PP2A-B'γ modulates foliar trans-methylation capacity and the formation of 4-methoxy-indol-3-yl-methyl glucosinolate in Arabidopsis leaves.

Glucosinolates (GSL) of cruciferous plants comprise a major group of structurally diverse secondary compounds which act as deterrents against aphids and microbial pathogens and have large commercial and ecological impacts. While the transcriptional regulation governing the biosynthesis and modification of GSL is now relatively well understood, post-translational regulatory components that specifically determine the structural variation of indole glucosinolates have not been reported. We show that the cytoplasmic protein phosphatase 2A regulatory subunit B'γ (PP2A-B'γ) physically interacts with indole glucosinolate methyltransferases and controls the methoxylation of indole glucosinolates and the formation of 4-methoxy-indol-3-yl-methyl glucosinolate in Arabidopsis leaves. By taking advantage of proteomic approaches and metabolic analysis we further demonstrate that PP2A-B'γ is required to control the abundance of oligomeric protein complexes functionally linked with the activated methyl cycle and the trans-methylation capacity of leaf cells. These findings highlight the key regulatory role of PP2A-B'γ in methionine metabolism and provide a previously unrecognized perspective for metabolic engineering of glucosinolate metabolism in cruciferous plants.

Interactions of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes at cold and ambient temperatures-surface morphology and single-molecule force measurements show phase separation, and reveal tertiary and quaternary associations.

Dehydrins (group 2 late embryogenesis abundant proteins) are intrinsically-disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Their roles include stabilizing cellular proteins and membranes, and sequestering metal ions. Here, we investigate the membrane interactions of the acidic dehydrin TsDHN-1 and the basic dehydrin TsDHN-2 derived from the crucifer Thellungiella salsuginea that thrives in the Canadian sub-Arctic. We show using compression studies with a Langmuir-Blodgett trough that both dehydrins can stabilize lipid monolayers with a lipid composition mimicking the composition of the plant outer mitochondrial membrane, which had previously been shown to induce ordered secondary structures (disorder-to-order transitions) in the proteins. Ellipsometry of the monolayers during compression showed an increase in monolayer thickness upon introducing TsDHN-1 (acidic) at 4°C and TsDHN-2 (basic) at room temperature. Atomic force microscopy of supported lipid bilayers showed temperature-dependent phase transitions and domain formation induced by the proteins. These results support the conjecture that acidic dehydrins interact with and potentially stabilize plant outer mitochondrial membranes in conditions of cold stress. Single-molecule force spectroscopy of both proteins pulled from supported lipid bilayers indicated the induced formation of tertiary conformations in both proteins, and potentially a dimeric association for TsDHN-2.

Methionine salvage and S-adenosylmethionine: essential links between sulfur, ethylene and polyamine biosynthesis.

Both Met (methionine) and SAM (S-adenosylmethionine), the activated form of Met, participate in a number of essential metabolic pathways in plants. The subcellular compartmentalization of Met fluxes will be discussed in the present review with respect to regulation and communication with the sulfur assimilation pathway, the network of the aspartate-derived amino acids and the demand for production of SAM. SAM enters the ethylene, nicotianamine and polyamine biosynthetic pathways and provides the methyl group for the majority of methylation reactions required for plant growth and development. The multiple essential roles of SAM require regulation of its synthesis, recycling and distribution to sustain these different pathways. A particular focus of the present review will be on the function of recently identified genes of the Met salvage cycle or Yang cycle and the importance of the Met salvage cycle in the metabolism of MTA (5'-methylthioadenosine). MTA has the potential for product inhibition of ethylene, nicotianamine and polyamine biosynthesis which provides an additional link between these pathways. Interestingly, regulation of Met cycle genes was found to differ between plant species as shown for Arabidopsis thaliana and Oryza sativa.

Recycling of methylthioadenosine is essential for normal vascular development and reproduction in Arabidopsis.

