Immunolocalization of mannitol dehydrogenase in celery plants and cells.

Immunolocalization of mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) suspension cells and plants showed that MTD is a cytoplasmic enzyme. MTD was found in the meristems of celery root apices, in young expanding leaves, in the vascular cambium, and in the phloem, including sieve-element/companion cell complexes, parenchyma, and in the exuding phloem sap of cut petioles. Suspension cells that were grown in medium with mannitol as the sole carbon source showed a high anti-MTD cross-reaction in the cytoplasm, whereas cells that were grown in sucrose-containing medium showed little or no cross-reaction. Gel-blot analysis of proteins from vascular and nonvascular tissues of mature celery petioles showed a strong anti-MTD sera cross-reactive band, corresponding to the 40-kD molecular mass of MTD in vascular extracts, but no cross-reactive bands in nonvascular extracts. The distribution pattern of MTD within celery plants and in cell cultures that were grown on different carbon sources is consistent with the hypothesis that the Mtd gene may be regulated by sugar repression. Additionally, a developmental component may regulate the distribution of MTD within celery plants.     

Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol-secreting fungal pathogen Alternaria alternata

Our previous observation that host plant extracts induce production and secretion of mannitol in the tobacco pathogen Alternaria alternata suggested that, like their animal counterparts, plant pathogenic fungi might produce the reactive oxygen quencher mannitol as a means of suppressing reactive oxygen-mediated plant defenses. The concurrent discovery that pathogen attack induced mannitol dehydrogenase (MTD) expression in the non-mannitol-containing host tobacco suggested that plants, unlike animals, might be able to counter this fungal suppressive mechanism by catabolizing mannitol of fungal origin. To test this hypothesis, transgenic tobacco plants constitutively expressing a celery Mtd cDNA were produced and evaluated for potential changes in resistance to both mannitol- and non-mannitol-secreting pathogens. Constitutive expression of the MTD transgene was found to confer significantly enhanced resistance to A. alternata, but not to the non-mannitol-secreting fungal pathogen Cercospora nicotianae. These results are consistent with the hypothesis that MTD plays a role in resistance to mannitol-secreting fungal plant pathogens.     

Is there leaderless protein secretion in plants?

The sugar alcohol mannitol and it’s catabolic enzyme mannitol dehydrogenase (MTD), in addition to welldocumented roles in metabolism and osmoprotection, may play roles in hostpathogen interactions. Research suggests that in response to the mannitol that pathogenic fungi secrete to suppress reactive oxygen-mediated host defenses, plants make MTD to catabolize fungal mannitol. Yet previous work suggested that pathogen-secreted mannitol is extracellular, while in healthy plants MTD is cytoplasmic. We have presented results showing that the normally cytoplasmic MTD is exported into the cell wall or extracellular space in response to the endogenous inducer of plant defense responses salicylic acid (SA). This SA-induced secretion is insensitive to brefeldin A, an inhibitor of Golgimediated protein transport. Together with the absence of MTD in Golgi stacks and the lack of a documented extracellular targeting sequence in the MTD protein, this suggests MTD is secreted by a non-Golgi, pathogen-activated secretion mechanism in plants. Here we discuss the potential significance of non-Golgi secretion in response to stress.Key words: protein secretion, mannitol metabolism, plant-pathogen interaction, extracellular space, apoplast     

Salicylic acid stimulates secretion of the normally symplastic enzyme mannitol dehydrogenase: a possible defense against mannitol-secreting fungal pathogens

The sugar alcohol mannitol is an important carbohydrate with well-documented roles in both metabolism and osmoprotection in many plants and fungi. In addition to these traditionally recognized roles, mannitol is reported to be an antioxidant and as such may play a role in host–pathogen interactions. Current research suggests that pathogenic fungi can secrete mannitol into the apoplast to suppress reactive oxygen-mediated host defenses. Immunoelectron microscopy, immunoblot, and biochemical data reported here show that the normally symplastic plant enzyme, mannitol dehydrogenase (MTD), is secreted into the apoplast after treatment with the endogenous inducer of plant defense responses salicylic acid (SA). In contrast, a cytoplasmic marker protein, hexokinase, remained cytoplasmic after SA-treatment. Secreted MTD retained activity after export to the apoplast. Given that MTD converts mannitol to the sugar mannose, MTD secretion may be an important component of plant defense against mannitol-secreting fungal pathogens such as Alternaria. After SA treatment, MTD was not detected in the Golgi apparatus, and its SA-induced secretion was resistant to brefeldin A, an inhibitor of Golgi-mediated protein transport. Together with the absence of a known extracellular targeting sequence on the MTD protein, these data suggest that a plant’s response to pathogen challenge may include secretion of selected defensive proteins by as yet uncharacterized, non-Golgi mechanisms.     

