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1.
Epistatic Interactions between Opaque2 Transcriptional Activator and Its Target Gene CyPPDK1 Control Kernel Trait Variation in Maize 总被引:1,自引:0,他引:1
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Domenica Manicacci Letizia Camus-Kulandaivelu Marie Fourmann Chantal Arar Stéphanie Barrault Agnès Rousselet No?l Feminias Luciano Consoli Lisa Francès Valérie Méchin Alain Murigneux Jean-Louis Prioul Alain Charcosset Catherine Damerval 《Plant physiology》2009,150(1):506-520
2.
Relatively little is known about the small subset of peroxisomal proteins with predicted protease activity. Here, we report that the peroxisomal LON2 (At5g47040) protease facilitates matrix protein import into Arabidopsis (Arabidopsis thaliana) peroxisomes. We identified T-DNA insertion alleles disrupted in five of the nine confirmed or predicted peroxisomal proteases and found only two—lon2 and deg15, a mutant defective in the previously described PTS2-processing protease (DEG15/At1g28320)—with phenotypes suggestive of peroxisome metabolism defects. Both lon2 and deg15 mutants were mildly resistant to the inhibitory effects of indole-3-butyric acid (IBA) on root elongation, but only lon2 mutants were resistant to the stimulatory effects of IBA on lateral root production or displayed Suc dependence during seedling growth. lon2 mutants displayed defects in removing the type 2 peroxisome targeting signal (PTS2) from peroxisomal malate dehydrogenase and reduced accumulation of 3-ketoacyl-CoA thiolase, another PTS2-containing protein; both defects were not apparent upon germination but appeared in 5- to 8-d-old seedlings. In lon2 cotyledon cells, matrix proteins were localized to peroxisomes in 4-d-old seedlings but mislocalized to the cytosol in 8-d-old seedlings. Moreover, a PTS2-GFP reporter sorted to peroxisomes in lon2 root tip cells but was largely cytosolic in more mature root cells. Our results indicate that LON2 is needed for sustained matrix protein import into peroxisomes. The delayed onset of matrix protein sorting defects may account for the relatively weak Suc dependence following germination, moderate IBA-resistant primary root elongation, and severe defects in IBA-induced lateral root formation observed in lon2 mutants.Peroxisomes are single-membrane-bound organelles found in most eukaryotes. Peroxin (PEX) proteins are necessary for various aspects of peroxisome biogenesis, including matrix protein import (for review, see Distel et al., 1996; Schrader and Fahimi, 2008). Most matrix proteins are imported into peroxisomes from the cytosol using one of two targeting signals, a C-terminal type 1 peroxisome-targeting signal (PTS1) or a cleavable N-terminal type 2 peroxisome-targeting signal (PTS2) (Reumann, 2004). PTS1- and PTS2-containing proteins are bound in the cytosol by soluble matrix protein receptors, escorted to the peroxisome membrane docking complex, and translocated into the peroxisome matrix (for review, see Platta and Erdmann, 2007). Once in the peroxisome, many matrix proteins participate in metabolic pathways, such as β-oxidation, hydrogen peroxide decomposition, and photorespiration (for review, see Gabaldon et al., 2006; Poirier et al., 2006).In addition to metabolic enzymes, several proteases are found in the peroxisome matrix. Only one protease, DEG15/Tysnd1, has a well-defined role in peroxisome biology. The rat Tysnd1 protease removes the targeting signal after PTS2-containing proteins enter the peroxisome and also processes certain PTS1-containing β-oxidation enzymes (Kurochkin et al., 2007). Similarly, the Arabidopsis (Arabidopsis thaliana) Tysnd1 homolog DEG15 (At1g28320) is a peroxisomal Ser protease that removes PTS2 targeting signals (Helm et al., 2007; Schuhmann et al., 2008).In contrast with DEG15, little is known about the other eight Arabidopsis proteins that are annotated as proteases in the AraPerox database of putative peroxisomal proteins (Reumann et al., 2004; Carter et al., 2004; Shimaoka et al., 2004), which, in combination with the minor PTS found in both of these predicted proteases (Reumann, 2004), suggests that these enzymes may not be peroxisomal. Along with DEG15, only two of the predicted peroxisomal proteases, an M16 metalloprotease (At2g41790), which we have named PXM16 for peroxisomal M16 protease, and a Lon-related protease (At5g47040/LON2; Ostersetzer et al., 2007), are found in the proteome of peroxisomes purified from Arabidopsis suspension cells (Eubel et al., 2008). DEG15 and LON2 also have been validated as peroxisomally targeted using GFP fusions (Ostersetzer et al., 2007; Schuhmann et al., 2008).
