Regulation of ammonium-induced proline accumulation in detached rice leaves

Accumulation of proline in response to NH₄Cl was studied indetached leaves of rice (Oryza sativa cv. Taichung Native1). Increasing concentrations of NH₄Cl from 50 to 200mMprogressively increased proline content and this was correlated with theincrease in ammonium content. Proline accumulation induced by NH₄Clwas related to proteolysis, an increase in ornithine--aminotransferaseactivity, a decrease in proline dehydrogenase activity, and a decrease inproline utilisation and could not be explained by NH₄Cl-inducedmodification in ¹-pyrroline-5-carboxylate reductase activity.The content of glutamic acid was decreased by NH₄Cl, whereas theincrease in arginine and ornithine contents was found to be associated with theincrease in proline content in NH₄Cl-treated detached rice leaves.     

Biochemical characterization of a spearmint mutant that resembles peppermint in monoterpene content

A radiation-induced mutant of Scotch spearmint (Mentha × gracilis) was shown to produce an essential oil containing principally C3-oxygenated p-menthane monoterpenes that are typical of peppermint, instead of the C6-oxygenated monoterpene family characteristic of spearmint. In vitro measurement of all of the enzymes responsible for the production of both the C3-oxygenated and C6-oxygenated families of monoterpenes from the common precursor (−)-limonene indicated that a virtually identical complement of enzymes was present in wild type and mutant, with the exception of the microsomal, cytochrome P-450-dependent (−)-limonene hydroxylase; the C6-hydroxylase producing (−)-trans-carveol in the wild type had been replaced by a C3-hydroxylase producing (−)-trans-isopiperitenol in the mutant. Additionally, the mutant, but not the wild type, could carry out the cytochrome P-450-dependent epoxidation of the α,β-unsaturated bond of the ketones formed via C3-hydroxylation. Although present in the wild type, the enzymes of the C3-pathway that convert trans-isopiperitenol to menthol isomers are synthetically inactive because of the absence of the key C3-oxygenated intermediate generated by hydroxylation of limonene. These results, which clarify the origins of the C3- and C6-oxygenation patterns, also allow correction of a number of earlier biogenetic proposals for the formation of monoterpenes in Mentha.     

Effect of Zn deficiency on net photosynthetic rate, 14C partitioning,and oil accumulation in leaves of peppermint

Changes in growth, CO₂ exchange rate, and distribution of photosynthetically fixed ¹⁴CO2 into the primary photosynthetic metabolic pool (sugars, amino acids and organic acids) and essential oil accumulation were determined in leaves (leaf positions 1-6 from apex) of developing peppermint grown in a solution culture at Zn concentrations of 0 and 0.05 g m-3. There was a significant decrease in ¹⁴C incorporation in total, ethanol-soluble and ethanol-insoluble fractions in Zn deficient plants at all leaf positions. 14C incorporated in essential oil and in sugars were significantly higher in leaf pairs 1 to 3 than in leaf pairs 4 to 6. ¹⁴C incorporation into amino acids and organic acids was higher in all leaf pairs in Zn deficient plants. Statistical analysis showed a positive significant association between Zn content of leaf and ¹⁴C incorporation into ethanol-soluble fraction and sugars and a negative correlation with ¹⁴C incorporation into amino acids and organic acids. Hence the content of sugars in leaves significantly influences essential oil accumulation under Zn stress. This revised version was published online in September 2006 with corrections to the Cover Date.     

Drought-stress-induced polypeptide accumulation in tomato leaves

Abstract. Tomato ( Lycopersicon esculentum Mill ev. Ailsa Craig) leaf polypeptides, radiolabelled with L-[³⁶S] methionine during drought stress, were separated by two-dimensional polyacrylamide gel electrophoresis. Three sets of polypeptides were distinguished: those that decreased, increased or transiently increased in response to drought stress. Three sets of polypeptides also could be distinguished during recovery from drought stress: those that increased, decreased rapidly or decreased slowly and could still be detected after 6 h of recovery. The set of polypeptides that decreased during drought stress was the same set that increased during recovery and those that increased during stress, decreased during recovery. The leaf ABA concentration increased rapidly in response to drought stress and decreased rapidly during recovery from drought stress, returning to the nonstress level after 6 h of rehydration. There was a correlation between the accumulation of some of the drought-induced polypeptides and the pattern of ABA accumulation during stress and recovery from stress.     