Formation of a hexokinase complex is associated with changes in energy utilization in celery organs and cells

We previously presented evidence that the hexose‐regulated repression of the mannitol catabolic enzyme mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) may be mediated by hexokinase (EC 2.7.1.1) (HK) [Prata et al. (1997) Plant Physiol 114: 307–314]. To see if differential regulation of HK forms might be involved in the sugar‐regulated repression of MTD we characterized two forms of HK with respect to their expression in various plant organs as well as in celery suspension cell cultures. We found that the vast majority of HK activity was membrane‐associated, whereas fructokinase (EC 2.7.1.4) was found largely in the soluble cell fraction. Gel filtration chromatography further revealed the differential expression of two molecular size classes of HK. One HK (HK‐L) chromatographed at 68 kDa, a typical size for a plant HK, while the second (HK‐H) chromatographed at 280 kDa. This unique 280 kDa HK was shown to be composed of a 50 kDa HK protein, possibly complexed with other, as yet unidentified, components. The HK‐L was present in all cells and organs analyzed, and thus may be a likely candidate for mediation of sugar repression. In contrast, the presence of the HK‐H complex was specific to certain organs and cells grown under certain conditions. Our analyses here showed no correlation between the presence of the HK‐H and MTD repression or derepression in celery cells. Instead, the HK‐H complex was present exclusively in rapidly growing organs and cells, but not in non‐growing celery storage tissues or in carbon‐depleted celery suspension‐cultured cells. Furthermore, the HK‐H complex was present when Glc in the growth media was replaced with 2‐deoxy Glc, a HK substrate that does not provide energy for growth and metabolism. These results imply that the HK‐H complex may have a potentially unique role in the metabolism of rapidly growing celery cells, in particular, in hexose phosphorylation. We also found that mitochondria prepared from Glc‐grown celery suspension‐cultured cells contained substantial HK activity, and that oxygen uptake of these mitochondria was stimulated by Glc. These results are consistent with the hypothesis that mitochondrial localization of celery HK may play a role in rapid recycling of adenylate.     

Overexpression of mannitol dehydrogenase in zonal geranium confers increased resistance to the mannitol secreting fungal pathogen Botrytis cinerea

The sugar alcohol mannitol is a carbohydrate with well-documented roles in both metabolism and osmoprotection in plants and fungi. In addition, however, mannitol is an antioxidant, and current research suggests that pathogenic fungi can secrete mannitol into the plant’s extracellular spaces during infection to suppress reactive oxygen-mediated host defenses. In response to pathogen attack, plants have been shown to secrete the normally symplastic enzyme, mannitol dehydrogenase (MTD). Given that MTD converts mannitol to the sugar mannose, extracellular MTD may be an important defense against mannitol-secreting fungal pathogens. Previous work demonstrated that overexpression of MTD in tobacco did, in fact, provide increased resistance to the mannitol-secreting fungal pathogen Alternaria alternata. In the present work we demonstrate that the fungal pathogen Botrytis cinerea also can secrete mannitol, and that overexpression of MTD in zonal geranium (Pelargonium × hortorum) in turn provides increased resistance to B. cinerea. These results are not only an important validation of previous work, but support the idea that MTD-overexpression might be used to engineer a broad variety of plants for resistance to mannitol-secreting fungal pathogens like B. cinerea for which specific resistance is lacking.     

果实中糖的运输、代谢与积累及其调控

叶片光合产物向果实运输的主要形态是蔗糖,但在木本蔷薇科果树中,光合产物的主要运输形态为山梨醇.糖从质外体空间跨膜运入共质体的过程由糖运输蛋白介导,而糖运输蛋白的基因表达伴随着果实糖的积累而增强.蔗糖代谢酶参与了细胞内外4个与糖运输有关的无效循环.己糖代谢抑制是果实糖快速积累的前提.在木本蔷薇科果实中,蔗糖代谢酶活力仍非常活跃,表明蔗糖可能与山梨醇在果实生长发育中都起重要的作用.糖作为信号分子,调节了承担糖运输与代谢的基因的表达.自然环境因子和栽培措施能有效调控糖运输、代谢与积累.反义抑制Ivr基因表达能提高番茄果实含糖量的实验结果表明遗传工程调控糖积累的潜力.阐明糖信号与其它信号互作对糖运输与代谢的调控机制是今后研究的重点.     

Biosynthesis of Sucrose and Mannitol as a Function of Leaf Age in Celery (Apium graveolens L.)

