Relationship between hexokinase and cytokinin in the regulation of leaf senescence and seed germination.

Arabidopsis hexokinase (AtHXK1), an enzyme that catalyses hexose phosphorylation, accelerates leaf senescence, whereas the plant hormone cytokinin inhibits senescence. Previous work in our laboratory has shown that isopentenyl transferase (IPT), a key gene in the biosynthesis of cytokinin, expressed under promoters of the senescence-associated genes SAG12 or SAG13 (P(SAG12)::IPT and P(SAG13)::IPT, respectively), inhibits leaf senescence in tomato plants. To study the relationship between hexokinase and cytokinin in the regulation of leaf senescence, we created and analysed double-transgenic tomato plants expressing both AtHXK1 and either P(SAG12)::IPT or P(SAG13)::IPT. We found that expression of IPT in the double-transgenic plants could not prevent the accelerated senescence induced by over-expression of AtHXK1. Since cytokinin inhibits senescence via an apoplastic invertase that produces extracellular hexoses, whereas AtHXK1 is an intracellular mitochondria-associated hexokinase, our results suggest that intracellular sugar sensing via AtHXK1 is dominant over extracellular sugar sensing with regard to leaf senescence. Interestingly, the heterologous SAG12 and SAG13 promoters are also expressed in germinating tomato seed, around the radicle penetration zone, suggesting that seed germination involves a senescence process that is probably necessary for radicle emergence. Indeed, seed expressing P(SAG12)::IPT and P(SAG13)::IPT exhibited delayed radicle emergence, possibly due to delayed endosperm senescence.

Effects of cytokinin production under two SAG promoters on senescence and development of tomato plants.

Two promoters of senescence-associated ARABIDOPSIS genes, SAG12 and SAG13, were used in tomato plants to express IPT that catalyzes the rate-limiting step in cytokinin biosynthesis. Expression of these heterologous promoters in tomato plants was analyzed using the reporter gene beta-glucuronidase. Both promoters are expressed in tomato leaves in a manner similar to their expression in ARABIDOPSIS plants. The SAG12 promoter is very specific to senescing leaves, whereas the SAG13 promoter is expressed in mature leaves prior to the onset of visible senescence and its expression increases in senescing leaves. Expression of both promoters in tomato tissues other than leaves was very low . IPT expressed under the control of SAG12 and SAG13 promoters ( PSAG12::IPT and PSAG13::IPT, respectively) resulted in suppression of leaf senescence and advanced flowering, as well as in a slight increase in fruit weight and fruit total soluble solids (TSS). However, expression of PSAG13::IPT also led to stem thickening, short internodal distances and loss of apical dominance. In contrast to the autoregulation of PSAG12::IPT, PSAG13::IPT is expressed at higher levels in mature leaves. This difference is likely due to PSAG13::IPT exhibiting two phases of expression - a senescence-independent expression prior to the onset of senescence that is not subjected to autoregulation by cytokinin, and enhanced expression throughout senescence which is autoregualted by cytokinin. This moderate different autoregulated behavior of PSAG12::IPT and PSAG13::IPT markedly influenced plant development, emphasizing the biological effects of cytokinin in addition to senescence inhibition.

Two newly identified membrane-associated and plastidic tomato HXKs: characteristics, predicted structure and intracellular localization.

Two new tomato hexokinase genes, LeHXK3 and LeHXK4, were cloned and characterized, placing tomato as the first plant with four characterized HXK genes. Based on their sequence, LeHXK3 is the third membrane-associated (type-B) and LeHXK4 is the first plastidic (type-A) HXK identified in tomato. Expression of HXK-GFP fusion proteins in protoplasts indicated that the LeHxk3 enzyme is associated with the mitochondria while LeHxk4 is localized in plastids. Furthermore, LeHxk4::GFP fusion protein is found within stromules, suggesting transport of LeHxk4 between plastids. Structure prediction of the various plant HXK enzymes suggests that unlike the plastidic HXKs, the predicted membrane-associated HXKs are positively charged near their putative N-terminal membrane anchor domain, which might enhance their association with the negatively charged membranes. LeHxk3 and LeHxk4 were analyzed following expression in yeast. Both enzymes have higher affinity for glucose relative to fructose and are inhibited by ADP. Yet, unlike the other HXKs, the stromal HXK has higher Vmax with glucose than with fructose. Expression analysis of the four HXK genes in tomato tissues demonstrated that LeHXK1 and LeHXK4 are the dominant HXKs in all tissues examined. Notably, the plastidic LeHXK4 is expressed in all tissues including starchless, non-photosynthetic sink tissues, such as pink and red fruits, implying phosphorylation of imported hexoses in plastids. It has been suggested that trehalose 6-phosphate (T6P) might inhibit HXK activity. However, none of the yeast-expressed tomato HXK genes was sensitive either to T6P or to trehalose, suggesting that unlike fungi HXKs, plant HXKs are not regulated by T6P.