Open in a separate windowaMajor PTS1 (Reumann, 2004).bMinor PTS1 (Reumann, 2004).cValidated PTS1 (Reumann et al., 2007).dMinor PTS2 (Reumann, 2004).PXM16 is the only one of the nine Arabidopsis M16 (pitrilysin family) metalloproteases (García-Lorenzo et al., 2006; Rawlings et al., 2008) containing a predicted PTS. M16 subfamilies B and C contain the plastid and mitochondrial processing peptidases (for review, see Schaller, 2004), whereas PXM16 belongs to M16 subfamily A, which includes insulin-degrading peptidases (Schaller, 2004). A tomato (Solanum lycopersicum) M16 subfamily A protease similar to insulin-degrading enzymes with a putative PTS1 was identified in a screen for proteases that cleave the wound response peptide hormone systemin (Strassner et al., 2002), but the role of Arabidopsis PXM16 is unknown.Arabidopsis LON2 is a typical Lon protease with three conserved domains: an N-terminal domain, a central ATPase domain in the AAA family, and a C-terminal protease domain with a Ser-Lys catalytic dyad (Fig. 1A; Lee and Suzuki, 2008). Lon proteases are found in prokaryotes and in some eukaryotic organelles (Fig. 1C) and participate in protein quality control by cleaving unfolded proteins and can regulate metabolism by controlling levels of enzymes from many pathways, including cell cycle, metabolism, and stress responses (for review, see Tsilibaris et al., 2006). Four Lon homologs are encoded in the Arabidopsis genome; isoforms have been identified in mitochondria, plastids, and peroxisomes (Ostersetzer et al., 2007; Eubel et al., 2008; Rawlings et al., 2008). Mitochondrial Lon protesases are found in a variety of eukaryotes (Fig. 1A) and function both as ATP-dependent proteases and as chaperones promoting protein complex assemblies (Lee and Suzuki, 2008). LON2 is the only Arabidopsis Lon isoform with a canonical C-terminal PTS1 (SKL-COOH; Ostersetzer et al., 2007) or found in the peroxisome proteome (Eubel et al., 2008; Reumann et al., 2009). Functional studies have been conducted with peroxisomal Lon isoforms found in the proteome of peroxisomes purified from rat hepatic cells (pLon; Kikuchi et al., 2004) and the methylotrophic yeast Hansenula polymorpha (Pln; Aksam et al., 2007). Rat pLon interacts with β-oxidation enzymes, and a cell line expressing a dominant negative pLon variant has decreased β-oxidation activity, displays defects in the activation processing of PTS1-containing acyl-CoA oxidase, and missorts catalase to the cytosol (Omi et al., 2008). H. polymorpha Pln is necessary for degradation of a misfolded, peroxisome-targeted version of dihydrofolate reductase and for degradation of in vitro-synthesized alcohol oxidase in peroxisomal matrix extracts, but does not contribute to degradation of peroxisomally targeted GFP (Aksam et al., 2007).Open in a separate windowFigure 1.Diagram of LON2 protein domains, gene models for LON2, PXM16, DEG15, PED1, PEX5, and PEX6, and phylogenetic relationships of LON family members. A, Organization of the 888-amino acid LON2 protein. Locations of the N-terminal domain conserved among Lon proteins, predicted ATP-binding Walker A and B domains (black circles), active site Ser (S) and Lys (K) residues (asterisks), and the C-terminal Ser-Lys-Leu (SKL) peroxisomal targeting signal (PTS1) are shown (Lee and Suzuki, 2008). B, Gene models for LON2, PXM16, DEG15, PED1, PEX5, and PEX6 and locations of T-DNA insertions (triangles) or missense alleles (arrows) used in this study. Exons are depicted by black boxes, introns by black lines, and untranslated regions by gray lines. C, Phylogenetic relationships among LON homologs. Sequences were aligned using MegAlign (DNAStar) and the ClustalW method. The PAUP 4.0b10 program (Swofford, 2001) was used to generate an unrooted phylogram from a trimmed alignment corresponding to Arabidopsis LON2 residues 400 to 888 (from the beginning of the ATPase domain to the end of the protein). The bootstrap method was performed for 500 replicates with distance as the optimality criterion. Bootstrap values are indicated at the nodes. Predicted peroxisomal proteins have C-terminal PTS1 signals in parentheses and are in light-gray ovals. Proteins in the darker gray oval have N-terminal extensions and include mitochondrial and chloroplastic proteins. Sequence identifiers are listed in Supplemental Table S2.In this work, we examined the roles of several putative peroxisomal proteases in Arabidopsis. We found that lon2 mutants displayed peroxisome-deficient phenotypes, including resistance to the protoauxin indole-3-butyric acid (IBA) and age-dependent defects in peroxisomal import of PTS1- and PTS2-targeted matrix proteins. Our results indicate that LON2 contributes to matrix protein import into Arabidopsis peroxisomes. 相似文献
Table I.