Cosuppression of limonene-3-hydroxylase in peppermint promotes accumulation of limonene in the essential oil

cDNA clones encoding limonene synthase and limonene-3-hydroxylase, both driven by the CaMV 35S promoter, were independently transformed into peppermint (Menthaxpiperita) to alter the production and disposition of (-)-limonene, the first committed intermediate of essential oil biosynthesis in this species. Although both genes were constitutively expressed in leaves of transformed plants, the corresponding enzyme activities were not significantly increased in the glandular trichome sites of essential oil biosynthesis; thus, there was no effect on oil yield or composition in the regenerated plants. Cosuppression of the hydroxylase gene, however, resulted in the accumulation of limonene (up to 80% of the essential oil compared to about 2% of the oil in wild type plants), without influence on oil yield. These results indicate that limonene does not impose negative feedback on the synthase, or apparently influence other enzymes of monoterpene biosynthesis in peppermint, and suggests that pathway engineering can be employed to significantly alter essential oil composition without adverse metabolic consequences.     

Regulation of sucrose-sucrose-fructosyltransferase in barley leaves

The activity of sucrose-sucrose-fructosyltransferase (SST), a vacuolar enzyme strongly induced by light in excised leaves of barley (Hordeum vulgare L.), rapidly declined even in continuous light upon feeding of cycloheximide (CHI). The rate of decline was similar to that observed in light-treated leaves that were placed into darkness, in the presence or absence of CHI. The protease inhibitor leupeptin totally stopped the decline in SST activity in the dark and caused a substantial increase in the rate of induction of SST activity by light. Feeding of sucrose prevented or even reversed the SST activity decay induced by darkness in the absence of CHI but did not stabilize SST activity in the presence of CHI. The results suggest that SST is continuously subjected to rapid, constant proteolytic degradation in the vacuole, and that the enhancement of SST activity in the light or upon feeding sucrose in the dark is due exclusively to de novo protein synthesis.     

Seasonal pattern of monoterpene synthase activities in leaves of the evergreen tree Quercus ilex

Monoterpene synthase activities were measured in current year and 1-year-old leaves of holm oak ( Quercus ilex L.). The monoterpene synthase activities of the leaves strongly changed with leaf development and leaf age. Enzyme activities increased rapidly in spring after leaf emergence, reaching maximum values in summer, which declined during the following winter period. In the next spring monoterpene synthase activities recovered in the old leaves to about one-third of values in the previous years and showed a similar seasonal variation as in young leaves. In both leaf age classes the pattern of enzymatic monoterpene formation was stable with α-pinene (33%), β-pinene (28%), and myrcene (26%) as prominent compounds followed by minor fractions of sabinene (10%) and limonene (3%). Monoterpene emission correlated with the activity of the synthetizing enzymes, indicating that monoterpene synthase activities in Q. ilex reflect the seasonal monoterpene emission potential of the leaves.     

Regulation of photosynthesis by end-product accumulation in leaves of plants storing starch, sucrose, and hexose sugars

In the present study, leaves of different plant species were girdled by the hot wax collar method to prevent export of assimilates. Photosynthetic activity of girdled and control leaves was evaluated 3 to 7 days later by two methods: (a) carbon exchange rate (CER) of attached leaves was determined under ambient CO₂ concentrations using a closed gas system, and (b) maximum photosynthetic capacity (A_max) was determined under 3% CO₂ with a leaf disc O₂ electrode. Starch, hexoses, and sucrose were determined enzymically. Typical starch storers like soybean (Glycine max L.) (up to 87.5 milligrams of starch per square decimeter in girdled leaves), cotton (Gossypium hirsutum L.), and cucumber (Cucumis sativus L.) responded to 7 days of girdling by increased (80-100%) stomatal resistance (r_s) and decreased A_max (>50%). On the other hand, spinach (Spinacia oleracea L.), a typical sucrose storer (up to 160 milligrams of sucrose per square decimeter in girdled leaves), showed only a slight reduction in CER and almost no change in A_max. Intermediate plants like tomato (Lycopersicon esculentum Mill.), sunflower (Helianthus annuus L.), broad bean (Vicia faba L.), bean (Phaseolus vulgaris L.), and pea (Pisum sativum L.), which upon girdling store both starch and sucrose, responded to the girdle by a considerable reduction in CER but only moderate inhibition of A_max, indicating that the observed reduction in CER was primarily a stomatal response. Both the wild-type tobacco (Nicotiana sylvestris) (which upon girdling stored starch and hexoses) and the starchless mutant (which stored only hexoses, up to 90 milligrams per square decimeter) showed 90 to 100% inhibition of CER and approximately 50% inhibition of A_max. In general, excised leaves (6 days) behaved like girdled leaves of the respective species, showing 50% reduction of A_max in wild-type and starchless N. sylvestris but only slight decline of A_max in spinach. The results of the present study demonstrate the possibility of the occurrence of end-product inhibition of photosynthesis in a large number of crop plants. The long-term inhibition of photosynthesis in girdled leaves is not confined to stomatal responses since the A_max declined up to 50%. The inhibition of A_max by girdling was strongest in starch storers, but starch itself cannot be directly responsible, because the starchless mutant of N. sylvestris was also strongly inhibited. Similarly, the inhibition cannot be attributed to hexose sugars either, because soybean, cotton, and cucumber are among the plants most strongly inhibited although they do not maintain a large hexose pool. Spinach, a sucrose storer, showed the least inhibition in both girdled and excised leaf systems, which indicates that sucrose is probably not directly responsible for the end-product inhibition of photosynthesis. The occurrence of strong end-product inhibition appears to be correlated with high acid-invertase activity in fully expanded leaves. The inhibition may be related to the nature of soluble sugar metabolism in the extrachloroplastic compartment and may be caused by a metabolite that has different rates of accumulation and turnover in sucrose storers and other plants.     