Labeling fibroblasts with biotin-BSA-GdDTPA-FAM for tracking of tumor-associated stroma by fluorescence and MR imaging.

Fibroblasts at the tumor-host interface can differentiate into myofibroblasts and pericytes, and contribute to the guidance and stabilization of endothelial sprouts. After intravenous administration of biotin-BSA-GdDTPA-FAM in mice with subcutaneous MLS human ovarian carcinoma tumors, the distribution of the macromolecular MRI/optical contrast material was confined to blood vessels in normal tissues, while it co-registered with alphaSMA-positive stroma tracks within the tumor. These alphaSMA-positive tumor-associated myofibroblasts and pericytes showed uptake of the contrast material into intracellular granules. We evaluated the use of this contrast material for in vitro labeling of tumor fibroblasts as an approach for tracking their involvement in angiogenesis. Fluorescence microscopy demonstrated internalization of the contrast material, and MRI revealed a significant increase in the R(1) relaxation rate of labeled fibroblasts. R(1) not only remained elevated for 2 weeks in culture, it also increased with cell proliferation, indicating prolonged retention of the contrast material and subsequent intracellular processing and redistribution of the material, and thereby enhancing MR contrast. Moreover, cells that were labeled ex vivo with MR contrast material and co-inoculated with tumor cells in mice were detected in vivo by MRI. Uptake of the contrast material was suppressed by nystatin, suggesting internalization by caveolae-mediated endocytosis. This study shows that labeling of fibroblasts with biotin-BSA-GdDTPA-FAM is feasible and would allow noninvasive in vivo tracking of fibroblasts during tumor angiogenesis and vessel maturation.

Characterization of native and yeast-expressed tomato fruit fructokinase enzymes.

Three fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.

Vesicle-associated membrane protein of Arabidopsis suppresses Bax-induced apoptosis in yeast downstream of oxidative burst.

Programmed cell death (PCD) in many systems is controlled by relative amounts of the apoptosis-regulating proteins Bax and Bcl-2 through homo- or heterodimerization. Here we show that Bax-induced PCD of yeast was suppressed by transformation with a vesicle-associated membrane protein from Arabidopsis (AtVAMP), which was isolated by screening a cDNA expression library against sugar-induced cell death in yeast. AtVAMP expression blocked Bax-induced PCD downstream of oxidative burst. AtVAMP also prevented H(2)O(2)-induced apoptosis in yeast and in Arabidopsis cells. Reduced oxidation of lipids and plasma membrane proteins was detected in the AtVAMP-transformed yeast, suggesting improved membrane repair. Inhibition of intracellular vesicle trafficking by brefeldin A induced apoptosis from a sublethal concentration of H(2)O(2). No protection occurred by overexpression of the yeast homolog SCN2. However, efficient suppression of yeast PCD occurred by expression of a chimeric gene, composed of the conserved domains from yeast, fused to the variable N-terminal domain from Arabidopsis, resulting in exchange of the proline-rich N-terminal domain of SCN2 with a proline-poor Arabidopsis sequence. Our results suggest that intracellular vesicle traffic can regulate execution of apoptosis by affecting the rate of membrane recycling and that the proline-rich N-terminal domain of VAMP inhibited this process.

Small is mighty: EmrE, a multidrug transporter as an experimental paradigm.

EmrE is a multidrug transporter from Escherichia coli that functions as a homooligomer and is unique in its small size. In each monomer there are four tightly packed transmembrane segments and one membrane-embedded charged residue. This residue provides the basis for the coupling mechanism as part of a binding site "time shared" by substrates and protons.

Precious things come in little packages.

