bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative and qualitative differences in C6‐volatile production from the lipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana
Authors: Bate Nicholas J. , Riley John C. M. , Thompson John E. , Rothstein Steven J.
Published: March 2003

Six‐carbon (C6) volatile products are released from the enzymatic action of hydroperoxide lyase (HPL), a component of the lipoxygenase (LOX) pathway and form the basis of the “green‐note” flavour characteristic of many consumed plant products. Arabidopsis leaf tissue contains the C6‐aldehydes hexanal, and trans‐2‐hexenal as well as the C6‐alcohols: hexanol, and 3‐hexenol. Interconversion between C6‐aldehydes and alcohols is thought to proceed through the action of alcohol dehydrogenase (ADH). Using an ADH mutant of Arabidopsis, we have shown that there are large quantitative and qualitative differences in the accumulation of C6‐volatiles in the absence of ADH activity. The total quantity of LOX‐derived volatiles is greater on a fresh weight basis in the ADH mutant. Qualitatively, hexanol and 3‐hexenol levels are approximately 62% and 51% lower in the mutant, respectively, whereas levels of hexenal are approximately 10‐fold higher. Hexanal accumulation, however, is unaffected in the mutant. The altered profile of LOX‐derived volatiles does not have an effect on the steady‐state levels of mRNA for allene oxide synthase (AOS) or LOX. HPL activity and mRNA quantity, however, are higher in the mutant relative to wild type, suggesting that altered product levels in the mutant affect HPL regulation.
Hierarchy of ozone scavenging reactions in the plant cell wall
Authors: Moldau Heino
Published: March 2003

To estimate protection of the plasmalemma against ozone by the cell apoplast, the decomposition network of ozone in the mesophyll cell wall is analysed in consideration of data on published bimolecular reaction rate constants and concentrations of the reactants involved. The effect of dimerization of ascorbate free radicals (AFR) on the stoichiometric ratio of ozone reduction by ascorbate is quantified over the range of cell wall acidity, pH = 5.0‐6.5. As the disproportionation of AFR decreases sharply towards higher pH, the flow of AFR through dimerization is low over the pH range 5.5‐6.5, allowing abstraction of the second electron from AFR and formation of dehydroascorbate with a nearly 1:1 stoichiometric ratio in relation to ozone. The direct reaction between ozone and ascorbate (AA) in cell walls 0.3‐0.5 µm thick and at an AA concentration of 0.5 mM is able to detoxify 50‐70% of the O3 that impinges on the wall surface. Generation of singlet oxygen and the hydroxyl radical, which are more reactive to AA than O3, decreases markedly the O3 flow to the plasmalemma. The question is raised whether cell wall alkalinization under ozone may hasten the decomposition of the pollutant due to the more rapid generation of hydroxyl radical by phenolic compounds.
Light‐independent thermoluminescence from thylakoids of greening barley leaves. Evidence for involvement of oxygen radicals and free chlorophyll
Authors: Marder Jonathan B. , Droppa Magdolna , Casp Varda , Raskin Victor I. , Horváth Gábor
Published: March 2003

A study was conducted to identify biophysical markers which change in response to changing chlorophyll organization during plant development. When heated to around 70°C in the dark, barley thylakoids emit red thermoluminescence (TL). This is a pure chemiluminescence signal and distinct from the lower‐temperature TL bands of thylakoids that are seen only with preillumination. The development of the light‐independent, 70°C TL band was investigated following transfer of dark‐grown barley leaves to the light. Because of the rapidly increasing chlorophyll content of the plastid membrane, the TL signal was normalized against either chlorophyll or tissue mass of the starting material. In either case, the extent of the TL signal reached a maximum in the early hours of greening and then declined. The drop in signal over 20 h was approximately 50% for TL per unit tissue mass, and well over 90% for TL per unit chlorophyll. Exposure of plastid membrane samples to hydrogen peroxide for several minutes caused a large increase in light‐independent TL, while addition of ascorbate caused substantial quenching.

The fluorescence profiles of dark‐grown barley leaves were recorded following transfer to the light. Basal fluorescence (F0) reaches a substantial level after just seconds of illumination. Over the next few hours, F0 increases only slightly and then starts to decline. The decline in F0 is correlated with an increase in variable fluorescence (Fv) which indicates the appearance of active photosystem II. It is concluded that the early peak in F0 reflects a state in which the leaves contain a maximum amount of disorganized chlorophyll.

Considering the TL and fluorescence data together, we propose the following: When chlorophyll first appears in the system, it is not properly assembled into the complexes that offer photochemical or non‐photochemical quenching of the excited state. Thus, fluorescence and parallel chlorophyll triplet formation are prevalent. The triplets cause generation of active oxygen resulting in lipid peroxidation and/or other radical‐generating processes. When the membranes are heated, increased interaction of the radicals with chlorophyll generates chemiluminescence. We thus conclude that light‐independent thermoluminescence is a marker for actual damage arising from poor chlorophyll organization and propose that this parameter might be usefully applied for assessing stress effects.
Characterization of the natural chemical and osmotic environment of early wheat embryogenesis
Authors: Maës O. , Comeau A. , Nadeau P. , Simard R. R. , St‐Pierre C. A.
Published: March 2003