Putative Arabidopsis proteases predicted or demonstrated to be peroxisomalAGI Identifier | Alias | Protein Class | T-DNA Insertion Alleles | PTS | Localization Evidence | Localization References |
---|---|---|---|---|---|---|
At1g28320 | DEG15 | PTS2-processing protease | SALK_007184 (deg15-1) | SKL>a | GFP | Reumann et al., 2004; Helm et al., 2007; Eubel et al., 2008; Schuhmann et al., 2008) |
Proteomics | ||||||
Bioinformatics | ||||||
At2g41790 | PXM16 | Peptidase M16 family protein | SALK_019128 (pxm16-1) | PKL>b | Proteomics | Reumann et al., 2004, 2009; Eubel et al., 2008) |
SALK_023917 (pxm16-2) | Bioinformatics | |||||
At5g47040 | LON2 | Lon protease homolog | SALK_128438 (lon2-1) | SKL>a | GFP | Reumann et al., 2004, 2009; Ostersetzer et al., 2007; Eubel et al., 2008) |
SALK_043857 (lon2-2) | Proteomics | |||||
Bioinformatics | ||||||
At2g18080 | Ser-type peptidase | SALK_020628 | SSI>c | Bioinformatics | (Reumann et al., 2004) | |
SALK_102239 | ||||||
At2g35615 | Aspartyl protease | SALK_090795 | ANL>b | Bioinformatics | (Reumann et al., 2004) | |
SALK_036333 | ||||||
At3g57810 | Ovarian tumor-like Cys protease | SKL>a | Bioinformatics | (Reumann et al., 2004) | ||
At4g14570 | Acylaminoacyl-peptidase protein | CKL>b | Bioinformatics (peroxisome) | (Reumann et al., 2004; Shimaoka et al., 2004) | ||
Proteomics (vacuole) | ||||||
At4g20310 | Peptidase M50 family protein | RMx5HLd | Bioinformatics | (Reumann et al., 2004) | ||
At4g36195 | Ser carboxypeptidase S28 family | SSM>b | Bioinformatics (peroxisome) | (Carter et al., 2004; Reumann et al., 2004) | ||
Proteomics (vacuole) |
3.
4.