5'-Methylthioadenosine (MTA) is the common by-product of polyamine (PA), nicotianamine (NA), and ethylene biosynthesis in Arabidopsis (Arabidopsis thaliana). The methylthiol moiety of MTA is salvaged by 5'-methylthioadenosine nucleosidase (MTN) in a reaction producing methylthioribose (MTR) and adenine. The MTN double mutant, mtn1-1mtn2-1, retains approximately 14% of the MTN enzyme activity present in the wild type and displays a pleiotropic phenotype that includes altered vasculature and impaired fertility. These abnormal traits were associated with increased MTA levels, altered PA profiles, and reduced NA content. Exogenous feeding of PAs partially recovered fertility, whereas NA supplementation improved fertility and also reversed interveinal chlorosis. The analysis of PA synthase crystal structures containing bound MTA suggests that the corresponding enzyme activities are sensitive to available MTA. Mutant plants that expressed either MTN or human methylthioadenosine phosphorylase (which metabolizes MTA without producing MTR) appeared wild type, proving that the abnormal traits of the mutant are due to MTA accumulation rather than reduced MTR. Based on our results, we propose that the key targets affected by increased MTA content are thermospermine synthase activity and spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A.

Gelation in protein extracts from cold acclimated and non-acclimated winter rye (Secale cereale L. cv Musketeer).

A protein gel is a three-dimensional network consisting of molecular interactions between biopolymers that entrap a significant volume of a continuous liquid phase (water). Molecular interactions in gels occur at junction zones within and between protein molecules through electrostatic forces, hydrogen bonding, hydrophobic associations (van der Waals attractions) and covalent bonding. Gels have the physicochemical properties of both solids and liquids, and are extremely important in the production and stability of a variety of foods, bioproducts and pharmaceuticals. In this study, gelation was induced in phenol extracted protein fractions from non-acclimated (NA) and cold-acclimated (CA) winter rye (Secale cereale L. cv Musketeer) leaf tissue after repeated freeze-thaw treatments. Gel formation only occurred at high pH (pH 12.0) and a minimum of 3-4 freeze-thaw cycles were required. The gel was thermally stable and only a specific combination of chemical treatments could disrupt the gel network. SDS-PAGE analysis identified ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) as the major protein component in the gel, although Rubisco itself did not appear to be a factor in gelation. Raman spectroscopy suggested changes in protein secondary structure during freeze-thaw cycles. Overall, the NA and CA gels were similar in composition and structure, with the exception that the CA gel appeared to be amyloidic in nature based on thioflavin T (ThT) fluorescence. Protein gelation, particularly in the apoplast, may confer protection against freeze-induced dehydration and potentially have a commercial application to improve frozen food quality.

Transcriptomic and metabolomic analysis of Yukon Thellungiella plants grown in cabinets and their natural habitat show phenotypic plasticity.

BACKGROUND: Thellungiella salsuginea is an important model plant due to its natural tolerance to abiotic stresses including salt, cold, and water deficits. Microarray and metabolite profiling have shown that Thellungiella undergoes stress-responsive changes in transcript and organic solute abundance when grown under controlled environmental conditions. However, few reports assess the capacity of plants to display stress-responsive traits in natural habitats where concurrent stresses are the norm. RESULTS: To determine whether stress-responsive changes observed in cabinet-grown plants are recapitulated in the field, we analyzed leaf transcript and metabolic profiles of Thellungiella growing in its native Yukon habitat during two years of contrasting meteorological conditions. We found 673 genes showing differential expression between field and unstressed, chamber-grown plants. There were comparatively few overlaps between genes expressed under field and cabinet treatment-specific conditions. Only 20 of 99 drought-responsive genes were expressed both in the field during a year of low precipitation and in plants subjected to drought treatments in cabinets. There was also a general pattern of lower abundance among metabolites found in field plants relative to control or stress-treated plants in growth cabinets. Nutrient availability may explain some of the observed differences. For example, proline accumulated to high levels in cold and salt-stressed cabinet-grown plants but proline content was, by comparison, negligible in plants at a saline Yukon field site. We show that proline accumulated in a stress-responsive manner in Thellungiella plants salinized in growth cabinets and in salt-stressed seedlings when nitrogen was provided at 1.0 mM. In seedlings grown on 0.1 mM nitrogen medium, the proline content was low while carbohydrates increased. The relatively higher content of sugar-like compounds in field plants and seedlings on low nitrogen media suggests that Thellungiella shows metabolic plasticity in response to environmental stress and that resource availability can influence the expression of stress tolerance traits under field conditions. CONCLUSION: Comparisons between Thellungiella plants responding to stress in cabinets and in their natural habitats showed differences but also overlap between transcript and metabolite profiles. The traits in common offer potential targets for improving crops that must respond appropriately to multiple, concurrent stresses.

Adenosine kinase contributes to cytokinin interconversion in Arabidopsis.

