Catalytic and structural properties of pheophytinase, the phytol esterase involved in chlorophyll breakdown.

During leaf senescence and fruit ripening, chlorophyll is degraded in a multistep pathway into linear tetrapyrroles called phyllobilins. A key feature of chlorophyll breakdown is the removal of the hydrophobic phytol chain that renders phyllobilins water soluble, an important prerequisite for their ultimate storage in the vacuole of senescent cells. Chlorophyllases had been considered for more than a century to catalyze dephytylation in vivo; however, this was recently refuted. Instead, pheophytinase was discovered as a genuine in vivo phytol hydrolase. While chlorophyllase acts rather unspecifically towards different porphyrin substrates, pheophytinase was shown to specifically dephytylate pheophytin, namely Mg-free chlorophyll. The aim of this work was to elucidate in detail the biochemical and structural properties of pheophytinase. By testing different porphyrin substrates with recombinant pheophytinase from Arabidopsis thaliana we show that pheophytinase has high specificity for the acid moiety of the ester bond, namely the porphyrin ring, while the nature of the alcohol, namely the phytol chain in pheophytin, is irrelevant. In silico modelling of the 3-dimensional structure of pheophytinase and subsequent analysis of site-directed pheophytinase mutant forms allowed the identification of the serine, histidine, and aspartic acid residues that compose the catalytic triad, a classical feature of serine-type hydrolases to which both pheophytinase and chlorophyllase belong. Based on substantial structural differences in the models of Arabidopsis pheophytinase and chlorophyllase 1, we discuss potential differences in the catalytic properties of these two phytol hydrolases.

Interaction of brassinosteroid functions and sucrose transporter SlSUT2 regulate the formation of arbuscular mycorrhiza.

Transgenic tomato plants with reduced expression of the sucrose transporter SlSUT2 showed higher efficiency of mycorrhization suggesting a sucrose retrieval function of SlSUT2 from the peri-arbuscular space back into the cell cytoplasm plant cytoplasm thereby limiting mycorrhiza fungal development. Sucrose uptake in colonized root cells requires efficient plasma membrane-targeting of SlSUT2 which is often retained intracellularly in vacuolar vesicles. Protein-protein interaction studies suggested a link between SISUT2 function and components of brassinosteroid biosynthesis and signaling. Indeed, the tomato DWARF mutant d(x) defective in BR synthesis (1) showed significantly reduced mycorrhization parameters. (2) The question has been raised whether the impact of brassinosteroids on mycorrhization is a general phenomenon. Here, we include a rice mutant defective in DIM1/DWARF1 involved in BR biosynthesis to investigate the effects on mycorrhization. A model is presented where brassinolides are able to impact mycorrhization by activating SUT2 internalization and inhibiting its role in sucrose retrieval.

The sucrose transporter SlSUT2 from tomato interacts with brassinosteroid functioning and affects arbuscular mycorrhiza formation.

Mycorrhizal plants benefit from the fungal partners by getting better access to soil nutrients. In exchange, the plant supplies carbohydrates to the fungus. The additional carbohydrate demand in mycorrhizal plants was shown to be balanced partially by higher CO2 assimilation and increased C metabolism in shoots and roots. In order to test the role of sucrose transport for fungal development in arbuscular mycorrhizal (AM) tomato, transgenic plants with down-regulated expression of three sucrose transporter genes were analysed. Plants that carried an antisense construct of SlSUT2 (SlSUT2as) repeatedly exhibited increased mycorrhizal colonization and the positive effect of plants to mycorrhiza was abolished. Grafting experiments between transgenic and wild-type rootstocks and scions indicated that mainly the root-specific function of SlSUT2 has an impact on colonization of tomato roots with the AM fungus. Localization of SISUT2 to the periarbuscular membrane indicates a role in back transport of sucrose from the periarbuscular matrix into the plant cell thereby affecting hyphal development. Screening of an expression library for SlSUT2-interacting proteins revealed interactions with candidates involved in brassinosteroid (BR) signaling or biosynthesis. Interaction of these candidates with SlSUT2 was confirmed by bimolecular fluorescence complementation. Tomato mutants defective in BR biosynthesis were analysed with respect to mycorrhizal symbiosis and showed indeed decreased mycorrhization. This finding suggests that BRs affect mycorrhizal infection and colonization. If the inhibitory effect of SlSUT2 on mycorrhizal growth involves components of BR synthesis and of the BR signaling pathway is discussed.