Morphology and monoterpene biosynthetic capabilities of secretory cell clusters isolated from glandular trichomes of peppermint (Mentha piperita L.)

Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (M_r=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the vacuole. The molecular-weight exclusion limit for this permeability was shown to be less than approx. 1800, based on exclusion of fluorescein-conjugated dextran (M_r 1800). Intact secretory cell clusters very efficiently incorporated [³H]geranyl pyrophosphate into monoterpenes. The addition of exogenous cofactors and redox substrates affected the distribution of monoterpenes synthesized from [³H]geranyl pyrophosphate, demonstrating that the cell clusters were permeable to these compounds and that the levels of endogenous cofactors and redox substrates were depleted in the isolated cells. When provided with the appropriate cofactors, such as NADPH, NAD⁺, ATP, ADP and coenzyme A, the isolated secretory cell clusters incorporated [¹⁴C]sucrose into monoterpenes, indicating that these structures are capable of the de-novo biosynthesis of monoterpenes from a primary carbon source, and that they maintain a high degree of metabolic competence in spite of their permeable nature.Abbreviations GLC gas liquid chromatography - LSCM laser scanning confocal microscopy - LY-IA Lucifer yellow iodoacetamide This investigation was supported in part by U.S. Department of Energy Grant DE-FG0688ER13869 and by Project 0268 from the Washington State University Agricultural Research Center. Light microscopy was carried out in the Plant Biology Light Microscopy and Image Analysis Facility (WSU) funded by the National Science Foundation (DIR9016138). We thank Greg Wichelns for growing the plants and Stephen Pfeiffer (BioRad Microsciences Division, Cambridge, Mass, USA), for help acquiring the confocal images.     

Bioactive monoterpene glycosides conjugated with gallic acid from the leaves of Eucalyptus globulus

Two monoterpene glycosides, conjugated with gallic acid [globulusin A (1) and B (2)], together with four known compounds, cypellocarpin A (3), eucaglobulin (4), cuniloside (5) and (1S, 2S, 4R)-trans-2-hydroxy-1,8-cineole beta-d-glucopyranoside (6), were isolated from hot-water extracts of the leaves of Eucalyptus globulus. The structures of compounds 1 and 2 were determined by 1D, 2D NMR and MS spectroscopic analyses. The absolute stereochemistry of 1 was determined by correlating the spectroscopic data with those of synthetic compound 6 with a known configuration. Globulusin A (1) and B (2), cypellocarpin A (3) and eucaglobulin (4), scavenged DPPH free radicals and globulusin A (1) showed a higher antioxidant activity than the other tested compounds, with an IC50 of 3.8microM. Globulusin A (1) and eucaglobulin (4) concentration-dependently suppressed inflammatory cytokine production, tumor-necrosis factor-alpha and interleukin-1beta in cultured human myeloma THP-1 cells co-stimulated with phorbol myristate acetate. These compounds also inhibited melanogenesis in cultured murine melanoma B16F1 cells, without any significant cytotoxicity. These results suggested that globulusin A (1) and eucaglobulin (4), which were isolated as antioxidants from E. globulus, also had anti-inflammatory as well as anti-melanogenesis activity.     

Brassinosteroid-induced accumulation of complex oxylipins in flax leaves

The effect of the steroid plant hormone 24-epibrassinolide on the content and composition of complex oxylipins in plants was studied for the first time, using flax as a model plant. In plants treated with the plant hormone, as well as in plants inoculated with Pectobacterium atrosepticum, the content of linolipins (galactolipids containing divinyl ether residues) increased. Hormonal pretreatment of the plants with subsequent infection resulted in a more significant accumulation of linolipins B (21-fold) and A (2.8-fold) as compared to the untreated (control) plants. The data suggest that brassinosteroids are involved in the regulation of the formation of complex oxylipins in pathogenesis of plants.     