Vincent W. Keng Barbara J. Ryan Kirk J. Wangensteen Darius Balciunas Christian Schmedt Stephen C. Ekker David A. Largaespada 《Genetics》2009,183(4):1565-1573
Insertional mutagenesis screens play an integral part in the annotating of functional data for all sequenced genes in the postgenomic era. Chemical mutagenesis screens are highly efficient but identifying the causative gene can be a laborious task. Other mutagenesis platforms, such as transposable elements, have been successfully applied for insertional mutagenesis screens in both the mouse and rat. However, relatively low transposition efficiency has hampered their use as a high-throughput forward genetic mutagenesis screen. Here we report the first evidence of germline activity in the mouse using a naturally active DNA transposon derived from the medaka fish called Tol2, as an alternative system for high-throughput forward genetic mutagenesis screening tool.THE Tol2 (transposable element of Oryzias latipes, number 2) element belongs to the hAT family (hobo of Drosophilia, Activator of maize and Tam3 of snapdragon) of transposons and was the first known autonomously active vertebrate type II transposable element (Koga et al. 1996; Kawakami et al. 1998). Unlike other DNA-type transposons like Sleeping Beauty (SB) (Ivics et al. 1997) or piggyBac (PB) (Fraser et al. 1996), Tol2 does not exhibit any known strong site specificity for integration nor does it exhibit any significant overexpression inhibition activity (Kawakami and Noda 2004; Balciunas et al. 2006) as seen in SB (Geurts et al. 2003). Recently, Tol2 was shown to effectively carry large DNA cargo of up to 10 kb in human and mouse cells without affecting its transposition efficiency (Balciunas et al. 2006). To date, Tol2 has also been demonstrated to transpose efficiently in zebrafish, frog, chicken, mouse cells, and human cells (Kawakami et al. 2000, 2004; Koga et al. 2003; Kawakami and Noda 2004; Balciunas et al. 2006; Hamlet et al. 2006; Sato et al. 2007).Germline mutagenesis using the SB transposon system has been demonstrated in both the mouse (Dupuy et al. 2001; Horie et al. 2001) and rat (Kitada et al. 2007; Lu et al. 2007). In addition, PB germline mutagenesis in mice has also been demonstrated (Ding et al. 2005; Wu et al. 2007). However, the relatively low germline transposition efficiency of both transposon systems reported so far has hampered their use in a high-throughput forward genetic mutagenesis screen (Keng et al. 2005; Kitada et al. 2007).
Open in a separate windowSB, Sleeping Beauty; PB, piggyBac; Tol2, transposable element of Oryzias latipes, number 2.In search of an alternative tool for high-throughput forward germline mutagenesis screen in mice, a Tol2 transposon insertional mutagenesis system was generated on the basis of a similar strategy used for the SB transposon system (Horie et al. 2003; Keng et al. 2005). In the present study, we successfully demonstrate the novel use of the Tol2 transposon system for germline mutagenesis in mouse. Our results indicate the potential use of this transposon system for a high-throughput, large-scale forward mutagenesis screen in the mouse germline. 相似文献
TABLE 1
Germline transposition frequency in various transposon systemsTransposon system | Average transposition events per gamete | Mouse strain | Reference |
---|---|---|---|
SB | 2 | FVB/N | Dupuy et al. (2001) |
SB | 1.25 | C3H and C57BL/6 | Horie et al. (2001) |
SB | 1.15 | C3H and C57BL/6 | Keng et al. (2005) |
PB | 1.1 | FVB/N | Ding et al. (2005) |
PB | 1 | C57BL/6 | Wu et al. (2007) |
Tol2 | 3 | FVB/N | Present study |
5.
Retrograde Intraflagellar Transport Mutants Identify Complex A Proteins With Multiple Genetic Interactions in Chlamydomonas reinhardtii
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The intraflagellar transport machinery is required for the assembly of cilia. It has been investigated by biochemical, genetic, and computational methods that have identified at least 21 proteins that assemble into two subcomplexes. It has been hypothesized that complex A is required for retrograde transport. Temperature-sensitive mutations in FLA15 and FLA17 show defects in retrograde intraflagellar transport (IFT) in Chlamydomonas. We show that IFT144 and IFT139, two complex A proteins, are encoded by FLA15 and FLA17, respectively. The fla15 allele is a missense mutation in a conserved cysteine and the fla17 allele is an in-frame deletion of three exons. The flagellar assembly defect of each mutant is rescued by the respective