Identification of a probable pore-forming domain in the multimeric vacuolar anion channel AtALMT9.

Aluminum-activated malate transporters (ALMTs) form an important family of anion channels involved in fundamental physiological processes in plants. Because of their importance, the role of ALMTs in plant physiology is studied extensively. In contrast, the structural basis of their functional properties is largely unknown. This lack of information limits the understanding of the functional and physiological differences between ALMTs and their impact on anion transport in plants. This study aimed at investigating the structural organization of the transmembrane domain of the Arabidopsis (Arabidopsis thaliana) vacuolar channel AtALMT9. For that purpose, we performed a large-scale mutagenesis analysis and found two residues that form a salt bridge between the first and second putative transmembrane α-helices (TMα1 and TMα2). Furthermore, using a combination of pharmacological and mutagenesis approaches, we identified citrate as an "open channel blocker" of AtALMT9 and used this tool to examine the inhibition sensitivity of different point mutants of highly conserved amino acid residues. By this means, we found a stretch within the cytosolic moiety of the TMα5 that is a probable pore-forming domain. Moreover, using a citrate-insensitive AtALMT9 mutant and biochemical approaches, we could demonstrate that AtALMT9 forms a multimeric complex that is supposedly composed of four subunits. In summary, our data provide, to our knowledge, the first evidence about the structural organization of an ion channel of the ALMT family. We suggest that AtALMT9 is a tetramer and that the TMα5 domains of the subunits contribute to form the pore of this anion channel.

Site directed mutagenesis of StSUT1 reveals target amino acids of regulation and stability.

Plant sucrose transporters (SUTs) are functional as sucrose-proton-cotransporters with an optimal transport activity in the acidic pH range. Recently, the pH optimum of the Solanum tuberosum sucrose transporter StSUT1 was experimentally determined to range at an unexpectedly low pH of 3 or even below. Various research groups have confirmed these surprising findings independently and in different organisms. Here we provide further experimental evidence for a pH optimum at physiological extrema. Site directed mutagenesis provides information about functional amino acids, which are highly conserved and responsible for this extraordinary increase in transport capacity under extreme pH conditions. Redox-dependent dimerization of the StSUT1 protein was described earlier. Here the ability of StSUT1 to form homodimers was demonstrated by heterologous expression in Lactococcus lactis and Xenopus leavis using Western blots, and in plants by bimolecular fluorescence complementation. Mutagenesis of highly conserved cysteine residues revealed their importance in protein stability. The accessibility of regulatory amino acid residues in the light of StSUT1's compartmentalization in membrane microdomains is discussed.

Post-translational regulation of sucrose transporters by direct protein-protein interactions.

Sucrose transporters are essential membrane proteins for the allocation of carbon resources in higher plants and protein-protein interactions play a crucial role in the post-translational regulation of sucrose transporters affecting affinity, transport capacity, oligomerization, localization, and trafficking. Systematic screening for protein interactors using sucrose transporters as bait proteins helped identifying several proteins binding to sucrose transporters from apple, Arabidopsis, potato, or tomato using the split ubiquitin system. This mini-review summarizes known sucrose transporter-interacting proteins and their potential function in plants. Not all of the identified interaction partners are postulated to be located at the plasma membrane, but some are predicted to be endoplasmic reticulum-residing proteins such as a protein disulfide isomerase and members of the cytochrome b5 family. Many of the SUT1-interacting proteins are secretory proteins or involved in metabolism. Identification of actin and actin-related proteins as SUT1-interacting proteins confirmed the observation that movement of SUT1-containing intracellular vesicles can be blocked by inhibition of actin polymerization using specific inhibitors. Manipulation of expression of these interacting proteins represents one possible way to modify resource allocation by post-translational regulation of sucrose transporters.

Photoperiodic regulation of the sucrose transporter StSUT4 affects the expression of circadian-regulated genes and ethylene production.