Soluble carbohydrates, amino acids, and major inorganic ions were quantified and compared with the total osmolarity of the ovule. Most of the analyzed components increased rapidly in concentration, reaching a peak at 1 day post anthesis (DPA) and decreasing quickly 1 day later. Within this time period, the osmolarity decreased from 1.49 to 1.18 MPa. The osmolarity then increased to 1.39 MPa at 4 DPA, and yet the overall concentration of analyzed molecules decreased. At 1 DPA, inorganic ions were observed at levels that could produce a high osmolarity (1.11 MPa), followed by the carbohydrates (0.37 MPa) and amino acids (0.07 MPa). Maltose was detected in ovules only from 0 to 2 DPA. Inositol was abundant in the ovules on the day of anthesis, but decreased to non‐detectable levels after 4 DPA. This suggested that ovules allow the rapid entry of solutes into the syncytial endosperm after fertilization, but are osmotically buffered through the increase and decrease of partly identified substances. Specific gravity tests on the liquid squeezed from young endosperm sukpported this new hypothesis. Osmolarity in ovules seems only indirectly related to morphogenetic regulation mechanisms, and it may play a lesser role than the fluctuations of certain specific substances. The observed chemical changes offer insights into the rapidly varying nutritional needs of proembryos. Thus, in this research yielded a useful complex amino acid formula, derived from knowledge of the natural environment of the proembryo, and also an improved proembryo and ovule culture medium.
Translocation of NH4+ in oilseed rape plants in relation to glutamine synthetase isogene expression and activity
Authors: Finnemann J. , Schjoerring J. K.
Published: March 2003

Translocation of NH4+ was studied in relation to the expression of three glutamine synthetase (GS, EC 6.3.1.2) isogenes and total GS activity in roots and leaves of hydroponically grown oilseed rape (Brassica napus). The concentration of NH4+ in the stem xylem sap of NO3−‐fed plants was 0.55–0.70 mM, which was ≈60% higher than that in plants deprived of external nitrogen for 2 days. In NH4+‐fed plants, xylem NH4+ concentrations increased linearly both with time of exposure to NH4+ and with increasing external NH4+ concentration. The maximum xylem NH4+ concentration was 8 mM, corresponding to 11% of the nitrogen translocated in the xylem. In the leaf apoplastic solution, the NH4+ concentration increased from 0.03 mM in N‐deprived plants to 0.20 mM in N‐replete plants. The corresponding values for leaf tissue water were 0.33 and 1.24 mM, respectively. The addition of either NO3− or NH4+ to N‐starved plants induced both cytosolic gs isogene expression and GS activity in the roots. In N‐replete plants, gs isogene expression and GS activity were repressed, probably due to carbon limitations, thereby protecting the roots against the excessive drainage of photosynthates. Repressed gs isogene expression and GS activity under N‐replete conditions caused enhanced NH4+ translocation to the shoots.
Galactosyl‐sucrose metabolism and UDP‐galactose pyrophosphorylase from Cucumis melo L. fruit
Authors: Studer Feusi Monika E. , Burton James D. , Williamson John D. , Mason Pharr D.
Published: March 2003

In muskmelon (Cucumis melo L.), sink tissues receive stachyose, raffinose and sucrose through phloem translocation of carbohydrates that are formed as products of leaf photosynthesis. Melon fruits accumulate sucrose massively during the final stages of maturation. This sucrose is derived partially from the catabolism of raffinose saccharides. Rapid galactose metabolism is required, because liberation of free galactose is the first step in the metabolic utilization of the raffinose sugars. The current study demonstrates that the enzyme UDP‐glucose‐hexose‐1‐P uridylyltransferase (EC 2.7.7.12), the central enzyme in the classical Lelior pathway, is not the central enzyme in galactose metabolism in muskmelon fruit. Rather, a broad substrate specificity UDP‐galactose pyrophosphorylase (PPase) serves the same functional role. This enzyme accepts either UDP‐galactose or UDP‐glucose as a substrate and is different from a UDP‐glucose PPase with more strict substrate specificity for UDP‐glucose that is also present in melon tissue. UDP‐galactose PPase was purified 113‐fold from melon tissue and was shown to be a 54 kDa (size exclusion chromatography) to 68 kDa (SDS‐PAGE) protein that is enzymatically active as a monomer. We also present evidence that the enzyme likely accepts UDP‐galactose and UDP‐glucose at the same catalytic site. Polyclonal antibodies prepared against this protein reacted with numerous other antigens in melon extracts, apparently as a result of the presence of common antigenic epitopes.
Effect of defoliation on fructan pattern and fructan metabolizing enzymes in young chicory plants (Cichorium intybus)
Authors: De Roover Joke , Van Laere André , Van den Ende Wim
Published: March 2003

Witloof chicory (Cichorium intybus L. var. foliosum cv. Flash) was sown in acid‐washed vermiculite in a controlled growth chamber. After 1 month of growth, one half of the chicory plants were defoliated whereas the intact chicory plants remained as a control. Twenty‐four hours after defoliation, a very sharp decrease in hexose, sucrose, and total fructan concentration was observed in the roots. This coincided with a strong decrease in sucrose:sucrose 1‐fructosyl transferase (1‐SST; EC 2.4.1.99) activity and a strong increase in fructan 1‐exohydrolase (1‐FEH; EC 3.2.1.80) activity. After day 5, 1‐SST activity increased and 1‐FEH activity decreased. However, from day 5 to 15, both the activities of 1‐SST and acid invertase (EC 3.2.1.26) remained significantly lower than in the control plants. From 10 days after defoliation, fructan synthesis resumed and hexose and sucrose concentrations increased. Up to now, 1‐FEH activity was believed to occur only in mature tissues (end of the growing season, storage, forcing, or sprouting). Therefore, the rather unexpected finding that 1‐FEH can also be induced in very young chicory roots after defoliation suggests that 1‐FEH can be considered a ‘survival’ enzyme that can be induced at any physiological stage when energy demands increase.
The oxidation of l‐ascorbic acid catalysed by pear tyrosinase
Authors: Espín Juan Carlos , Veltman Rob H. , Wichers Harry J.
Published: March 2003