transgenes. In fla15 and fla17 mutants, bulges form in the distal one-third of the flagella at the permissive temperature and this phenotype is also rescued by the transgenes. These bulges contain the complex B component IFT74/72, but not α-tubulin or p28, a component of an inner dynein arm, which suggests specificity with respect to the proteins that accumulate in these bulges. IFT144 and IFT139 are likely to interact with each other and other proteins on the basis of three distinct genetic tests: (1) Double mutants display synthetic flagellar assembly defects at the permissive temperature, (2) heterozygous diploid strains exhibit second-site noncomplemention, and (3) transgenes confer two-copy suppression. Since these tests show different levels of phenotypic sensitivity, we propose they illustrate different gradations of gene interaction between complex A proteins themselves and with a complex B protein (IFT172).CILIA and flagella are microtubule-based organelles that are found on most mammalian cells. They provide motility to cells and participate in many sensory processes. Defects in or loss of cilia/flagella cause a variety of human diseases that include polycystic kidney disease, retinal degeneration, infertility, obesity, respiratory defects, left–right axis determination, and polydactyly (Fliegauf et al. 2007). Mouse mutants demonstrate that cilia are essential for viability, neural tube closure, and bone development (Eggenschwiler and Anderson 2007; Fliegauf et al. 2007). Cilia and flagella are also present in protists, algae, moss, and some fungi.The assembly and maintenance of cilia and flagella require intraflagellar transport (IFT) (Kozminski et al. 1995). IFT involves the movement of 100- to 200-nm-long protein particles from the basal body located in the cell body to the tip of the flagella using the heterotrimeric kinesin-2 (anterograde movement) (Kozminski et al. 1995) and movement back to the cell body (retrograde movement) using the cytoplasmic dynein complex (Pazour et al. 1999; Porter et al. 1999). IFT particles change their direction of movement as well as their size, speed, and frequency at the ends of the flagella as they switch from anterograde to retrograde movement (Iomini et al. 2001). Biochemical isolation of IFT particles reveals that they are composed of at least 16 proteins and that these particles can be dissociated into two complexes in vitro by changing the salt concentration (Cole et al. 1998; Piperno et al. 1998). Recent genetic and bioinformatics analysis adds at least 7 more proteins to the IFT particle (Follit et al. 2009) (Eggenschwiler and Anderson 2007).
Open in a separate window—, no mutant found to date in Chlamydomonas.A collection of temperature-sensitive mutant strains that fail to assemble flagella at the restrictive temperature of 32° was isolated in Chlamydomonas (Huang et al. 1977; Adams et al. 1982; Piperno et al. 1998; Iomini et al. 2001). Analysis of the flagella at 21° permits the measurement of the velocity and frequency of IFT particles in the mutant strains. This analysis suggested that assembly has four phases: recruitment to the basal body, anterograde movement (phases I and II), retrograde movement, and return to the cytoplasm (phases III and IV) (Iomini et al. 2001). Different mutants were classified as defective in these four phases. However, because different alleles of FLA8 were classified as defective in different phases (Iomini et al. 2001; Miller et al. 2005), we combined mutants with IFT defects into just two classes. The first group (phases I and II) includes mutant strains that show decreased anterograde velocities, a decreased ratio of anterograde to retrograde particles, and an accumulation of complex A proteins at the basal body. This group includes mutations in the FLA8 and FLA10 genes, which encode the two motor subunits of kinesin-2 (Walther et al. 1994; Miller et al. 2005), as well as mutations in three unknown genes (FLA18, FLA27, and FLA28). The second group includes mutant strains that show the reciprocal phenotype (phases III and IV); these phenotypes include decreased retrograde velocities, an increased ratio of anterograde to retrograde particles, and an accumulation of complex B proteins in the flagella. With the exception of the FLA11 gene, which encodes IFT172, a component of complex B (Pedersen et al. 2005), the gene products in this class are unknown (FLA2, FLA15, FLA16, FLA17, and FLA24). One might predict that mutations in this group would map to genes that encode complex