In celery (Apium graveolens L.), the two major translocated carbohydrates are sucrose and the acyclic polyol mannitol. Their metabolism, however, is different and their specific functions are uncertain. To compare their roles in carbon partitioning and sink-source transitions, developmental changes in ¹⁴CO₂ labeling, pool sizes, and key enzyme activities in leaf tissues were examined. The proportion of label in mannitol increased dramatically with leaf maturation whereas that in sucrose remained fairly constant. Mannitol content, however, was high in all leaves and sucrose content increased as leaves developed. Activities of mannose-6-P reductase, cytoplasmic and chloroplastic fructose-1,6-bisphosphatases, sucrose phosphate synthase, and sucrose synthase increased with leaf maturation and decreased as leaves senesced. Ribulose bisphosphate carboxylase and nonreversible glyceraldehyde-3-P dehydrogenase activities rose as leaves developed but did not decrease. Thus, sucrose is produced in all photosynthetically active leaves whereas mannitol is synthesized primarily in mature leaves and stored in all leaves. Onset of sucrose export in celery may result from sucrose accumulation in expanding leaves, but mannitol export is clearly unrelated to mannitol concentration. Mannitol export, however, appears to coincide with increased mannitol biosynthesis. Although mannitol and sucrose arise from a common precursor in celery, subsequent metabolism and transport must be regulated separately.     

Expression of a celery mannose 6-phosphate reductase in Arabidopsis thaliana enhances salt tolerance and induces biosynthesis of both mannitol and a glucosyl-mannitol dimer

Mannitol, a sugar alcohol that may serve as a compatible solute to cope with salt stress, is synthesized via the action of a mannose‐6‐phosphate reductase (M6PR) in celery (Apium graveolens L). In contrast to previous approaches that have used a bacterial gene to engineer mannitol biosynthesis in plants and other organisms, Arabidopsis thaliana, a non‐mannitol producer, was transformed with the celery leaf M6PR gene under control of the CaMV 35S promotor. In all independent Arabidopsis M6PR transformants, mannitol accumulated throughout the plants in amounts ranging from 0·5 to 6 µmol g^?1 fresh weight. A novel compound, not found in either celery or Arabidopsis, 1‐O‐β‐d ‐glucopyranosyl‐d ‐mannitol, also accumulated in vegetative tissues of mature plants in amounts up to 4 µmol g^?1 fresh weight, but not in flowers and seeds. In the absence of NaCl, all transformants were phenotypically the same as the wild type; however, in the presence of NaCl, mature transgenic plants showed a high level of salt tolerance, i.e. growing, completing normal development, flowering, and producing seeds in soil irrigated with 300 mm NaCl in the nutrient solution. These results demonstrate a major role in developing salt‐tolerant plants by means of introducing mannitol biosynthesis using M6PR.     

Sugar Repression of Mannitol Dehydrogenase Activity in Celery Cells

We present evidence that the activity of the mannitol-catabolizing enzyme mannitol dehydrogenase (MTD) is repressed by sugars in cultured celery (Apium graveolens L.) cells. Furthermore, this sugar repression appears to be mediated by hexokinases (HKs) in a manner comparable to the reported sugar repression of photosynthetic genes. Glucose (Glc)-grown cell cultures expressed little MTD activity during active growth, but underwent a marked increase in MTD activity, protein, and RNA upon Glc starvation. Replenishment of Glc in the medium resulted in decreased MTD activity, protein, and RNA within 12 h. Addition of mannoheptulose, a competitive inhibitor of HK, derepressed MTD activity in Glc-grown cultures. In contrast, the addition of the sugar analog 2-deoxyglucose, which is phosphorylated by HK but not further metabolized, repressed MTD activity in mannitol-grown cultures. Collectively, these data suggest that HK and sugar phosphorylation are involved in signaling MTD repression. In vivo repression of MTD activity by galactose (Gal), which is not a substrate of HK, appeared to be an exception to this hypothesis. Further analyses, however, showed that the products of Gal catabolism, Glc and fructose, rather than Gal itself, were correlated with MTD repression.     