The ability of partially purified pear tyrosinase (PPO) to catalyse the oxidation of l‐ascorbic acid (AA) has been reported here for the first time. The ascorbate oxidase activity of PPO was studied by oxymetric assays. The activity was linearly related to the enzyme concentration with a Michaelis constant (Km) for AA of 0.55±0.03 mM at pH 7. The stoichiometry was found to be 1:2 (O2:AA). The action of the PPO inhibitors tropolone and sodium chloride was studied to exclude a possible interference of endogenous pear ascorbate oxidase in the oxidation of AA. A possible role of the ‘AA/PPO’ system in the browning of pears is proposed.
AM fungi might affect the root morphology of maize by increasing indole‐3‐butyric acid biosynthesis
Authors: Kaldorf Michael , Ludwig‐Müller Jutta
Published: March 2003

Inoculation of maize (Zea mays L.) with the arbuscular mycorrhizal (AM) fungus Glomusintraradices resulted in a distinct root phenotype ca 10 days after inoculation. Although the fresh weight of inoculated and control roots was about the same, the AM‐inoculated roots showed a significant increase in the percentage of lateral fine roots. This increase coincided with an increase in free indole‐3‐butyric acid (IBA) as well as an increase in IBA synthesis. At later time points (31 days after inoculation), the free IBA content was not increased in infected roots; however, the fraction of bound IBA increased compared to controls. The phenotype of mycorrhizal maize roots could be mimicked by IBA applied exogenously to non‐mycorrhizal roots. Addition of trifluoro‐IBA (TFIBA), an inhibitor of IBA‐induced root growth and lateral root induction, simultaneously with IBA resulted in a phenotype resembling that of untreated controls. In roots treated with TFIBA the inoculation with AM fungi did not increase the formation of fine roots. The TFIBA treatment also reduced endogenous free IBA and the AM infection rate in mycorrhizal roots. The results are discussed with respect to a possible role of IBA in the establishment of AM symbiosis.
Changes in cis‐zeatin and cis‐zeatin riboside levels and biological activity during potato tuber dormancy
Authors: Suttle Jeffrey C. , Banowetz G. M.
Published: March 2003

The effects of postharvest storage duration and temperature on endogenous cis‐zeatin (cis‐Z) and cis‐zeatin riboside (cis‐ZR) levels in potato (Solanum tuberosum L.) tubers were determined in relation to tuber bud dormancy. The tubers used in these studies were completely dormant for at least 81 days of storage. Thereafter, tuber bud dormancy diminished gradually and after 165 days of postharvest storage, the tubers were completely non‐dormant. Immediately after harvest, endogenous levels of cis‐Z and cis‐ZR were approximately 25 pmol (g fresh weight)−1 and 8 pmol (g fresh weight)−1, respectively. In tubers exiting dormancy but stored at a growth‐inhibiting temperature (3°C), endogenous levels of cis‐Z rose over threefold after 25 days of storage and remained elevated for the duration of the study. Levels of cis‐ZR remained essentially constant during this same period. In tubers transferred to a growth permissive temperature (20°C) prior to use, the rise in endogenous cis‐Z was less dramatic and more protracted; increasing twofold after 53 days of storage. No change in cis‐Z riboside content was observed in these tubers during this period. Dose‐response studies using either cis‐Z or trans‐Z demonstrated a time‐dependent increase in cytokinin sensitivity during postharvest storage. Immediately after harvest, dormant tubers were insensitive to both zeatin isomers. Thereafter, tubers exhibited a dose‐dependent increase in premature sprouting following injection with either cytokinin isomer. After injection into dormant tubers, cis‐[8‐14C]‐zeatin was metabolized primarily to adenine/adenosine and cis‐Z riboside. Seven days after injection, less than 10% of the recovered radioactivity was associated with trans‐ZR. These results are consistent with a role for endogenous cis‐Z (and its derivatives) in the regulation of potato tuber dormancy.
Factors affecting ‘Hass’ avocado fruit size: Carbohydrate, abscisic acid and isoprenoid metabolism in normal and phenotypically small fruit
Authors: Richings Evelyn W. , Cripps Ryan F. , Cowan A. Keith
Published: March 2003

