of PCs has been reported to date. Here we present evidence that PC formation indeed contributes significantly to Zn²⁺ detoxification in Arabidopsis and enhances Zn accumulation.