bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in tonoplast protein and density with the development of pear fruit
Authors: Shiratake Katsuhiro , Kanayama Yoshinori , Maeshima Masayoshi , Yamaki Shohei
Published: March 2003

Protoplasts isolated from pear fruit at the end of the cell‐division stage, 30 days after flowering (DAF), had already formed a large central vacuole and the vacuole occupied most of the protoplast. The changes in protein composition and density of the tonoplast (vacuolar membrane) were investigated during fruit development. After a linear sucrose density gradient centrifugation, the distribution of tonoplasts at 30 and 48 DAF was broad and began to narrow with further fruit development. This suggests that the tonoplast of young fruit is heterogeneous and becomes homogeneous with fruit development. The apparent density of the tonoplast at 30 DAF was approximately 1.12 g ml−1; it decreased with fruit development and was finally 1.09 g ml−1 in mature fruit. The phospholipid amount on the basis of tonoplast protein was 0.80 mg mg−1 at 30 DAF. It increased with fruit development, and finally reached 7.49 mg mg−1. This result indicates that the decrease in the density of the tonoplast was caused by the increase in the ratio of phospholipid to membrane protein. The protein composition of the tonoplast at each stage was quite different. The level of polypeptides of 94, 70, 61, 52, 48 and 41 kDa was low in young fruit and high in the middle or later stages of fruit development. In contrast, the level of a 76‐kDa polypeptide was high in young fruit and decreased with fruit development. Although their functions are still unclear, these tonoplast proteins may play important roles in fruit development.
Internode length in Pisum. The lrs mutation reduces gibberellin response and levels
Authors: MacKenzie‐Hose Alasdair K. , Sherriff Leanne J. , Ross John J. , Reid James B.
Published: March 2003

We describe a new mutation, lrs, which reduces internode length in Pisum sativum L. The mutation appears to act by reducing both GA synthesis and the response to GA1. The levels of the 13‐hydroxylated GAs, GA53, GA44, GA19, GA20, GA1, and GA8 in the lrs mutant were greatly reduced compared with the wild‐type. The extent of the reduction in GA1 content in the apical tissues would, at least in part, account for the dwarf phenotype of the mutant. The reduced GA responsiveness of the new mutant was indicated by the inability of applied GA1 to remove the difference in elongation between lrs and LRS plants. The lrs mutant appears to be unique amongst internode length genotypes, possessing characteristics of both GA synthesis and GA response mutants.
Cloning and expression of a hypoxic and nitrogen inducible maize alanine aminotransferase gene
Authors: Muench Douglas G. , Christopher Mary E. , Good Allen G.
Published: March 2003

Alanine aminotransferase (ALT, EC 2.6.1.2) is regulated by hypoxia in the roots of several plant species, and by light and nitrogen stress in the leaves of the C4 plant, broomcorn millet (Panicum miliaceum). In order to more fully characterize the regulation of ALT, we isolated a maize alt genomic clone that has high sequence homology to the coding regions of both the barley (Hordeum vulgare) and P. miliaceum alt cDNA clones. This is the first plant alt gene isolated to date. The alt gene consists of 15 exons, and has regions of the promoter that are similar to the maize Anaerobic Responsive Element, and to the maize 27‐kDa zein promoter. ALT activity increased 2.1‐fold in roots after 96 h of hypoxic stress, but did not increase significantly in either root or leaf tissue when the plant was subjected to anaerobic conditions. Northern analysis of hypoxic root tissue showed an 18‐fold increase in a single alt mRNA band after 8 h of hypoxic stress, followed by a continual decline in mRNA levels. ALT activity and mRNA levels were also found to increase in root tissue after recovery from nitrogen stress but not after salt, cold or heat stress conditions. These interesting patterns of ALT expression in maize roots as a result of exposure to hypoxia and nitrogen stress are discussed.
Adenosine diphosphate glucose pyrophosphorylase, a rate‐limiting step in starch biosynthesis
Authors: Green Thomas W. , Hannah L. Curtis
Published: March 2003