Several recent publications reported different subcellular localization of the sucrose transporters belonging to the SUT4 subfamily. The physiological function of the SUT4 sucrose transporters requires clarification, because down-regulation of the members of the SUT4 clade had different effects in rice, poplar, and potato. Here, we provide new data for the localization and function of the Solanaceous StSUT4 protein, further elucidating involvement in the onset of flowering, tuberization and in the shade avoidance syndrome of potato plants. Induction of an early flowering and a tuberization in the SUT4-inhibited potato plants correlates with increased sucrose export from leaves and increased sucrose and starch accumulation in terminal sink organs, such as developing tubers. SUT4 affects expression of the enzymes involved in gibberellin and ethylene biosynthesis, as well as the rate of ethylene biosynthesis in potato. In the SUT4-inhibited plants, the ethylene production no longer follows a diurnal rhythm. Thus it was concluded that StSUT4 controls circadian gene expression, potentially by regulating sucrose export from leaves. Furthermore, SUT4 expression affects clock-regulated genes such as StFT, StSOC1, and StCO, which might be also involved in a photoperiod-dependent tuberization. A model is proposed in which StSUT4 controls a phloem-mobile signaling molecule generated in leaves, which together with enhanced sucrose export affects developmental switches in apical meristems. SUT4 seems to link photoreceptor-perceived information about the light quality and day length with phytohormone biosynthesis and the expression of circadian-regulated genes.

The potato sucrose transporter StSUT1 interacts with a DRM-associated protein disulfide isomerase.

Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A. We therefore aimed to identify SUT1-interacting proteins that might be involved in dimerization, endocytosis, or targeting of SUT1 to raft-like microdomains. Therefore, we identified potato membrane proteins, which are associated with the detergent-resistant membrane (DRM) fraction. Among the proteins identified, we clearly confirmed StSUT1 as part of DRM in potato source leaves. We used the yeast two-hybrid split ubiquitin system (SUS) to systematically screen for interaction between the sucrose transporter StSUT1 and other membrane-associated or soluble proteins in vivo. The SUS screen was followed by immunoprecipitation using affinity-purified StSUT1-specific peptide antibodies and mass spectrometric analysis of co-precipitated proteins. A large overlap was observed between the StSUT1-interacting proteins identified in the co-immunoprecipitation and the detergent-resistant membrane fraction. One of the SUT1-interacting proteins, a protein disulfide isomerase (PDI), interacts also with other sucrose transporter proteins. A potential role of the PDI as escort protein is discussed.

Sucrose transporter regulation at the transcriptional, post-transcriptional and post-translational level.

Sucrose transporters are crucial to carbon partitioning in higher plants. With their role in loading of sucrose into the phloem they control sucrose distribution throughout the whole plant and drive the osmotic flow system in the phloem. Recently, first insight was obtained on the coordination of sucrose transporter action with plant growth and development. The analysis of transgenic plants with reduced or enhanced expression of sucrose transporters helped to elucidate their physiological function and regulation in detail and connections to light and hormone signalling pathways were discovered. Whereas members of the SUT1 subfamily of sucrose transporters seem to be tightly controlled at the transcriptional and post-translational level in solanaceous plants, other family members show primarily post-transcriptional control of their mRNA stability. Post-translational regulation of sucrose transporters might be affected by direct protein-protein interactions or by recycling of sucrose transporters at the plasma membrane. A model is proposed showing cell-to-cell movement of both the SUT1 mRNA as well as the SUT1 protein via the desmotubule connecting companion cells where transcription of sucrose transporters occurs, and the neighbouring sieve elements. We provide an overview over sucrose transporter regulation in Solanum species at the transcriptional, post-transcriptional and post-translational level with emphasis on the many old and new questions surrounding the topic and how they could be answered.

Proteasomal degradation of Sfp1 contributes to the repression of ribosome biogenesis during starvation and is mediated by the proteasome activator Blm10.

The regulation of ribosomal protein (RP) gene transcription is tightly linked to the nutrient status of the cell and is under the control of metabolic signaling pathways. In Saccharomyces cerevisiae several transcriptional activators mediate efficient RP gene transcription during logarithmic growth and dissociate from RP gene promoters upon nutrient limitation. Repression of RP gene transcription appears to be regulated predominantly by posttranslational modification and cellular localization of transcriptional activators. We report here that one of these factors, Sfp1, is degraded by the proteasome and that the proteasome activator Blm10 is required for regulated Sfp1 degradation. Loss of Blm10 results in the stabilization and increased nuclear abundance of Sfp1 during nutrient limitation, increased transcription of RP genes, increased levels of RPs, and decreased rapamycin-induced repression of RP genes. Thus we conclude that proteasomal degradation of Sfp1 is mediated by Blm10 and contributes to the repression of ribosome biogenesis under nutrient depletion.