The 110-amino acid multidrug transporter from E. coli, EmrE, is a member of the family of MiniTexan or Smr drug transporters. EmrE can transport acriflavine, ethidium bromide, tetraphenylphosphonium (TPP+), benzalkonium and several other drugs with relatively high affinities. EmrE is an H+/drug antiporter, utilizing the proton electrochemical gradient generated across the bacterial cytoplasmic membrane by exchanging two protons with one substrate molecule. The EmrE multidrug transporter is unique in its small size and hydrophobic nature. Hydropathic analysis of the EmrE sequence predicts four alpha-helical transmembrane segments. This model is experimentally supported by FTIR studies that confirm the high alpha-helicity of the protein and by high-resolution heteronuclear NMR analysis of the protein structure. The TMS of EmrE are tightly packed in the membrane without any continuous aqueous domain, as was shown by Cysteine scanning experiments. These results suggest the existence of a hydrophobic pathway through which the substrates are translocated. EmrE is functional as a homo-oligomer as suggested by several lines of evidence, including co-reconstitution experiments of wild-type protein with inactive mutants in which negative dominance has been observed. EmrE has only one membrane embedded charged residue, Glu-14, that is conserved in more than fifty homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions. We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons. The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes. Because of some of its properties and its size, EmrE provides a unique system to understand mechanisms of substrate recognition and translocation.

Cloning and characterization of a cDNA encoding hexokinase from tomato.

Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransferase, EC 2.7.1.1) were isolated from tomato (Lycopersicon esculentum) by RT-PCR using degenerate primers. Southern blot analysis suggested the existence of two divergent HXK genes. A complete cDNA of one HXK was isolated by screening a cDNA library prepared from young cherry tomato fruit. The 1770 bp cDNA of LeHXK2 contained an open reading frame encoding a 496 amino acid protein that has 69% identity with the two Arabidopsis HXKs, 83 and 85% identity with potato StHXK1 and tobacco NtHXK, respectively. However, this clone had 97% amino acid identity with potato StHXK2 and, therefore, was named LeHXK2. LeHXK2 cDNA was expressed in a triple mutant yeast (Saccharomyces cerevisiae) strain which lacked the ability to phosphorylate glucose and fructose and, therefore, was unable to grow on these sugars as carbon sources. Mutant cells expressing LeHXK2 grew on both glucose and fructose with shorter doubling time on glucose. The kinetic properties of LeHXK2 expressed in yeast were determined after the purification of LeHXK2 by HPLC-ion exchange chromatography, confirming the identity of LeHXK2 as hexokinase with higher affinity to glucose. LeHXK2 mRNA was detected by RT-PCR expression analysis in all organs and tissues and at all stages of fruit development. However, semi-quantitative RT-PCR analysis showed that LeHXK2 was most highly expressed in flowers.

Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence.

Sugars are key regulatory molecules that affect diverse processes in higher plants. Hexokinase is the first enzyme in hexose metabolism and may be a sugar sensor that mediates sugar regulation. We present evidence that hexokinase is involved in sensing endogenous levels of sugars in photosynthetic tissues and that it participates in the regulation of senescence, photosynthesis, and growth in seedlings as well as in mature plants. Transgenic tomato plants overexpressing the Arabidopsis hexokinase-encoding gene AtHXK1 were produced. Independent transgenic plants carrying single copies of AtHXK1 were characterized by growth inhibition, the degree of which was found to correlate directly to the expression and activity of AtHXK1. Reciprocal grafting experiments suggested that the inhibitory effect occurred when AtHXK1 was expressed in photosynthetic tissues. Accordingly, plants with increased AtHXK1 activity had reduced chlorophyll content in their leaves, reduced photosynthesis rates, and reduced photochemical quantum efficiency of photosystem II reaction centers compared with plants without increased AtHXK1 activity. In addition, the transgenic plants underwent rapid senescence, suggesting that hexokinase is also involved in senescence regulation. Fruit weight, starch content in young fruits, and total soluble solids in mature fruits were also reduced in the transgenic plants. The results indicate that endogenous hexokinase activity is not rate limiting for growth; rather, they support the role of hexokinase as a regulatory enzyme in photosynthetic tissues, in which it regulates photosynthesis, growth, and senescence.

Scanning cysteine accessibility of EmrE, an H+-coupled multidrug transporter from Escherichia coli, reveals a hydrophobic pathway for solutes.

EmrE is a 12-kDa Escherichia coli multidrug transporter that confers resistance to a wide variety of toxic reagents by actively removing them in exchange for hydrogen ions. The three native Cys residues in EmrE are inaccessible to N-ethylmaleimide (NEM) and a series of other sulfhydryls. In addition, each of the three residues can be replaced with Ser without significant loss of activity. A protein without all the three Cys residues (Cys-less) has been generated and shown to be functional. Using this Cys-less protein, we have now generated a series of 48 single Cys replacements throughout the protein. The majority of them (43) show transport activity as judged from the ability of the mutant proteins to confer resistance against toxic compounds and from in vitro analysis of their activity in proteoliposomes. Here we describe the use of these mutants to study the accessibility to NEM, a membrane permeant sulfhydryl reagent. The study has been done systematically so that in one transmembrane segment (TMS2) each single residue was replaced. In each of the other three transmembrane segments, at least four residues covering one turn of the helix were replaced. The results show that although the residues in putative hydrophilic loops readily react with NEM, none of the residues in putative transmembrane domains are accessible to the reagent. The results imply very tight packing of the protein without any continuous aqueous domain. Based on the findings described in this work, we conclude that in EmrE the substrates are translocated through a hydrophobic pathway.