Comparative studies of sucrose and mannitol utilization in celery (Apium graveolens)

Utilization of sucrose and mannitol, the major forms of translocatable assimilate in celery ( Apium graveolens L. cv. Giant Pascal), was investigated in intact plants, excised leaves and leaf discs by estimating the soluble carbohydrate pools, starch levels and oxidation of [¹⁴C]-sucrose or mannitol in the light and after extended dark treatments. In detached mature fully-expanded leaves, mannitol pools remained constant, while sucrose decreased during a 48 h dark treatment. In attached leaves on plants trimmed to a single compound leaf, however, mannitol levels decreased after a dark treatment. In leaf discs floated on bathing solutions containing [¹⁴C]-sucrose or [¹⁴C]-mannitol, oxidation of mannitol was restricted to young leaf tissues, whereas sucrose was metabolized to CO₂ regardless of leaf age. Uptake of labelled mannitol, however, was greater than that of sucrose in the light in leaves of every age. Although both mannitol and sucrose are translocated out of leaf tissues, leaf age differences indicate that, unlike sucrose, mannitol utilization is restricted to active sink tissues. The results suggest different roles for mannitol and sucrose with mannitol representing a more rigorously sequestered transport carbohydrate.     

Kanamycin reveals the role played by glutamate receptors in shaping plant resource allocation

Ionotropic glutamate receptors (iGluRs) play important roles in neurotransmission in animals. There is growing evidence that iGluRs also play important roles in plants. Using a chemical genetics approach, which combined a pH-homeostasis mutant of Arabidopsis thaliana (de-etiolated3), several different iGluR agonists, molecular modelling, and reporter gene expression in transgenic plants, we provide evidence that iGluR agonism can induce dramatic changes in plant development and metabolism. Systematic hypothesis testing revealed a signalling circuit that integrates amino acid and sugar signals to affect elongation growth and the deposition of carbon into starch and lignins. The data show that aminoglycoside antibiotics, such as kanamycin, and polyamines impinge upon this circuit. These findings provide a mechanism for the conversion of amino acid and sugar signals into an appropriate response at the gene expression level, and underline the similarities in iGluR agonism between animals and plants.     

Mannose-6-Phosphate Reductase,a Key Enzyme in Photoassimilate Partitioning,Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves

Mannitol, a major photosynthetic product and transport carbohydrate in many plants, accounts for approximately 50% of the carbon fixed by celery (Apium graveolens L.) leaves. Previous subfractionation studies of celery leaves indicated that the enzymes for mannitol synthesis were located in the cytosol, but these data are inconsistent with that published for the sites of sugar alcohol synthesis in other families and taxa, including apple (Malus) and a brown alga (Fucus). Using antibodies to a key synthetic enzyme, NADPH-dependent mannose-6-phosphate reductase (M6PR), and immunocytochemical techniques, we have resolved both the inter-cellular and intracellular sites of mannitol synthesis. In leaves, M6PR was found only in cells containing ribulose-1,5-bisphosphate carboxylase/oxygenase. M6PR was almost exclusively cytosolic in these cells, with the nucleus being the only organelle to show labeling. The key step in transport carbohydrate biosynthesis that is catalyzed by M6PR displays no apparent preferential association with vascular tissues or with the bundle sheath. These results show that M6PR and, thus, mannitol synthesis are closely associated with the distribution of photosynthetic carbon metabolism in celery leaves. The principal role of M6PR is, therefore, in the assimilation of carbon being exported from the chloroplast, and it seems unlikely that this enzyme plays even an indirect role in phloem loading of mannitol.     

The function of trehalose biosynthesis in plants

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranoside) occurs in a large variety of organisms, ranging from bacteria to invertebrate animals, where it serves as an energy source or stress protectant. Until recently, only few plant species, mainly desiccation-tolerant 'resurrection' plants, were considered to synthesise trehalose. Instead of trehalose, most other plants species accumulate sucrose as major transport sugar and during stress. The ability to synthesize sucrose has probably evolved from the cyanobacterial ancestors of plastids and may be linked to photosynthetic function. Although most plant species do not appear to accumulate easily detectable amounts of trehalose, the discovery of genes for trehalose biosynthesis in Arabidopsis and in a range of crop plants suggests that the ability to synthesise trehalose is widely distributed in the plant kingdom. The apparent lack of trehalose accumulation in these plants is probably due to the presence of trehalase activity. After inhibition of trehalase, trehalose synthesis can be detected in Arabidopsis. Since trehalose induces metabolic changes, such as an accumulation of storage carbohydrates, rapid degradation of trehalose may be required to prevent detrimental effects of trehalose on the regulation of plant metabolism. In addition, the precursor of trehalose, trehalose-6-phosphate, is probably involved in the regulation of developmental and metabolic processes in plants.     