Carbohydrate and abscisic acid (ABA) metabolism were investigated in normal and phenotypically small ‘Hass’ avocado (Persea americana Mill.) fruit in an attempt to link alterations in sugar and ABA content with changes in 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) activity and fruit size. The small‐fruit phenotype was characterized by reduced seed HMGR activity, increased seed insoluble acid invertase (β‐
d‐fructofuranosidase, EC 3.2.1.26), decreased sucrose synthase (SS; UDP‐
d‐glucose:
d‐fructose‐2‐α‐glucosyl‐transferase, EC 2.4.1.13) activity, decreased sucrose content, and increased glucose as a proportion of the total soluble sugar. Sucrose phosphate synthase (SPS; UDP‐
d‐glucose:
d‐fructose 6‐phosphate 2‐α‐
d‐glucosyltransferase, EC 2.4.1.14) activity was unaffected in seed but reduced in mesocarp of the small fruit. In addition, the small‐fruit variant displayed enhanced respiration and both seed and mesocarp tissue showed increased ABA metabolism. Applied ABA caused an increase in insoluble acid invertase activity in seed tissue of normal fruit while mevastatin reduced HMGR activity in this tissue, caused sucrose depletion and increased the proportion of glucose from 5 to 57% of total soluble sugars. Exogenous glucose suppressed HMGR activity in seed tissue whereas in mesocarp tissue, HMGR activity was reduced to 38% of the control after 6 h but enhanced by 46% by 48 h. Glucose increased ABA biosynthesis and turnover in competent tissues. These results suggest that ABA turnover is mediated, in part, by carbohydrate content and composition which also affects HMGR activity. It is proposed that sugar and ABA signals act in concert to modulate expression and/or activity of HMGR in the control of ‘Hass’ avocado fruit growth and final fruit size.
Molecular control of ethylene production by cyanide in Arabidopsis thaliana
Authors: McMahon Smith Jennifer , Arteca Richard N.
Published: March 2003

Although cyanide has long been recognized as a co‐product of ethylene synthesis, little attention has been given to its potential physiological and molecular roles. In the present work, the long‐term effects of cyanide on growth and development were observed in Arabidopsis thaliana. Two days after a single 20‐min application of cyanide, plants demonstrated visible signs of stress. Long‐term detrimental effects on growth and photosynthetic capabilities were noted, including low chlorophyll accumulation and stunted growth. Because of the relationship between cyanide and ethylene production, we chose to evaluate the results of cyanide treatment on genes encoding proteins involved in ethylene synthesis. We have found that only the 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase gene, ACS6, is rapidly activated in response to cyanide treatment, while other ACS genes were unaffected. This same gene has previously been shown to be transcriptionally activated in response to touch and other environmental stimuli. Cyanide was capable of activating ACS6 transcription within 10 min of treatment, and the amount of transcript correlated positively with the cyanide dosage. Due to the toxic nature of cyanide, plant in vivo concentrations are generally maintained lower than 10 μM, but can increase under certain stresses. In the present work, we observed that physiologically relevant concentrations as low as 1 μM HCN, considered metabolically ‘safe’, were capable of initiating ACS6 transcription. ACS6 transcripts were not substantially reduced as a result of multiple cyanide treatments, which is in contrast with the effects of mechanical stimulation on transcription. Our results suggest a relationship between cyanide production during ethylene synthesis and the molecular control of ethylene synthesis. This work corresponds with earlier experiments that have demonstrated that ethylene and cyanide can elicit some similar physiological responses. It is possible that cyanide may play an active role in ethylene regulation under conditions where rapid cyanide accumulation occurs. Since cyanide can rapidly activate ethylene synthesis, it is possible that it is involved in the positive‐feedback regulation of ethylene that occurs in some plant tissues.
Morphological, physiological and dormancy responses of three Vitis genotypes to short photoperiod
Authors: Wake Carol M. F. , Fennell Anne
Published: March 2003

Dormancy is initiated by decreasing photoperiod or temperature in many woody species. We investigated the effect of photoperiod on the induction of dormancy in Vitis. Three genotypes (Vitis riparia, Vitis spp. cv. Seyval Blanc and a F1 hybrid [V. riparia×‘Seyval Blanc’]) were monitored for morphological and physiological dormancy responses during 6 weeks of short (8 h, SD) and long (15 h, LD) photoperiod treatments. V. riparia became dormant after 4 weeks of SD as indicated by several morphologic changes. Cane elongation ceased, terminal meristems began to abscise, and primary dormant buds failed to grow after pruning and returning to LD. There was also a pronounced decrease in bud water content in response to SD. ‘Seyval Blanc’, in contrast, maintained actively growing meristems with no terminal abscission throughout SD treatment, and little delay in budbreak after pruning and returning to LD. Moderate decreases in bud water content were observed after 6 weeks of SD treatment in ‘Seyval Blanc’. F1 plants displayed intermediate responses to SD treatment, but more closely paralleled that of the V. riparia parent. Growth cessation, terminal meristem abscission and absence of budbreak occurred in the F1 after 6 weeks of SD. Ten percent discontinuous SDS‐PAGE protein profiles demonstrated SD‐induced changes in polypeptide accumulation. A 19‐kDa polypeptide increased 2–3‐fold in response to SD in all genotypes. A 17‐kDa polypeptide appeared at 4 weeks of SD, and represented 2.5–5% of all proteins visualized on the gels by 6 weeks of SD. These increases in polypeptides appear to be a general response to SD. This experimental system with heritable morphological and physiological variations in SD responsiveness will be valuable for studying dormancy initiation and maintenance in Vitis.
Phenoloxidase of peach (Prunus persica) endocarp: Its relationship with peroxidases and lignification
Authors: Alba Claudia Mónica , De Forchetti Silvia Milrad , Tigier Horacio A.
Published: March 2003