Starch represents the major component of virtually all plant‐derived foods consumed by man and animal. Hence, a thorough understanding of the starch biosynthetic pathway is critically important not only in understanding the biosynthesis of a major plant storage product, but also in allowing the genetic manipulation of both starch quality and quantity for human benefit. A major goal in these studies has been the identification of key steps in controlling starch levels. Evidence from a number of independent approaches clearly points to the enzyme adenosine diphosphate glucose pyrophosphorylase (AGPase) as a key regulatory step in starch synthesis. Here we highlight and summarize our understanding of this important enzyme.
Isolation, characterization and significance of papaya β‐galactanases to cell wall modification and fruit softening during ripening
Authors: Ali Zainon Mohd , Ng Shu‐Yih , Othman Roohaida , Goh Lee‐Yin , Lazan Hamid
Published: March 2003

β‐Galactosidases (EC 3.2.1.23) from ripe papaya (Carica papaya L. cv. Eksotika) fruits having galactanase activities were fractionated by a combination of cation exchange and gel‐filtration chromatography into three isoforms, viz., β‐galactosidase I, II and III. The native proteins of the respective isoforms have apparent molecular masses of 67, 67 and 55 kDa, each showing one predominant polypeptide upon SDS‐PAGE of about 31 and 33 kDa for β‐galactosidases I and III, respectively, and of 67 kDa for β‐galactosidase II. The β‐galactosidase I protein, which was undetectable in immature fruits, appeared to be specifically accumulated during ripening. The β‐galactosidase II protein was present in developing fruits, but its level seemed to decrease with ripening. β‐Galactosidase I seemed to be an important softening enzyme; its activity increased dramatically (4‐ to 8‐fold) to a peak early during ripening and correlated closely with differential softening as related to position in the fruit tissue. The inner mesocarp tissue was softer, and its wall pectins were modified earlier and firmness decreased more rapidly during ripening compared to the outer mesocarp tissue. β‐Galactosidase II also may contribute significantly to softening because of its ability to catalyse increased solubility and depolymerization of pectins as well as through its ability to modify the alkali‐soluble hemicellulose fraction of the cell wall. The physiological significance of both β‐galactosidase isoforms may partly be attributed to their functional capacity as β‐(1,4)‐galactanases.
The internal rotenone‐insensitivae NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria
Authors: Agius Stephanie C. , Bykova Natalia V. , Igamberdiev Abir U. , Møller Ian M.
Published: March 2003

Inside‐out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone‐sensitive oxidation of deamino‐NADH, NDin(NADPH) catalyzes the rotenone‐insensitive and Ca2+‐dependent oxidation of NADPH and NDin(NADH) catalyzes the rotenone‐insensitive and Ca2+‐independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, NDin(NADPH) and NDin (NADH) activity with a Ki of 3.7, 0.17 and 63 µM, respectively, and the 400‐fold difference in Ki between the two NDin made possible the use of DPI inhibition to estimate NDin (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O2 mg−1 min−1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate‐removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD+ or NADP+) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP+ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of NDin(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.
Tolerance to atmospheric ozone in transgenic tobacco over‐expressing glutathione synthetase in plastids
Authors: Wellburn Florence A. M. , Creissen Gary P. , Lake Janice A. , Mullineaux Phil M. , Wellburn Alan R.
Published: March 2003

A cross between transgenic tobacco (Nicotiana tabacum L.) plants which over‐expressed either γ‐glutamylcysteine synthetase (cpGSHI) or glutathione synthetase (cpGSHII) in their chloroplasts was used to compare the consequences of over‐expression of components of the glutathione synthetic pathway on tolerance to atmospheric O3 or paraquat. A high proportion (50%) of those progeny which carried the cpGSHII transgene alone showed tolerance to atmospheric O3 but not to paraquat. Progeny of an additional two, independent, self‐pollinated primary transgenic lines, which segregated in a Mendelian fashion for the presence of the cpGSHII transgene and therefore included both transformed and non‐transformed (recessive, wild‐type) plants, were also challenged by fumigation with O3. Again, in both cases, about 50% of the plants expressing the epGSHII transgene were found to be O3‐tolerant on the basis of reduced ethylene emissions and increased or unchanged total pigment concentrations. However, this tolerance was not due to specific changes in stomatal densities.
Free radical involvement in the generation of trans‐root potential
Authors: Vuletić M. , Radotić K. , Vućini Ž.
Published: March 2003