A or retrograde motor subunits. Interestingly, IFT particles isolated from fla11, fla15, fla16, and fla17-1 flagella show depletion of complex A polypeptides (Piperno et al. 1998; Iomini et al. 2001). The inclusion of IFT172 in this class is explained by the observations that IFT172 plays a role in remodeling the IFT particles at the flagellar tip to transition from anterograde to retrograde movement (Pedersen et al. 2005). The remaining mutant strains do not show obvious defects in velocities, ratios, or accumulation at 21° and may reflect a less severe phenotype at the permissive temperature or a non-IFT role for these genes.Direct interactions occur between components of complex B. IFT81 and IFT74/72 interact to form a scaffold required for IFT complex B assembly (Lucker et al. 2005). IFT57 and IFT20 also interact with each other and kinesin-2 (Baker et al. 2003). While physical interactions are being used to define IFT particle architecture, genetic interactions among loci encoding IFT components should be instructive regarding their function as well. To probe retrograde movement and its function, we have identified the gene products encoded by two retrograde defective mutant strains. They are FLA15 and FLA17 and encode IFT144 and IFT139, respectively. The genetic interactions of these loci provide interesting clues about the assembly of the IFT particles and possible physical interactions in the IFT particles. 相似文献
TABLE 1
Proteins and gene names for the intraflagellar transport particles in Chlamydomonas, C. elegans, and mouseProtein | Motif | Chlamydomonas gene | C. elegans gene | Mouse gene | References to worm and mouse genes |
---|---|---|---|---|---|
Complex A | |||||
IFT144 | WD | FLA15 | |||
IFT140 | WD | — | che-11 | Qin et al. (2001) | |
IFT139 | TRP | FLA17 | dyf-2 | THM1 | Efimenko et al. (2006); Tran et al. (2008) |
IFT122 | WD | — | IFTA-1 | Blacque et al. (2006) | |
IFT121 | WD | — | daf-10 | Bell et al. (2006) | |
IFT43 | — | ||||
Complex B | |||||
IFT172 | WD | FLA11 | osm-1 | Wimple | Huangfu et al. (2003); Pedersen et al. (2005); Bell et al. (2006) |
IFT88 | TRP | IFT88 | osm-5 | Tg737/Polaris | Pazour et al. (2000); Qin et al. (2001) |
IFT81 | Coil | — | ift-81 | CDV1 | Kobayashi et al. (2007) |
IFT80 | WD | — | che-2 | Wdr56 | Fujiwara et al. (1999) |
IFT74/72 | Coil | — | ift-74 | Cmg1 | Kobayashi et al. (2007) |
IFT57/55 | Coil | — | che-13 | Hippi | Haycraft et al. (2003) |
IFT54 | Microtubule binding domain MIP-T3 | — | dyf-11 | Traf3IP1 | Kunitomo and Iino (2008); Li et al. (2008); Omori et al. (2008); Follit et al. (2009) |
IFT52 | ABC type | BLD1 | osm-6 | Ngd2 | Brazelton et al. (2001); Bell et al. (2006) |
IFT46 | IFT46 | dyf-6 | Bell et al. (2006); Hou et al. (2007) | ||
IFT27 | G protein | — | Not present | Rabl4 | |
IFT25 | Hsp20 | — | Not present | HSP16.1 | Follit et al. (2009) |
IFT22 | G protein | — | IFTA-2 | Rabl5 | Schafer et al. (2006) |
IFT20 | Coil | — | Follit et al. (2006) | ||
FAP22 | Cluamp related protein | — | dyf-3 | Cluamp1 | Murayama et al. (2005); Follit et al. (2009) |
DYF13 | — | dyf-13 | Ttc26 | Blacque et al. (2005) |
6.
Hendrik Küpper Birgit G?tz Ana Mijovilovich Frithjof C. Küpper Wolfram Meyer-Klaucke 《Plant physiology》2009,151(2):702-714
The amphibious water plant Crassula helmsii is an invasive copper (Cu)-tolerant neophyte in Europe. It now turned out to accumulate Cu up to more than 9,000 ppm in its shoots at 10 μm (=0.6 ppm) Cu2+ in the nutrient solution, indicating that it is a Cu hyperaccumulator. We investigated uptake, binding environment, and toxicity of Cu in this plant under emerged and submerged conditions. Extended x-ray absorption fine structure measurements on frozen-hydrated samples revealed that Cu was bound almost exclusively by oxygen ligands, likely organic acids, and not any sulfur ligands. Despite significant differences in photosynthesis biochemistry and biophysics between emerged and submerged plants, no differences in Cu ligands were found. While measurements of tissue pH confirmed the diurnal acid cycle typical for Crassulacean acid metabolism, Δ13C measurements showed values typical for regular C3 photosynthesis. Cu-induced inhibition of photosynthesis mainly affected the photosystem II (PSII) reaction center, but with some unusual features. Most obviously, the degree of light saturation of electron transport increased during Cu stress, while maximal dark-adapted PSII quantum yield did not change and light-adapted quantum yield of PSII photochemistry decreased particularly