A preliminary characterization of a phenoloxidase from extracts of soluble and ionically‐bound cell wall proteins of peach (Prunus persica L. Batsch, cv. Redhaven) endocarp is described in the present study to establish differences with peroxidases from the same plant tissue. The phenoloxidase activity was detected mainly in the first stage of peach fruit growth, while peroxidase activity and lignin content increased along the second stage of growth. There were clear differences between the two enzymes. The phenoloxidase had a pI value of 5.6, different from those of peroxidases isoenzymes with various pIs ranging from 3.6 to 9.6. The oxidase molecular mass was 112 kDa, similar to other phenoloxidases described in the literature, while all peroxidase isoenzymes showed a molecular mass of around 40 kDa. The specific activities of phenoloxidase against different substrates and its inhibition by various effectors suggest that the endocarp oxidase described here is probably a metal‐dependent polyphenol oxidase, displaying attributes of both catechol oxidase (EC 1.10.3.1) and laccase (EC 1.10.3.2).
Effects of rho‐independent terminators on the expression of the prokaryotic β‐glucuronidase gene in tobacco
Authors: Jeng Shih‐Tong , Yen Chia‐Yu
Published: March 2003

The threonine (thr) attenuator with a dyad symmetrical structure is from the regulatory region of the thr operon of Escherichia coli, and encodes RNA with a stem‐and‐loop structure followed by a stretch of uridine residues. The thr attenuator and its variants were subcloned into the region between the 35S promoter and β‐glucuronidase (GUS) coding region, and the transient expression of GUS gene in tobacco protoplast was treated as a reporter for gene regulation in plants. Results from the 14 variants in the stem region of the thr attenuator indicated that both base pairing and sequence specificity in the G+C‐rich region of the stem were important for the GUS expression, but 1 base mismatch in the A+U‐rich region of stem did not affect the GUS expression in plants. Seven variants with nested deletion in the stretch of uridine residues were also analyzed, and the results suggested that the variants with the shorter uridine stretch produced more GUS protein than those with the longer stretch. Transgenic tobacco plants with the thr attenuator and its variants located between the 35S promoter and GUS coding region were also generated, and their steady state RNAs were hybridized with 2 radioactive antisense RNA probes which bound 5′ and 3′ of the thr hairpin, respectively. After the digestion of S1 nuclease, the amount of the nuclease‐resistant transcript from the protection of the 5′ antisense RNA probe was much more than that from the protection of the 3′ probe in all tested variants. This result suggests that these dyad symmetries may affect transcription of plant RNA polymerase II.
Stress, as assessed by the appearance of sHsp transcripts, is required but not sufficient to initiate androgenesis
Authors: Smykal P. , Pechan P. M.
Published: March 2003

A number of abiotic stresses can elicit young pollen grains of Brassica napus to irreversibly alter their development to androgenic (embryo formation) pathway. In our experiments, temperature and colchicine were used as abiotic stresses capable of inducing androgenesis. The most effective temperature was 32–33°C. This absolute temperature requirement, but only in combination with a temperature differential of at least 5–10°C between the donor plant and in vitro culture conditions, had a major stimulatory effect to elicit androgenesis in young pollen grains. Colchicine teatment also resulted in induction of androgenesis but at lower levels than the temperature treatment. In both instances, application of abiotic stresses resulted in the elicitation of a general stress response in young pollen grains as indicated by the appearance of sHsp transcripts. The appearance of sHsp transcripts can be used as a marker to estimate the level of stress necessary to change the differentiation pathway of young pollen grains that leads to induction of androgenesis since androgenesis never took place without the sHsps gene transcription. The need for the presence of sHsp transcripts is supported by observation that a mutant form of B. napus cv. Topas, producing approximately 50 times fewer embryos, had a substantially reduced expression of sHsps in young pollen grains subjected to the androgenesis inductive heat stress. It remains to be determined to what extent sHsps are directly involved in induction of androgenesis as, under stress, sHsps gene products were also detected in microspores and pollen grains developmentally too young or old to undergo the process. Nevertheless, it can be concluded that application of stress and the subsequent stress response, which includes the transcription of sHsps, are some of the major events associated with changes in the pollen differentiation process that can lead to androgenesis. Other factors, such as the pollen developmental stage and the phase of the cell cycle at the time of stress imposition, play an important role as well.
Identification and characterization of soluble and insoluble myrosinase isoenzymes in different organs of Sinapis alba
Authors: Eriksson Susanna , Ek Bo , Xue Jiaping , Rask Lars , Meijer Johan
Published: March 2003

Extraction of Sinapis alba seeds under native conditions solubilized 3 myrosinase isoforms, pool I, II and III, which could be separated by ion exchange chromatography. Sequencing of numerous peptides of the I and III isoforms showed that they belonged to the Myrosinase A (MA) family of myrosinases and that they were encoded by different genes. Western blot analysis of S. alba seed proteins, extracted with a sodium dodecyl sulphate‐containing buffer, using an anti‐myrosinase monoclonal antibody, showed the presence of two additional myrosinase isoforms with approximate molecular sizes of 62 and 59 kDa. These myrosinases, which only could be solubilized from seeds by inclusion of denaturing agents in the extraction buffer, were by sequence analysis identified as MB myrosinases. These isoenzymes or very similar forms were also present in seedling cotyledons. However, from this tissue, they could be extracted with non‐denaturing buffers. In addition, cotyledons contained a 65‐kDa MB myrosinase not found in seeds. In contrast, seedling cotyledons contained only minute amounts of pool I and no pool III MA myrosinases, emphasizing the tissue‐specific expression of the corresponding gene families. Sequence analysis of myrosinase cDNAs generated cDNA by reversed transcription‐polymerase chain reaction using degenerate primers with mRNA isolated from seeds, cotyledons and leaves confirmed the result that the MA isoforms were expressed only in seed tissue, while MB myrosinases were found in all tissues investigated. Furthermore, seed and leaf contained unique MB myrosinase transcripts, suggesting organ‐specific expression of individual MB genes.
The effect of high temperature and high atmospheric CO2 on carbohydrate changes in bell pepper (Capsicum annuum) pollen in relation to its germination
Authors: Aloni Beny , Peet Mary , Pharr Mason , Karni Leah
Published: March 2003