Transport and sorting of the solanum tuberosum sucrose transporter SUT1 is affected by posttranslational modification.

The plant sucrose transporter SUT1 from Solanum tuberosum revealed a dramatic redox-dependent increase in sucrose transport activity when heterologously expressed in Saccharomyces cerevisiae. Plant plasma membrane vesicles do not show any change in proton flux across the plasma membrane in the presence of redox reagents, indicating a SUT1-specific effect of redox reagents. Redox-dependent sucrose transport activity was confirmed electrophysiologically in Xenopus laevis oocytes with SUT1 from maize (Zea mays). Localization studies of green fluorescent protein fusion constructs showed that an oxidative environment increased the targeting of SUT1 to the plasma membrane where the protein concentrates in 200- to 300-nm raft-like microdomains. Using plant plasma membranes, St SUT1 can be detected in the detergent-resistant membrane fraction. Importantly, in yeast and in plants, oxidative reagents induced a shift in the monomer to dimer equilibrium of the St SUT1 protein and increased the fraction of dimer. Biochemical methods confirmed the capacity of SUT1 to form a dimer in plants and yeast cells in a redox-dependent manner. Blue native PAGE, chemical cross-linking, and immunoprecipitation, as well as the analysis of transgenic plants with reduced expression of St SUT1, confirmed the dimerization of St SUT1 and Sl SUT1 (from Solanum lycopersicum) in planta. The ability to form homodimers in plant cells was analyzed by the split yellow fluorescent protein technique in transiently transformed tobacco (Nicotiana tabacum) leaves and protoplasts. Oligomerization seems to be cell type specific since under native-like conditions, a phloem-specific reduction of the dimeric form of the St SUT1 protein was detectable in SUT1 antisense plants, whereas constitutively inhibited antisense plants showed reduction only of the monomeric form. The role of redox control of sucrose transport in plants is discussed.

Dimerization and endocytosis of the sucrose transporter StSUT1 in mature sieve elements.

The sucrose transporter StSUT1 from Solanum tuberosum was shown to be regulated post-translationally by redox reagents. Its activity is increased at least 10-fold in the presence of oxidizing agents if expressed in yeast. Oxidation has also an effect on plasma membrane targeting and dimerization of the protein. In response to oxidizing agents, StSUT1 is targeted to lipid raft-like microdomains and SUT1 protein is detectable in the detergent resistant membrane fraction of plant plasma membranes. Interestingly, StSUT1 treated with brefeldin A seems to aggregate in endocytic compartments in mature sieve elements.1 Further analysis of SUT1 targeting will certainly provide more information about the putative involvement of lipid raft-like microdomains in endocytic events. We provide here additional information on the dimerization and endocytosis of the SUT1 protein. The oligomerization of overexpressed SoSUT1 from Spinacia oleracea in transgenic potato plants was analyzed by two-dimensional gel electrophoresis and endocytosis of the StSUT1 protein was confirmed by immunogold labeling.

Sucrose transporter StSUT4 from potato affects flowering, tuberization, and shade avoidance response.

Sucrose (Suc) transporters belong to a large gene family. The physiological role of SUT1 proteins has been intensively investigated in higher plants, whereas that of SUT4 proteins is so far unknown. All three known Suc transporters from potato (Solanum tuberosum), SUT1, SUT2, and SUT4, are colocalized and their RNA levels not only follow a diurnal rhythm, but also oscillate in constant light. Here, we examined the physiological effects of transgenic potato plants on RNA interference (RNAi)-inactivated StSUT4 expression. The phenotype of StSUT4-RNAi plants includes early flowering, higher tuber production, and reduced sensitivity toward light enriched in far-red wavelength (i.e. in canopy shade). Inhibition of StSUT4 led to tuber production of the strict photoperiodic potato subsp. andigena even under noninductive long-day conditions. Accumulation of soluble sugars and Suc efflux from leaves of transgenic plants are modified in StSUT4-RNAi plants, leading to modified Suc levels in sink organs. StSUT4 expression of wild-type plants is induced by gibberellins and ethephon, and external supply of gibberellic acid leads to even more pronounced differences between wild-type and StSUT4-RNAi plants regarding tuber yield and internode elongation, indicating a reciprocal regulation of StSUT4 and gibberellins.