in the first 50 s after onset of actinic irradiance. This combination of changes, which were strongest in submerged cultures, shows a decreasing number of functional reaction centers relative to the antenna in a system with high antenna connectivity. Nonphotochemical quenching, in contrast, was modified by Cu mainly in emerged cultures. Pigment concentrations in stressed plants strongly decreased, but no changes in their ratios occurred, indicating that cells either survived intact or died and bleached quickly.Heavy metals such as cadmium (Cd), copper (Cu), manganese, nickel (Ni), and zinc (Zn) are well known to be essential microelements for the life of plants (for Cd, see Lane and Morel, 2000). On the other hand, elevated concentrations of these metals induce inhibition of various processes in plant metabolism (for review, see Prasad and Hagemeyer, 1999; Küpper and Kroneck, 2005). Cu can occur in very high concentrations that are detrimental or even lethal to most plants. It is widely used as a pesticide in agriculture, and field runoff may easily reach concentrations of several micromolar (Gallagher et al., 2001). Photosynthetic reactions, both photochemical and biochemical ones, belong to the most important sites of inhibition by many heavy metals and in particular Cu. In the thylakoids, PSII has frequently been identified to be the main target. The exact location of its damage, however, strongly depends on the irradiance conditions, as shown originally by Cedeno-Maldonado et al. (1972) and later by Küpper et al. (1996b, 1998, 2002). The latter authors found that in low irradiance including a dark phase, the inhibition of PSII is largely due to the impairment of the correct function of the light-harvesting antenna; this mechanism was termed “shade reaction.” It results from the substitution by heavy metals of the Mg2+ ion in the chlorophyll (Chl) molecules of the light-harvesting complex II. In high irradiance, direct damage to the PSII reaction center (RC) occurs instead, which most likely involves insertion of Cu2+ into the Pheo a of the PSII RC. This was named “sun reaction” (Küpper et al., 1996b, 1998, 2002). Also, oxidative stress has often been described as a result of Cu stress; recent data have shown that in photosynthetic organisms, it is mainly a consequence of an inhibition of the photosynthetic light reactions (Rocchetta and Küpper, 2009).Plants developed a number of strategies to resist the toxicity of heavy metals, as reviewed by Cobbett and Goldsbrough (2002) and Küpper and Kroneck (2005). Such strategies include efflux pumps, sequestration in cells and intracellular compartments where metals do least harm, and binding of heavy metals inside the cells by strong ligands like phytochelatins or free amino acids. A majority of the heavy metal-resistant plants, called “excluders,” prevent the accumulation of heavy metals inside their tissues (Baker, 1981). Other resistant plants actively take up heavy metals, translocate them into the shoot, and sequester them to certain parts of the plant, where they are stored in a harmless state. These plants, which accumulate up to several percent of heavy metals in the dry mass of their aboveground parts, are called “hyperaccumulators” (Brooks et al., 1977). In their natural habitats, metal-rich soils in many parts of the world, this type of heavy metal accumulation serves as a defense against pathogens and herbivores (Boyd and Martens, 1994; Martens and Boyd, 1994; Boyd et al., 2002; Hanson et al., 2003; Jhee et al., 2005). They can now be used for the decontamination (“phytoremediation”) of anthropogenically heavy metal-contaminated soils and in some cases also for the commercial extraction (“phytomining”) of high-value metals (mainly Ni) from metal-rich soils (Baker et al., 1994; McGrath and Zhao, 2003; Chaney et al., 2005).The mechanisms by which hyperaccumulator plants accumulate the enormous amounts of heavy metals in their shoots and prevent phytotoxicity of these metals have been the subject of many studies. Nevertheless, many of these mechanisms are still under debate (Pollard et al., 2002; Küpper and Kroneck, 2005), and a short overview is given in our companion article (Mijovilovich et al., 2009) on Cu in the Cd/Zn model hyperaccumulator plant Thlaspi caerulescens. Studies of arsenic, Cd, Ni, and Zn binding in hyperaccumulators (Krämer et al., 1996; Sagner et al., 1998; Salt et al., 1999, Wang et al., 2002; Küpper et al., 2004) indicated that in such plants most of the metals are coordinated