Pollen viability and germination are known to be sensitive to high temperature (HT). However, the mode by which high temperature impairs pollen functioning is not yet clear. In the present study, we investigated the effect of high temperature on changes occurring in carbohydrate of bell pepper (Capsicum annuum L. cv. Mazurka) pollen in order to find possible relations between these changes and pollen germination under heat stress. When pepper plants were maintained under a moderate HT regime (32/26°C, day/night) for 8 days before flowers have reached anthesis, pollen count at anthesis was similar to that found in plants grown under normal temperatures (NT 28/22°C). However, the in vitro germination, carried out at 25°C, of pollen from HT plants was greatly reduced. This effect matched the marked reduction in the number of seeds per fruit in the HT plants. Maintaining the plants at high air CO2 concentration (800 μmol mol−1 air) in both temperature treatments did not affect the in vitro germination of pollen from NT plants, but restored germination to near the normal level in pollen from HT plants. Under NT conditions, starch, which was negligible in pollen at meiosis (8 days before anthesis, A−8) started to accumulate at A−4 and continued to accumulate until A−2. From that stage until anthesis, starch was rapidly degraded. On the other hand, sucrose concentration rose from stage A−4 until anthesis. Acid invertase (EC 3.2.1.26) activity rose parallel with the increase of sucrose. In pollen from HT plants, sucrose and starch concentrations were significantly higher at A−1 pollen than in that of NT plants. Under high CO2 conditions, the sucrose concentration in the pollen of HT plants was reduced to levels similar to those in NT pollen. In accordance with the higher sucrose concentration in HT pollen, the acid invertase activity in these pollen grains was lower than in NT pollen. The results suggest that the higher concentrations of sucrose and starch in the pollen grains of HT plants may result from reduction in their metabolism under heat stress. Elevated CO2 concentration, presumably by increasing assimilate availability to the pollen grain, may alleviate the inhibition of sucrose and starch metabolism, thereby increasing their utilization for pollen germination under the HT stress. Acid invertase may have a regulatory role in this system.
Response to mild water stress in transgenic Pssu‐ipt tobacco
Authors: Synková Helena , Valcke Roland
Published: March 2003

The response of antioxidant enzymes to cyclic drought was studied in control non‐transformed tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) and two types of transgenic Pssu‐ipt tobacco (grafted on wild rootstock and poorly rooted progeny of F1 generation) grown under different conditions of irradiation (greenhouse, referred as high light, versus growth chamber, referred as low light). Water stress cycles started with plants at two contrasting developmental stages, i.e., at the stage of vegetative growth (young) and at the onset of flowering (old). Drought reduced the growth of SR1 plants compared with transgenic ones, particularly, when treatment started in earlier stage of plant development. Relative leaf water content was significantly lower (below 70%) in all transgenic grafts and plants compared with the wild type, irrespective of age, drought, and growth conditions. The response of antioxidant enzymes was significantly dependent on plant type and plant age; nevertheless, growth conditions and water stress also affected enzyme activities. Contrary to non‐transgenic tobacco, where about half of glutathione reductase activity was found in older plants, both transgenic types exhibited unchanged activities throughout plant development and stress treatment. No differences were found in catalase activity, although the growth in the greenhouse caused a moderate increase in all older plants. In contrast to non‐transgenic and Pssu‐ipt rooted plants, peroxidase activities (ascorbate, guaiacol, and syringaldazine peroxidase) in older Pssu‐ipt grafts were up to four times higher, irrespective of growth and stress, nevertheless, the effect seemed to be age‐dependent. Superoxide dismutase (SOD) activity was affected particularly by plant age but also by growth conditions. Unlike in older plants, water stress caused an increase of SOD activities in all younger plants. The differences observed in activities of enzymes of intermediary metabolism (i.e., malic enzyme and glucose‐6‐phosphate dehydrogenase) revealed that transgenic grafts probably compensated differently for a decrease of ATP and NADPH than control and transgenic rooted plants under stress.
Up‐regulation of sucrose metabolizing enzymes in Oncidium goldiana grown under elevated carbon dioxide
Authors: Li Chang Run , Sun Wendell Q. , Hew Choy Sin
Published: March 2003