Phytochrome-mediated accumulation of chloroplast DNA in pea leaves

Pea seedlings grown for 5 days in the dark were treated with red light for 5 min and grown for 2 more days in the dark. Effects of the red light on chloroplast DNA levels in the pea leaves were examined using probe DNA of the chloroplast-coded large subunit and nuclear-coded small subunit of ribulosebisphosphate carboxylase/oxygenase. The gene dosage of the large subunit, but not of the small subunit, was increased by red light. The increase was inhibited by subsequent far-red light treatment. These results indicate that accumulation of chloroplast DNA in the cell is mediated by phytochrome. Probably the replication of chloroplast DNA is mediated by phytochrome.     

Regulation of nitrate reduction in wheat leaves

Wheat leaves exposed to 710 nm monochromatic light, when only photosystem 1 operates, reduced small but significant amount of nitrate to nitrite. This could be due to partial inhibition of mitochondrial oxidation of NADH, brought about by cyclic photo-phosphorylation. Under dark aerobic conditions, citric acid cycle intermediates only slightly stimulated nitrate reduction. Under dark anaerobic conditions, when maximum reduction of nitrate occurred, the time course showed a 1:1 stoichiometry between nitrite and CO₂. It is suggested that for maximum reduction of nitrate under physiological conditions, CO₂ fixation and export of ATP via triose phosphate shuttle is essential.     

Light and temperature regulated terpene biosynthesis: hepatoprotective monoterpene picroside accumulation in Picrorhiza kurrooa

Picrorhiza (Picrorhiza kurrooa) is an endangered medicinal plant with well-known hepatoprotective activity attributed to monoterpenoid picrosides. The present article details on regulatory genes of terpenoid metabolism, 3-hydroxy-3-methylglutaryl coenzyme A reductase (pkhmgr) and 1-deoxy-D-xylulose-5-phosphate synthase (pkdxs) from picrorhiza. Since no molecular information was available, these genes were cloned to full-length by degenerate primers and rapid amplification of cDNA ends, followed by cloning of the upstream sequences that showed the presence of core sequences for light and temperature responsiveness. Electrophoretic mobility shift assay confirmed binding of protein to these motifs. Expression of pkhmgr and pkdxs was up-regulated at 15°C as compared to at 25°C as well as under light as compared to dark conditions. Picrosides content exhibited the trend similar to gene expression. To rule out the possible limitation of carbon pool under dark condition, plantlets of picrorhiza were raised in vitro in Murashige and Skoog medium supplemented with 3% sucrose. Results showed similar up-regulation of both the genes and the higher picrosides content in in vitro raised plantlets in the presence of light. Data suggested the important roles played by light and temperature in regulating pkhmgr and pkdxs, and the picrosides level in picrorhiza.     

Abscisic Acid accumulation is not required for proline accumulation in wilted leaves

Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone-grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt.     

tie-dyed1 Regulates carbohydrate accumulation in maize leaves

Acquisition of cell identity requires communication among neighboring cells. To dissect the genetic pathways regulating cell signaling in later leaf development, a screen was performed to identify mutants with chloroplast pigmentation sectors that violate cell lineage boundaries in maize (Zea mays) leaves. We have characterized a recessive mutant, tie-dyed1 (tdy1), which develops stable, nonclonal variegated yellow and green leaf sectors. Sector formation requires high light, occurs during a limited developmental time, and is restricted to leaf blade tissue. Yellow tdy1 sectors accumulate excessive soluble sugars and starch, whereas green sectors appear unaffected. Significantly, starch accumulation precedes chlorosis in cells that will become a yellow sector. Retention of carbohydrates in tdy1 leaves is associated with a delay in reproductive maturity, decreased stature, and reduced yield. To explain the tdy1 sectoring pattern, we propose a threshold model that incorporates the light requirement and the hyperaccumulation of photoassimilates. A possible function consistent with this model is that TDY1 acts as a sugar sensor to regulate an inducible sugar export pathway as leaves develop under high light conditions.     

Regulation of compatible solute accumulation in bacteria

In their natural habitats, microorganisms are often exposed to osmolality changes in the environment. The osmotic stress must be sensed and converted into an activity change of specific enzymes and transport proteins and/or it must trigger their synthesis such that the osmotic imbalance can be rapidly restored. On the basis of the available literature, we conclude that representative Gram-negative and Gram-positive bacteria use different strategies to respond to osmotic stress. The main focus of this paper is on the initial response of bacteria to hyper- and hypo-osmotic conditions, and in particular the osmosensing devices that allow the cell to rapidly activate and/or to synthesize the transport systems necessary for uptake and excretion of compatible solutes. The experimental data allow us to discriminate the transport systems by the physicochemical parameter that is sensed, which can be a change in external osmotic pressure, turgor pressure, membrane strain, internal osmolality and/or concentration of specific signal mmicule. We also evaluate the mmicular basis for osmosensing by reviewing the unique structural features of known osmoregulated transport systems.