by organic acids, which are commonly found in plant vacuoles. Nonaccumulator plants, in contrast, are well known to bind heavy metals by strong sulfur ligands such as phytochelatins (mainly for Cd) and metallothioneins (mainly for Cu), as reviewed by Cobbett and Goldsbrough (2002).While hundreds of species have been found to hyperaccumulate Ni and about two dozen to hyperaccumulate Zn, true Cu hyperaccumulation in the sense of reaching thousands of ppm in the shoot dry weight has rarely been confirmed. Most species reported to be Cu hyperaccumulators before were later found to be false positives due to Cu adsorption on the leaf surface, et cetera; actually, none of the species recently revisited had a bioaccumulation factor larger than 1, which is commonly regarded as a necessary prerequisite of true hyperaccumulation (Faucon et al., 2007). But it is important in terms of the general understanding of metal metabolism in plants to identify how plants can cope with Cu toxicity other than excluding it from their metabolism and how far the mechanisms of Cu detoxification and Cu stress differ in Cu-resistant and -accumulating plants from Cu excluders and Cu-sensitive plants. Such questions are important also for breeding better Cd/Zn hyperaccumulators, since such plants (e.g. T. caerulescens) turned out to be Cu sensitive, limiting their phytoremediation potential on soils with mixed contamination (Walker and Bernal, 2004). We now analyzed Cu accumulation and Cu stress in a so far not well-characterized species, the amphibious Crassula helmsii, an aggressively invasive plant in Europe (Küpper et al., 1996a). We chose this plant because in a previous study it had turned out to be much more Cu resistant than all other investigated species (Küpper et al., 1996b), but more in summer than in winter. Moreover, preliminary experiments indicated that under high temperatures and salinity, C. helmsii switches to circadian acid metabolism (CAM), which might cause its elevated Cu resistance in summer due to the enhanced availability of malate as a Cu ligand. CAM metabolism was first reported for C. helmsii by Newman and Raven (1995).In this study, we investigated physiological mechanisms of Cu-induced inhibition of photosynthesis, Crassulacean acid metabolism induction, and Cu accumulation and complexation in C. helmsii. The most important method for our investigations of Cu stress was the two-dimensional (imaging) and spectrally resolved microscopic in vivo measurement of the transients of Chl variable fluorescence in the fluorescence kinetic microscope (FKM; Küpper et al., 2000a, 2007a). Cu ligands were investigated via EXAFS (Technical Term Definition/Explanation Antenna connectivity The likelihood of energy transfer between antennae of different photosystems (PSII and/or PSI) CA Component analysis. In this study, we use this term for the fitting of EXAFS spectra with a linear combination of the EXAFS spectra of model compounds. EXAFS Extended x-ray absorption fine structure F0 Minimal fluorescence yield of a dark-adapted sample, fluorescence in nonactinic measuring light Fm Maximum fluorescence yield of a dark-adapted sample after supersaturating irradiation pulse Fm′ Maximum fluorescence yield of a light-adapted sample after supersaturating irradiation pulse Fv/Fm (Fm − F0)/Fm = maximal dark-adapted quantum yield of PSII photochemistry Fp Fluorescence yield at the P level of the induction curve after the onset of actinic light exposure Light saturation Measured by the increased amplitude of Fp relative to Fm after subtraction of F0. (Fp − F0)/(Fm − F0) is mostly dependent on the ratio of functional antenna molecules to functional RCs and electron transport chains. Under constant actinic irradiance for measuring Fp, a large antenna capturing photons and delivering them to its RC will cause more of the “electron traffic jam” that leads to Fp than a small antenna. ΦPSII Φe = (Fm′ − Ft′)/Fm′ = the light-acclimated efficiency of PSII (Genty et al., 1989). In this article, the use of this parameter is extended to the relaxation period after the end of actinic light to analyze the return of the system to its dark-acclimated state as measured by Fv/Fm. NPQ Nonphotochemical quenching, in this article used as an acronym for the name of this phenomenon. In this article, we measure nonphotochemical quenching as qCN = (Fm − Fm′)/Fm = “complete nonphotochemical quenching of Chl fluorescence,” i.e. with normalization to Fm. Pheo Pheophytin XAS X-ray absorption spectroscopy Z Atomic number