Experiments were conducted in controlled growth chambers to evaluate how increase in CO2 concentration affected sucrose metabolizing enzymes, especially sucrose phosphate synthase (SPS; EC 2.4.1.14) and sucrose synthase (SS; EC 2.4.1.13), as well as carbon metabolism and partitioning in a tropical epiphytic orchid species (Oncidium goldiana). Response of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) to elevated CO2 was determined along with dry mass production, photosynthesis rate, chlorophyll content, total nitrogen and total soluble protein content. After 60 days of growth, there was a 80% and 150% increase in dry mass production in plants grown at 750 and 1 100 μl l−1 CO2, respectively, compared with those grown at ambient CO2 (about 370 μl l−1). A similar increase in photosynthesis rate was detected throughout the growth period when measured under growth CO2 conditions. Concomitantly, there was a decline in leaf Rubisco activity in plants in elevated CO2 after 10 days of growth. Over the growth period, leaf SPS and SS activities were up‐regulated by an average of 20% and 40% for plants grown at 750 and 1100 μl l−1 CO2, respectively. Leaf sucrose content and starch content were significantly higher throughout the growth period in plants grown at elevated CO2 than those at ambient CO2. The partitioning of photosynthetically fixed carbon between sucrose and starch appeared to be unaffected by the 750 μl l−1 CO2 treatment, but it was favored into starch under the 1 100 μl l−1 CO2 condition. The activities of SPS and SS in leaf extracts were closely associated with photosynthetic rates and with partitioning of carbon between starch and sucrose in leaves. The data are consistent with the hypothesis that the up‐regulation of leaf SPS and SS might be an acclimation response to optimize the utilization and export of organic carbon with the increased rate of inorganic‐carbon fixation in elevated CO2 conditions.
Molecular cloning of a Brassica napus thiohydroximate S‐glucosyltransferase gene and its expression in Escherichia coli
Authors: Marillia Elizabeth‐France , MacPherson Jim M. , Tsang Edward W. T. , Van Audenhove Katrien , Keller Wilf A. , GrootWassink Jan W. D.
Published: March 2003

A genomic clone encoding a thiohydroximate S‐glucosyltransferase (S‐GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173‐bp intron. At least two copies of the S‐GT gene were present in B. napus. The full‐length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51‐kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose‐binding domain. The recombinant protein was expressed in Escherichia coli, and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S‐GT activity detected from the recombinant protein confirmed that this clone was indeed a S‐glucosyltransferase.
Purification and catalytic properties of polygalacturonase isoforms from ripe avocado (Perseaamericana) fruit mesocarp
Authors: Wakabayashi Kazuyuki , Huber Donald J.
Published: March 2003

Endo‐polygalacturonase (PG; EC 3.2.1.15) was recovered from the cell walls of avocado mesocarp (Persea americana Mill cv. Lula) tissue and purified by sequential ion exchange and gel permeation chromatography. Two isoforms (S‐I and S‐II) were recovered, exhibiting molecular masses of about 41 kD on size exclusion media and about 48 (S‐I) and 46 (S‐II) kDa as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Both isoforms exhibited maximum activity at pH 6.0 against polygalacturonic acid (PGA) and hydrolyzed PGA of about 180 kDa to polymers of about 4 kDa. The catalytic activity of the 48‐kDa isoform against PGA was slightly higher than that of the 46‐kDa isoform. The purified PGs catalyzed significant molecular mass downshifts in the polyuronides of pre‐ripe avocados; however, the capacity of the enzymes to solubilize polyuronides from cell walls of pre‐ripe fruit was limited.
The effect of a microgravity (space) environment on the expression of expansins from the peg and root tissues of Cucumis sativus
Authors: Link Bruce M. , Wagner Edward R. , Cosgrove Daniel J.
Published: March 2003

In young cucumber seedlings, the peg is a polar outgrowth of tissue that functions by snagging the seed coat, thereby freeing the cotyledons. The development of the peg is thought to be gravity‐dependent and has become a model system for plant‐gravity response. Peg development requires rapid cell expansion, a process thought to be catalyzed by α‐expansins, and thus was a good system to identify expansins that were regulated by gravity. This study identified 7 new α‐expansin cDNAs from cucumber seedlings (Cucumis sativus L. cv Burpee Hybrid II) and examined their expression patterns. Two α‐expansins (CsExp3 and CsExp4) were more highly expressed in the peg and the root. Earlier reports stated that pegs tend not to form in the absence of gravity, so the expression levels were compared in the pegs of seedlings grown in space (STS‐95), on a clinostat, and on earth (1 g). Pegs were observed to form at high frequency on clinostat and space‐grown seedlings, yet on clinostats there was more than a 4‐fold reduction in the expression of CsExp3 in the pegs of seedlings grown on clinostats vs. those grown at 1 g, while the CsExp4 gene appeared to be turned off (below detection limits). There were no detectable differences in expansin gene expression levels for the pegs of seedlings grown in space or in the orbiter environmental simulator (OES) (1 g) at NASA. The microgravity environment did not affect the expression of CsExp3 or CsExp4, and the clinostat did not simulate the microgravity environment well.
Changes in phenol content during strawberry (Fragaria×ananassa, cv. Chandler) callus culture
Authors: López Arnaldos Tomás , Muñoz Romualdo , Ferrer María Angeles , Calderón Antonio A.
Published: March 2003