Purine salvage enzymes have been implicated, but not proven, to be involved in the interconversion of cytokinin (CK) bases, ribosides, and nucleotides. Here, we use Arabidopsis (Arabidopsis thaliana) lines silenced in adenosine kinase (ADK) expression to understand the contributions of this enzyme activity to in vivo CK metabolism. Both small interfering RNA- and artificial microRNA-mediated silencing of ADK led to impaired root growth, small, crinkled rosette leaves, and reduced apical dominance. Further examination of ADK-deficient roots and leaves revealed their irregular cell division. Root tips had uneven arrangements of root cap cells, reduced meristem sizes, and enlarged cells in the elongation zone; rosette leaves exhibited decreased cell size but increased cell abundance. Expression patterns of the cyclinB1;1::ß-glucuronidase and Arabidopsis Response Regulator5::ß-glucuronidase reporters in the ADK-deficient background were consistent with altered cell division and an increase in CK activity, respectively. In vivo feeding of ADK-deficient leaves with radiolabeled CK ribosides of isopentenyladenosine and zeatin showed a decreased flux into the corresponding CK nucleotides. Comprehensive high-performance liquid chromatography-tandem mass spectrometry analysis detected significantly higher levels of active CK ribosides in both sense ADK and artificial microADK. Taken together, these metabolic and phenotypic analyses of ADK-deficient lines indicate that ADK contributes to CK homeostasis in vivo.

Seed development, seed germination and seedling growth in the R50 (sym16) pea mutant are not directly linked to altered cytokinin homeostasis.

R50 (sym16) is a pea nodulation mutant that accumulates cytokinin (CK) in its vegetative organs. Total CK content increases as the plant ages because of the low activity of the enzyme cytokinin oxidase/dehydrogenase (CKX) responsible for CK degradation. R50 exhibits a large seed with high relative water content, and its seedling establishes itself slowly. Whether these two traits are linked to abnormal CK levels was considered here. R50 was found to have a similar germination rate but a much slower epicotyl emergence than Sparkle, its wild-type (WT). At the onset of emergence, the starch grains in R50 cotyledons were larger than those of WT; furthermore, they did not degrade as fast as in WT because of low amylase activity. No differences between the pea lines were observed in the CK forms identified during seed embryogenesis. However, while CK content compared to that of WT was reduced early in R50 embryogenesis, it was elevated later on in its dry seeds where CKX activity was low, although CKX transcript abundance remained high. Transcripts of the two known PsCKX isoforms exhibited tissue- and development-specific profiles with no detectable PsCKX2 expression in cotyledons. There were more of both transcripts in R50 roots than in WT roots, but less of PsCKX2 than PsCKX1 in R50 shoots compared to WT shoots. Thus, although there is a definite CKX post-transcriptional defect in R50 dry seeds, an abnormal CK homeostasis is not the basis of the delay in R50 seedling establishment, which we linked to abnormal amylase activity early in development.

Phosphorylation of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 facilitates cation-induced conformational changes and actin assembly.

Group 2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperatures. These proteins are characterized by the presence of at least one conserved, lysine-rich K-segment and sometimes by one or more serine-rich S-segments that are phosphorylated. Dehydrins may stabilize proteins and membrane structures during environmental stress and can sequester and scavenge metal ions. Here, we investigate how the conformations of two dehydrins from Thellungiella salsuginea, denoted as TsDHN-1 (acidic) and TsDHN-2 (basic), are affected by pH, interactions with cations and membranes, and phosphorylation. Both TsDHN-1 and TsDHN-2 were expressed as SUMO fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and structural analysis by circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy. We show that the polyproline II conformation can be induced in the dehydrins by their environmental conditions, including changes in the concentration of divalent cations such as Ca(2+). The assembly of actin by these dehydrins was assessed by sedimentation assays and viewed by transmission electron and atomic force microscopy. Phosphorylation allowed both dehydrins to polymerize actin filaments. These results support the hypothesis that dehydrins stabilize the cytoskeleton under stress conditions and further that phosphorylation may be an important feature of this stabilization.

Mechanism of substrate specificity in 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidases.