The identity of the naturally occurring compounds that accept electrons from plasma membrane‐bound redox systems in vivo is obscure. We analysed the effect of ascorbate, oxygen, iron, as well as their free radical forms, and also the free radical‐generating and ‐quenching systems on the trans‐root electrical potential, which had previously been shown to be coupled to plasma membrane‐bound redox systems. The material was the primary root of 8‐day‐old maize (Zea mays L.) seedlings. Trans‐root electrical potential difference was measured across excised roots. Different ascorbate (ascorbate, dehydroascorbate and ascorbate free radical) and oxygen redox forms (superoxide and hydroxide radicals and hydrogen peroxide), as well as scavenging agents of oxygen species (superoxide dismutase, catalase, mannitol), and ferric and ferrous ions were added to the solution flowing around the root. Ascorbate free radical induced the greatest depolarization of the trans‐root potential when compared to other ascorbate redox forms, which is consistent with its suggested role as a natural electron acceptor. Addition of xanthine oxidase, with or without xanthine, also produced depolarizing effects. The presence of SOD magnified this effect both with ascorbate free radical and xanthine oxidase. When ferric or ferrous chloride and ferric EDTA were applied to the bathing medium, only free ferric ion produced a very pronounced depolarization. The magnitude and kinetics of trans‐root potential depolarization, induced by the ascorbate redox forms and systems for the generation and scavenging of oxygen species, argue in favour of the mutually competing electron transfer role of ascorbate free radicals and superoxide radicals in the extracellular space of the root. These results provide evidence that at least a part of the electrical potential difference occurring across plant roots arises from current flow from the symplast, via the plasma membrane‐bound redox systems, to naturally occurring compounds in the apoplast, and that this transfer is achieved through the mediation of their free radical forms.
Increased auxin efflux in the IAA‐overproducingsur1mutant ofArabidopsis thaliana: A mechanism of reducing auxin levels?
Authors: Delarue Marianne , Muller Philimppe , Bellini Catherine , Delbarre Alain
Published: March 2003

With the aim of investigating the mechanisms that maintain auxin homeostasis in plants, we have monitored the net uptake and metabolism of exogenously supplied indole‐3‐acetic acid (IAA) and naphthalene‐1‐acetic acid (NAA) in seedlings of wild type and the IAA‐overproducing mutantsur1ofArabidopsis thaliana. Tritiated IAA and NAA entered the seedling tissues within minutes and were mostly accumulated as metabolites, probably amino acid and sugar conjugates. The mutant seedlings were marked by a strong increase of [3H]IAA metabolism and a reduction of the accumulation levels of both free [3H]IAA and [3H]NAA. The same characteristics were observed in wild‐type seedlings grown on 5 μMpicloram. We measured [3H]NAA uptake in the presence of high concentrations of unlabeled NAA or the auxin efflux carrier inhibitor naphthylphthalamic acid (NPA). This abolished the difference in free [3H]NAA accumulation between the mutant or picloram‐treated seedlings and wild‐type seedlings. These data indicated that active auxin efflux carriers were present inArabidopsisseedling tissues. Picloram‐treated seedlings and seedlings of the IAA‐overproducing mutantsur1displayed increased auxin efflux carrier activity as well as elevated conjugation of IAA. There is previous evidence to suggest that conjugation is a means to remove excess IAA in plant cells. Here, we discuss the possibility of efflux constituting an additional mechanism for regulating free IAA levels in the face of an excess auxin supply.
Development‐related changes of protein ubiquitination in pollen from male and female kiwifruit (Actinidia deliciosa)
Authors: Scoccianti Valeria , Speranza Anna , Crinelli Rita , Calzoni Gian Lorenzo , Biasi Rita , Altamura Maria Maddalena , Bagni Nello
Published: March 2003