Total soluble phenols, soluble flavanols, (+)‐catechin, ferulic acid and 1‐O‐feruloyl‐β‐d‐glucose were analyzed during the development of a strawberry (Fragaria×ananassa, cv. Chandler) callus culture. The time‐course changes of the different phenols assayed were well correlated with callus growth and morphology. The changes in polyphenol oxidase (EC 1.10.3.1‐2) and β‐glucosidase (EC 3.2.1.21) activities in the callus were also examined. The total phenol, soluble flavanols and (+)‐catechin contents were high during the preexponential and exponential phases of growth. The subsequent decrease in (+)‐catechin concentration coincided with high levels of polyphenol oxidase activity. The 1‐O‐feruloyl‐β‐d‐glucose content was highest as callus growth ceased, and its subsequent decrease was accompanied by the increased production of ferulic acid. This increase in ferulic acid was accompanied by an increase in β‐glucosidase activity. The ferulic acid content decreased at the end of culture, when callus growth had stopped and showed clear symptoms of senescence. This decrease in the ferulic acid concentration was accompanied by an increase in the levels of ferulic acid bound to cell wall components.
Molecular cloning and in situ hybridization of α‐l‐arabinofuranosidase from carrot cells
Authors: Tanaka Reiji , Ikeda Minoru , Funatsuki Kenzo , Yukioka Hideo , Katoh Kenji , Konno Haruyoshi
Published: March 2003

The cDNA of extracellular α‐l‐arabinofuranosidase (α‐l‐AFase, EC 3.2.1.55) secreted from suspension‐cultured carrot cells (Daucus carota L. cv. Kintoki) was isolated and characterized. The nucleotide sequence of the cDNA (2.4 kb) revealed an open reading frame consisting of 655 amino acid residues. Sequence homology research showed 28.4% identity to the α‐l‐AFase A protein of Aspergillus niger. The genomic DNA was cloned by PCR, and the nucleotide ligature sequence showed 18 exons and 17 introns. The first intron was upstream of the initiation codon. In situ hybridization revealed that the α‐l‐AFase gene is expressed in the root meristem, elongation zone and the root hair of carrot seedlings, indicating that this enzyme may participate in cell proliferation and development of carrot root cells.
Sustaining S‐adenosyl‐l‐methionine‐dependent methyltransferase activity in plant cells
Authors: Moffatt Barbara A. , Weretilnyk Elizabeth A.
Published: March 2003

Many biochemical reactions in plants involve the transfer of a methyl group from S‐adenosyl‐l‐methionine (SAM). The transfer of the methyl group from SAM generates S‐adenosyl‐l‐homocysteine (SAH), a potent inhibitor of SAM‐dependent methyltransferases (MTs). To mitigate the toxic effects of SAH on MT activity, SAH is removed by SAH hydrolase (SAHH, EC 3.3.1.1) in a reaction generating homocysteine and adenosine (Ado). However, SAHH catalyzes a reversible reaction that is favored to move in the direction of SAH hydrolysis only by removal of these products. Removal of Ado is reported to exert a greater influence on promoting SAH hydrolysis. Whereas animals appear to rely upon Ado deaminase (EC 3.5.4.4) to catabolize Ado, plants appear to use adenosine kinase (EC 2.7.1.20) for this important role.  Compounds undergoing methylation represent a broad spectrum of chemically diverse substrates ranging from nucleic acids, lipids and cell wall components to comparatively simpler amines, alcohols and metal halides. Given the diverse nature of methyl acceptor compounds, it is very likely that the demand for SAM synthesis and SAH removal changes both temporally and spatially during the course of plant growth and development. Plants also use SAM as a precursor for the synthesis of ethylene, polyamines, biotin and nicotianamine. These uses are also expected to undergo changes reflective of the metabolic activities of different plants, plant organs, or cells. This review examines the various uses of SAM in plants and addresses how they allocate this resource to satisfy potentially competing needs.
Quantitative changes in secondary metabolites of dark‐leaved willow (Salix myrsinifolia) exposed to enhanced ultraviolet‐B radiation
Authors: Tegelberg Riitta , Julkunen‐Tiitto Riitta
Published: March 2003

This is a study of the impact of increased ultraviolet‐B (UV‐B) radiation on the secondary chemistry of Salix myrsinifolia (dark‐leaved willow). For nearly two decades, the loss of stratospheric ozone above the high latitudes of the Northern Hemisphere has increased UV‐B radiation (280–320 nm) over the long‐term mean. Willows (Salicaceae) are widely distributed in these northern regions. To determine the effects of increased UV‐B radiation on willows, the plantlets of three clones of S. myrsinifolia were grown under ambient (3.6 kJ m−2 day−1) or enhanced (7.18 kJ m−2 day−1) UV‐B irradiance. After the 2‐week indoor experiment, the concentrations of UV‐B‐screening phenolics (flavonoids and phenolic acids) and low‐UV‐B‐screening phenolics (salicylates and condensed tannins) in fresh leaves were investigated and the biomass of leaves, stems and roots was determined. As expected, the total amount of flavonoids in willow leaves clearly increased when plantlets were exposed to higher UV‐B irradiation. However, the degree of increase of individual compounds varied: luteolin‐7‐glucoside, monomethyl‐monocoumaryl‐luteolin‐7‐glucoside and one myricetin derivative increased significantly, while the apigenin‐7‐glucuronide increased only slightly. The enhanced UV‐B also increased the amount of p‐hydroxycinnamic acid derivative. The UV‐B effects on other phenolic acids and tannins were minor. In contrast to the other phenolics, the amounts of two salicylates, salicin and saligenin, decreased under enhanced UV‐B irradiation. Our results indicate that the concentrations of both UV‐B‐screening and low‐UV‐B‐screening phenolic compounds in leaves of S.myrsinifolia may vary in response to elevated UV‐B radiation. However, while the UV‐B protective flavonoids and phenolic acids accumulate during UV‐B exposure, the concentrations of certain salicylates decrease.