Gamma Aminobutyric Acid (GABA) and Plant Responses to Stress

4-aminobutyrate (GABA) is a non-protein amino acid that is widely distributed throughout the biological world. In animals, GABA functions as the predominant inhibitory neurotransmitter in the central nervous system by acting through the GABA receptors. The neuromuscular system enables animals to escape from environmental stresses. Being nonmotile, plants have evolved chemical responses to mitigate stress. Mechanisms by which GABA may facilitate these responses are discussed in this review. Environmental stresses increase GABA accumulation through two different mechanisms. Stresses causing metabolic and/or mechanical disruptions, resulting in cytosolic acidification, induce an acidic pH-dependent activation of glutamate decarboxylase and GABA synthesis. Extremely marked declines in cytosolic pH occur under oxygen deprivation, which is the primary stress factor in flooded soils, and this stress induces the greatest accumulation of GABA. Other stresses, including cold, heat, salt, and mild or transient environmental factors, such as touch, wind, rain, etc. rapidly increase cellular levels of Ca²⁺. Increased cytosolic Ca²⁺ stimulates calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. A review of the kinetics of GABA accumulation in plants reveals a stress-specific pattern of accumulation that is consistent with a physiological role for GABA in stress mitigation. Recent physiological and genetic evidence indicates that plants may possess GAB A-like receptors that have features in common with the animal receptors. The mechanism of action of animal GABA receptors suggests a model for rapid amplification of ion-mediated signals and GABA accumulation in response to stress. Metabolic pathways that link GABA to stress-related metabolism and plant hormones are identified. The survival value of stress-related metabolism is dependent on metabolic changes occurring before stress causes irreversible damage to plant tissue. Rapid accumulation of GABA in stressed tissue may provide a critical link in the chain of events leading from perception of environmental stresses to timely physiological responses.     

Phospholipases C and D modulate proline accumulation in Thellungiella halophila/salsuginea differently according to the severity of salt or hyperosmotic stress

Proline accumulation is one of the most common responses of plants to environmental constraints. Thellungiella halophila/salsuginea, a model halophyte, accumulates high levels of proline in response to abiotic stress and in the absence of stress. Recently, lipid signaling pathways have been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. Here we investigated the relationship between lipid signaling enzymes and the level of proline in T. salsuginea. Inhibition of phospholipase C (PLC) enzymes by the specific inhibitor U73122 demonstrated that proline accumulation is negatively controlled by PLCs in the absence of stress and under moderate salt stress (200 mM NaCl). The use of 1-butanol to divert some of the phospholipase D (PLD)-derived phosphatidic acid by transphosphatidylation revealed that PLDs exert a positive control on proline accumulation under severe stress (400 mM NaCl or 400 mM mannitol) but have no effect on its accumulation in non-stress conditions. This experimental evidence shows that positive and negative lipid regulatory components are involved in the fine regulation of proline metabolism. These signaling pathways in T. salsuginea are regulated in the opposite sense to those previously described in A. thaliana, revealing that common signaling components affect the physiology of closely related glycophyte and salt-tolerant plants differently.     

Gas Exchange and Carbon Partitioning in the Leaves of Celery (Apium graveolens L.) at Various Levels of Root Zone Salinity

Both mannitol and sucrose (Suc) are primary photosynthetic products in celery (Apium graveolens L.). In other biological systems mannitol has been shown to serve as a compatible solute or osmoprotectant involved in stress tolerance. Although mannitol, like Suc, is translocated and serves as a reserve carbohydrate in celery, its role in stress tolerance has yet to be resolved. Mature celery plants exposed to low (25 mM NaCl), intermediate (100 mM NaCl), and high (300 mM NaCl) salinities displayed substantial salt tolerance. Shoot fresh weight was increased at low NaCl concentrations when compared with controls, and growth continued, although at slower rates, even after prolonged exposure to high salinities. Gas-exchange analyses showed that low NaCl levels had little or no effect on photosynthetic carbon assimilation (A), but at intermediate levels decreases in stomatal conductance limited A, and at the highest NaCl levels carboxylation capacity (as measured by analyses of the CO2 assimilation response to changing internal CO2 partial pressures) and electron transport (as indicated by fluorescence measurements) were the apparent prevailing limits to A. Increasing salinities up to 300 mM, however, increased mannitol accumulation and decreased Suc and starch pools in leaf tissues, e.g. the ratio of mannitol to Suc increased almost 10-fold. These changes were due in part to shifts in photosynthetic carbon partitioning (as measured by 14C labeling) from Suc into mannitol. Salt treatments increased the activity of mannose-6-phosphate reductase (M6PR), a key enzyme in mannitol biosynthesis, 6-fold in young leaves and 2-fold in fully expanded, mature leaves, but increases in M6PR protein were not apparent in the older leaves. Mannitol biosynthetic capacity (as measured by labeling rates) was maintained despite salt treatment, and relative partitioning into mannitol consequently increased despite decreased photosynthetic capacity. The results support a suggested role for mannitol accumulation in adaptation to and tolerance of salinity stress.