Saccharomyces cerevisiae cells harboring the gene encoding sarcotoxin IA secrete a peptide that is toxic to plant pathogenic bacteria.

Sarcotoxin IA is a cecropin-type antibacterial protein produced by the flesh fly, Sarcophaga peregrina. Similar to other bactericidal small proteins produced by insects, sarcotoxin IA is released into the hemolymph of larvae and nymphs upon mechanical injury or bacterial infection. The gene (sarco) that encodes this toxin was introduced into Saccharomyces cerevisiae yeast cells and was expressed under a constitutive yeast promoter. The transformed yeast cells were grown in a liquid medium, and a peptide with a similar molecular size to that of the mature sarcotoxin IA was detected in the medium by Western blot analysis. The secreted sarcotoxin-like peptide (SLP) had a potent cytotoxic effect against several bacteria, including plant pathogenic bacteria, similar to the toxic effects of the authentic sarcotoxin IA. Erwinia carotovora was more susceptible to the toxic medium than Pseudomonas solanacearum and Pseudomonas syringae pv. lachrymans. Thus, yeast may be used in the production of such proteins for employment against various bacterial pathogens.

Tomato fructokinases exhibit differential expression and substrate regulation

Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expressed in tomato (Lycopersicon esculentum L.) and have now been further characterized with regard to their spatial expression and the enzymic properties of the encoded proteins. Frk1 and Frk2 mRNA levels were coordinately induced by exogenous sugar, indicating that both belong to the growing class of sugar-regulated genes. However, in situ hybridization indicated that Frk1 and Frk2 were expressed in a spatially distinct manner, with Frk2 mRNA primarily localized in cells of the fruit pericarp, which store starch, and Frk1 mRNA distributed ubiquitously in pericarp tissue. To evaluate the biochemical characteristics of the products of the Frk1 and Frk2 genes, each cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line defective in hexose phosphorylation and unable to grow on glucose or fructose (Fru). Both Frk1 and Frk2 proteins expressed in yeast conferred the ability to grow on Fru and exhibited fructokinase activity in vitro. Although both Frk1 and Frk2 both utilized Fru as a substrate, only Frk2 activity was inhibited at high Fru concentrations. These results indicate that Frk2 can be distinguished from Frk1 by its sensitivity to substrate inhibition and by its temporal and spatial pattern of expression, which suggests that it plays a primary role in plant cells specialized for starch storage.

Divergent fructokinase genes are differentially expressed in tomato.

Two cDNA clones (Frk1 and Frk2) encoding fructokinase (EC 2.7.1.4) were isolated from tomato (Lycopersicon esculentum). The Frk2 cDNA encoded a deduced protein of 328 amino acids that was more than 90% identical with a previously characterized potato (Solanum tuberosum) fructokinase. In contrast, the Frk1 cDNA encoded a deduced protein of 347 amino acids that shared only 55% amino acid identity with Frk2. Both deduced proteins possessed and ATP-binding motif and putative substrate recognition site sequences identified in bacterial fructokinases. The Frk1 cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line, which lacks the ability to phosphorylate glucose and fructose and is unable to grow on glucose or fructose. Mutant cells expressing Frk1 were complemented to grow on fructose but not glucose, indicating that Frk1 phosphorylates fructose but not glucose, and this activity was verified in extracts of transformed yeast. The mRNA corresponding to Frk2 accumulated to high levels in young, developing tomato fruit, whereas the Frk1 mRNA accumulated to higher levels late in fruit development. The results indicate that fructokinase in tomato is encoded by two divergent genes, which exhibit a differential pattern of expression during fruit development.

Sugar induced cell death in yeast is dependent on the rate of sugar phosphorylation as determined by Arabidopsis thaliana hexokinase.

Sugars like glucose and fructose induce death of yeast cells within a few hours, in the absence of additional nutrients to support growth, while cells incubated in water remain viable for weeks. This sugar-induced cell death (SICD) by glucose and fructose required glucose or fructose phosphorylation since yeast cells deficient in hexose phosphorylation did not die. However, when hexose phosphorylation is restored by complementation with Arabidopsis thaliana hexokinase, the cells died. The affinity of A. thaliana hexokinase is about 400 times higher for glucose than for fructose, therefore, A. thaliana hexokinase was further utilized to study the role of hexose phosphorylation in SICD. The rate of SICD of hexokinase-deficient yeast cells expressing A. thaliana hexokinase was significantly slower in fructose than in glucose, indicating that SICD is determined by the rate of hexose phosphorylation. The significance of hexose phosphorylation and its role in SICD is discussed.