5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTAN) plays a key role in the methionine-recycling pathway of bacteria and plants. Despite extensive structural and biochemical studies, the molecular mechanism of substrate specificity for MTAN remains an outstanding question. Bacterial MTANs show comparable efficiency in hydrolyzing MTA and SAH, while the plant enzymes select preferentially for MTA, with either no or significantly reduced activity towards SAH. Bacterial and plant MTANs show significant conservation in the overall structure, and the adenine- and ribose-binding sites. The observation of a more constricted 5'-alkylthio binding site in Arabidopsis thalianaAtMTAN1 and AtMTAN2, two plant MTAN homologues, led to the hypothesis that steric hindrance may play a role in substrate selection in plant MTANs. We show using isothermal titration calorimetry that SAH binds to both Escherichia coli MTAN (EcMTAN) and AtMTAN1 with comparable micromolar affinity. To understand why AtMTAN1 can bind but not hydrolyze SAH, we determined the structure of the protein-SAH complex at 2.2Å resolution. The lack of catalytic activity appears to be related to the enzyme's inability to bind the substrate in a catalytically competent manner. The role of dynamics in substrate selection was also examined by probing the amide proton exchange rates of EcMTAN and AtMTAN1 via deuterium-hydrogen exchange coupled mass spectrometry. These results correlate with the B factors of available structures and the thermodynamic parameters associated with substrate binding, and suggest a higher level of conformational flexibility in the active site of EcMTAN. Our results implicate dynamics as an important factor in substrate selection in MTAN.

Inhibition of 5'-methylthioadenosine metabolism in the Yang cycle alters polyamine levels, and impairs seedling growth and reproduction in Arabidopsis.

The methionine or Yang cycle recycles Met from 5'-methylthioadenosine (MTA) which is produced from S-adenosyl-L-methionine (SAM) as a by-product of ethylene, polyamines, and nicotianamine (NA) synthesis. MTA nucleosidase is encoded by two genes in Arabidopsis thaliana, MTN1 and MTN2. Analysis of T-DNA insertion mutants and of wt revealed that MTN1 provides approximately 80% of the total MTN activity. Severe knock down of MTN enzyme activity in the mtn1-1 and mtn1-2 allelic lines resulted in accumulation of SAM/dSAM (decarboxylated SAM) and of MTA in seedlings grown on MTA as sulfur source. While ethylene and NA synthesis were not altered in mtn1-1 and mtn1-2 seedlings grown on MTA, putrescine and spermine were elevated. By contrast, mtn2-1 and mtn2-2 seedlings with near wt enzyme activity had wt levels of SAM/dSAM, MTA, and polyamines. In addition to the metabolic phenotypes, mtn1-1 and mtn1-2 seedlings were growth retarded, while seedlings of wt, mtn2-1, and mtn2-2 showed normal growth on 500 microm MTA. The double knock down mutant mtn1-1/mtn2-1 was sterile. In conclusion, the data presented identify MTA as a crucial metabolite that acts as a regulatory link between the Yang cycle and polyamine biosynthesis and identifies MTA nucleosidase as a crucial enzyme of the Yang cycle.

Zinc induces disorder-to-order transitions in free and membrane-associated Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2: a solution CD and solid-state ATR-FTIR study.

Dehydrins are intrinsically unstructured proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Although their role is not completely understood, it has been suggested that they stabilize proteins and membrane structures during environmental stress and also sequester metals such as zinc. Here, we investigate two dehydrins (denoted as TsDHN-1 and TsDHN-2) from Thellungiella salsuginea. This plant is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and experiences periods of both extreme cold and drought. We show using circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy that ordered secondary structure is induced and stabilized in these proteins, both in free and vesicle-bound form, by association with zinc. In membrane-associated form, both proteins have an increased proportion of ß-strand conformation induced by the cation, in addition to the amphipathic α-helices formed by their constituent K-segments. These results support the hypothesis that dehydrins stabilize plant plasma and organellar membranes in conditions of stress, and further that zinc may be an important co-factor in stabilization. Whereas dehydrins in the cytosol of a plant cell undergoing dehydration or temperature stress form bulk hydrogels and remain primarily disordered, dehydrins with specific membrane- or protein-associations will have induced ordered secondary structures.

Interactions of intrinsically disordered Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes - synergistic effects of lipid composition and temperature on secondary structure.

Dehydrins are intrinsically disordered (unstructured) proteins that are expressed in plants experiencing stressful conditions such as drought or low temperature. Dehydrins are typically found in the cytosol and nucleus, but also associate with chloroplasts, mitochondria, and the plasma membrane. Although their role is not completely understood, it has been suggested that they stabilize proteins or membrane structures during environmental stress, the latter association mediated by formation of amphipathic α-helices by conserved regions called the K-segments. Thellungiella salsuginea is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and experiences periods of both extreme cold and drought. We have cloned and expressed in Escherichia coli two dehydrins from this plant, denoted TsDHN-1 (acidic) and TsDHN-2 (basic). Here, we show using transmission-Fourier transform infrared (FTIR) spectroscopy that ordered secondary structure is induced and stabilized in these proteins by association with large unilamellar vesicles emulating the lipid compositions of plant plasma and organellar membranes. Moreover, this induced folding is enhanced at low temperatures, lending credence to the hypothesis that dehydrins stabilize plant outer and organellar membranes in conditions of cold.

Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d.

BACKGROUND: Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins. RESULTS: The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. CONCLUSIONS: Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

Evolutionary conservation and post-translational control of S-adenosyl-L-homocysteine hydrolase in land plants.

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH), which essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and post-translational control of SAHH in land plants. We provide evidence suggesting that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants and that the predominant protein complex is composed by a tetramer of the enzyme. Analysis of light-stress-induced adjustments of SAHH in Arabidopsis thaliana and Physcomitrella patens further suggests that regulatory actions may take place on the levels of protein complex formation and phosphorylation of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.

Differential Mechanisms of Photosynthetic Acclimation to Light and Low Temperature in Arabidopsis and the Extremophile Eutrema salsugineum.

Photosynthetic organisms are able to sense energy imbalances brought about by the overexcitation of photosystem II (PSII) through the redox state of the photosynthetic electron transport chain, estimated as the chlorophyll fluorescence parameter 1-qL, also known as PSII excitation pressure. Plants employ a wide array of photoprotective processes that modulate photosynthesis to correct these energy imbalances. Low temperature and light are well established in their ability to modulate PSII excitation pressure. The acquisition of freezing tolerance requires growth and development a low temperature (cold acclimation) which predisposes the plant to photoinhibition. Thus, photosynthetic acclimation is essential for proper energy balancing during the cold acclimation process. Eutrema salsugineum (Thellungiella salsuginea) is an extremophile, a close relative of Arabidopsis thaliana, but possessing much higher constitutive levels of tolerance to abiotic stress. This comparative study aimed to characterize the photosynthetic properties of Arabidopsis (Columbia accession) and two accessions of Eutrema (Yukon and Shandong) isolated from contrasting geographical locations at cold acclimating and non-acclimating conditions. In addition, three different growth regimes were utilized that varied in temperature, photoperiod and irradiance which resulted in different levels of PSII excitation pressure. This study has shown that these accessions interact differentially to instantaneous (measuring) and long-term (acclimation) changes in PSII excitation pressure with regard to their photosynthetic behaviour. Eutrema accessions contained a higher amount of photosynthetic pigments, showed higher oxidation of P700 and possessed more resilient photoprotective mechanisms than that of Arabidopsis, perhaps through the prevention of PSI acceptor-limitation. Upon comparison of the two Eutrema accessions, Shandong demonstrated the greatest PSII operating efficiency (ΦPSII) and P700 oxidizing capacity, while Yukon showed greater growth plasticity to irradiance. Both of these Eutrema accessions are able to photosynthetically acclimate but do so by different mechanisms. The Shandong accessions demonstrate a stable response, favouring energy partitioning to photochemistry while the Yukon accession shows a more rapid response with partitioning to other (non-photochemical) strategies.

Acquisition of freezing tolerance in Arabidopsis and two contrasting ecotypes of the extremophile Eutrema salsugineum (Thellungiella salsuginea).

Eutrema salsugineum (Thellungiella salsuginea) is an extremophile, a close relative of Arabidopsis, but possessing much higher constitutive levels of tolerance to abiotic stress. This study aimed to characterize the freezing tolerance of Arabidopsis (Columbia ecotype) and two ecotypes of Eutrema (Yukon and Shandong) isolated from contrasting geographical locations. Under our growth conditions, maximal freezing tolerance was observed after two- and three-weeks of cold acclimation for Arabidopsis and Eutrema, respectively. The ecotypes of Eutrema and Arabidopsis do not differ in their constitutive level of freezing tolerance or short-term cold acclimation capacity. However Eutrema remarkably outperforms Arabidopsis in long-term acclimation capacity suggesting a wider phenotypic plasticity for the trait of freezing tolerance. The combination of drought treatment and one-week of cold acclimation was more effective than long-term cold acclimation in achieving maximum levels of freezing tolerance in Eutrema, but not Arabidopsis. Furthermore, it was demonstrated growth conditions, particularly irradiance, are determinates of the level of freezing tolerance attained during cold acclimation suggesting a role for photosynthetic processes in adaptive stress responses.

Adenine phosphoribosyltransferase isoforms of Arabidopsis and their potential contributions to adenine and cytokinin metabolism.

Adenine phosphoribosyltransferase (APT; EC 2.4.2.7) is a constitutively expressed enzyme involved in the one-step salvage of adenine to AMP. The Arabidopsis thaliana genome contains five sequences annotated as encoding APT or APT-like enzymes. Three of these have now been cloned, over-expressed and compared using kinetic analyses. At a cytosolic pH, all bind adenine efficiently based on their Km values (0.8-2.6 &mgr;M), although APT1 metabolizes adenine at a rate 31-53 times faster than APT2 and APT3, respectively. Since APT also has a possible role in the interconversion of cytokinin bases to nucleotides, we characterized the activity of each isoform on zeatin, isopentenyladenine and benzyladenine. Based on their Km values, APT2 and APT3 had much higher affinities than APT1 for all three cytokinins (15-440 &mgr;M for APT2 and 3 vs. 1.8-2.5 mM for APT1); conversely the Vmax values for APT2 and APT3 on these CK substrates showed the opposite trend, being 4- to 19-fold lower than those of APT1. Anti-peptide antibodies for APT1, APT2, and APT3 were prepared and used to examine the subcellular localization of each isoform. Based on these results, APT1 and APT3 appear to be cytosolic, while the localization of APT2 was inconclusive although sequence analysis implies that APT2 is also cytosolic. Each isoform was modelled against the crystal structure of APT from Leishmania donovani, and structural differences in substrate specificity-determining domains have been found. The estimated kinetic activities of these APTs suggest that they contribute primarily to adenine recycling, although an involvement in cytokinin interconversion cannot be discounted.

Genes encoding chitinase-antifreeze proteins are regulated by cold and expressed by all cell types in winter rye shoots.

One group of antifreeze proteins (AFPs) is composed of two chitinases that accumulate in the apoplast of winter rye leaves during cold acclimation. In this study, the 28- and 35-kDa chitinase-AFPs were localized in nonacclimated and cold-acclimated rye leaves by immunoelectron microscopy with an antiserum produced against the purified winter rye 35-kDa chitinase-AFP. In cold-acclimated winter rye leaves, labelled chitinase-AFPs were abundant in the walls of epidermal, parenchymal sheath and mesophyll cells and xylem vessels, while less label was present in walls of vascular parenchyma cells. In contrast, chitinase labelling was essentially absent in the nonacclimated cells except in xylem vessels. As shown by RNA blotting, the transcripts of chitinase-AFPs accumulated to a high level in rye leaves during cold acclimation, to a lesser extent in crowns and were not detectable in roots. mRNA transcripts of the 28-kDa chitinase-AFP were localized in rye leaves by in situ hybridization. The chitinase-AFP transcripts were found in the same cell types as the protein itself. We conclude that all metabolically active cell types in cold-acclimated winter rye leaves and crowns are able to synthesize chitinase-AFPs and secrete them into adjacent cell walls, where they may interact with ice to delay its propagation through the plant and modify its growth.

Nuclear targeting of methyl-recycling enzymes in Arabidopsis thaliana is mediated by specific protein interactions.

Numerous transmethylation reactions are required for normal plant growth and development. S-adenosylhomocysteine hydrolase (SAHH) and adenosine kinase (ADK) act coordinately to recycle the by-product of these reactions, S-adenosylhomocysteine (SAH) that would otherwise competitively inhibit methyltransferase (MT) activities. Here, we report on investigations to understand how the SAH produced in the nucleus is metabolized by SAHH and ADK. Localization analyses using green fluorescent fusion proteins demonstrated that both enzymes are capable of localizing to the cytoplasm and the nucleus, although no obvious nuclear localization signal was found in their sequences. Deletion analysis revealed that a 41-amino-acid segment of SAHH (Gly(150)-Lys(190)) is required for nuclear targeting of this enzyme. This segment is surface exposed, shows unique sequence conservation patterns in plant SAHHs, and possesses additional features of protein-protein interaction motifs. ADK and SAHH interact in Arabidopsis via this segment and also interact with an mRNA cap MT. We propose that the targeting of this complex is directed by the nuclear localization signal of the MT; other MTs may similarly target SAHH/ADK to other subcellular compartments to ensure uninterrupted transmethylation.