The 5' untranslated region of Arabidopsis thaliana calmodulin cDNA is an independent cDNA containing an open reading frame.

A cDNA clone, pAUK1, with an open reading frame (ORF) coding for a hypothetical 164-amino-acid protein was isolated from an Arabidopsis thaliana (L.) Heynh cDNA library. The clone was attached, tail to tail, to the 3' end of A. thaliana hexokinase cDNA. An almost identical sequence had been previously described as the 5' untranslated region (5' UTR) of A. thaliana calmodulin cDNA (ACaM-2). Sequence comparison with three additional A. thaliana truncated cDNA clones which appear in a database (GenBank) supports the conclusion that pAUK1 is identical to the 5' UTR of ACaM-2 and that the 5' UTR of ACaM-2 is an independent cDNA artificially linked to A. thaliana calmodulin cDNA.

PCR-generated molecular markers for the invertase gene and sucrose accumulation in tomato.

The green-fruited tomato species, Lycopersicon hirsutum, unlike the domesticated red-fruited species, L. esculentum, accumulates sucrose during the final stages of fruit development, concomitant with the loss of soluble acid invertase activity. In order to study the genetic linkage of sucrose accumulation to the invertase gene, part of the invertase gene from L. hirsutum was cloned, sequenced and the sequence compared with the invertase sequence of the red-fruited L. esculentum. Several base changes were found in the coding region of the two invertase genes. Based on these base -pair differences, we developed a species-specific PCR assay capable of determining, in a single PCR reaction, the origin of the invertase gene in segregating seedlings of an interspecific cross. Our results indicate that the invertase gene is genetically linked to sucrose accumulation in the green-fruited L. hirsutum.

Carbon source induces growth of stationary phase yeast cells, independent of carbon source metabolism.

Nutrients regulate the proliferation of many eukaryotic cells: in the absence of sufficient nutrients vegetatively growing cells will enter stationary (G0 like) phase; in the presence of sufficient nutrients non-proliferative cells will begin growth. Previously we have shown that glucose is the critical nutrient which stimulates a variety of growth-related events in the yeast Saccharomyces cerevisiae (Granot and Snyder, 1991). This paper describes six new aspects of the induction of cell growth events by nutrients in S. cerevisiae. First, all carbon sources tested, both fermentable and non-fermentable, induce growth-related events in stationary phase cells, suggesting that the carbon source is the critical nutrient which stimulates growth. Second, the continuous presence of glucose is not necessary for the induction of growth events, but rather a short 'pulse' of glucose followed by an incubation period in water will induce growth events. Third, growth stimulation by glucose occurs in the absence of the SNF3 high affinity glucose transporter. Fourth, growth stimulation occurs independent of carbon source phosphorylation and carbon source metabolism. Fifth, growth induction by carbon source does not require protein synthesis or extracellular calcium. Sixth, following stimulation by carbon source, the cells remain induced for more than 2 h after removal of the carbon source. We suggest a general model in which different carbon sources act as signals to induce the earliest growth events during or following its entry into the cell and that these growth events do not depend upon metabolism of the carbon source.

Glucose induces cAMP-independent growth-related changes in stationary-phase cells of Saccharomyces cerevisiae.

Nutrients play a critical role in the decision to initiate a new cell cycle. Addition of nutrients to arrested cells such as stationary-phase cells and spores induces them to begin growth. We have analyzed the nutrients required to induce early cellular events in yeast. When stationary-phase cells or spores are incubated in the presence of only glucose, morphological and physiological changes characteristic of mitotically growing cells are induced and, in the absence of additional nutrients to support growth, the cells rapidly lose viability. Preincubation of stationary-phase cells in the presence of glucose decreases the time required to reach bud emergence upon the subsequent addition of rich medium. These processes are specifically induced by D-glucose and not by other components such as nitrogen source or L-glucose. The glucose-induced events are independent of the adenylate cyclase pathway, since strains with a temperature-sensitive mutation in either the adenylate cyclase gene (CDC35) or its regulator (CDC25) undergo glucose-induced cellular changes when incubated at the restrictive temperature. We suggest that glucose triggers events in the induction of a new mitotic cell cycle and that these events are either prior to the adenylate cyclase pathway or are in an alternative pathway.