Kiwifruit (Actinidia deliciosa) is a dioecious vine whose staminate and pistillate flowers nonetheless develop non‐functional reproductive structures of the ompposite sex. Ubiquitin is a small, highly conserved protein found in all eucaryotes: a covalent ATP‐dependent attachment of ubiquitin marks proteins for degradation. In the present paper, we used immunoblotting to investigate the presence of free ubiquitin and ubiquitin conjugates during pollen development in male (androfertile) and in female (androsterile) genotypes of kiwifruit. In the male, several high molecular mass protein conjugates were present throughout development. On the contrary, such a pattern characterized only early stages of pollen from the female genotype, where conjugates progressively disamppeared, until they were detectable only in trace amounts at anthesis. The highest content of conjugates in the male genotype was observed when microspores were ampproaching the first mitosis. Free ubiquitin increased continuously during development of the male microgametophyte so that mature pollen contained considerable amounts of the ubiquitin monomer at the time of its release from the anther. By contrast, only low levels were detectable in the degenerating microspores in the pistillate flowers. In vitro experiments using labeled ubiquitin indicated that early‐uninucleate microspores of the female genotype had a much higher conjugation rate than those of the male genotype at the same stage. However, after feeding α‐lactalbumin as exogenous substrate, the rate of ubiquitin conjugation strongly increased and was quite similar in both sexes. Nuclear features of pollen development in both genotypes are also described. The nucleus progressively degenerated in the microspores of the pistillate flowers starting from the early‐uninucleate stage, in parallel with the progressive decrease in ubiquitin content and activity. At anthesis, the microspores in the pistillate flowers either had no nucleus or showed only traces of chromatin. Thus, the ubiquitin system seems to play an important role in protein turnover occurring during the normal developmental pathway of the kiwifruit microgametophyte, while it was mainly involved in regressive events related to microspore degeneration in the female genotype.
Pectic epitopes are differentially distributed in the cell walls of potato (Solanum tuberosum) tubers
Authors: Bush Maxwell S. , McCann Maureen C.
Published: March 2003

Six monoclonal antibodies (mAbs) were used to map the distribution of pectic epitopes in the cell walls of potato (Solanum tuberosum L. cvs Kardal and Karnico) tuber tissue in both light and electron microscopes. Unesterified (mAb JIM 5 epitope) and methyl‐esterified (mAb JIM 7 epitope) pectins were abundant and equally distributed in all parenchymal and vascular cell walls. Homogalacturonans (HGAs) involved in Ca2+‐cross‐linking (mAb 2F4 epitope) were localised to the middle lamella and abundant at cell corners. The tuber cortex was densely labelled, but parenchymal cell walls in the perimedullary region contained few epitopes of calcium pectate except at corners and pit fields. In contrast, pectic side‐chains were not detectable in the middle lamella of all parenchymal cell walls, except in the cortex where mAb LM6 (arabinan epitope) labelled the entire wall. The galactan epitope (mAb LM5) was localised to a zone very close to the plasmalemma in cortical cell walls and was also less abundant at pit fields and in vascular cell walls. MAb CCRC‐M2 (rhamnogalacturonan I epitope) did not cross‐react. Our results show that the cell walls of potato tubers are not homogeneous structures and that the pectic composition of the walls is spatially regulated.
Partial characterization of potato F0F1‐ATPase Γ‐subunit cDNA and its regulation during fungal infection and elicitor treatment
Authors: Madrid Enrique Alberto , Laxalt Ana María , Beligni Verónica , Lamattina Lorenzo
Published: March 2003

Mitochondrial F0F1‐ATP synthase from dicotyledonous plants contains six different subunits named α, β, Γ, Δ, Δ′ and ɛ. A key subunit in the coupling mechanism is the Γ‐subunit, which appears as a single copy in the complex interacting with multiple α and β subunits that contain the catalytic sites. One 0.8‐kb potato cDNA clone sharing 88% homology with the 3′‐end of sweet potato Γ‐ATPase has been isolated by reverse transcriptase‐polymerase chain reaction (RT‐PCR) amplification from potato leaves infected with the pathogenic fungus Phytophthora infestans. Northern blot experiments revealed that the potato Γ‐subunit mRNA level increased 2‐ and 3‐fold in infected potato leaves 48 and 72 h after infection, respectively. The enzyme activity of F0F1‐ATPase was 2‐fold higher in 48‐h‐infected potato leaves than in mock‐inoculated ones. The fungal elicitors eicosapentanoic acid (EPA) and glucans from P. infestans cell wall were used in induction experiments on potato tuber discs. After 48 h of treatment, the Γ‐ATPase mRNA levels were respectively increased by 1.8, 1.4 and 2.2‐fold in EPA, glucans and EPA plus glucan treatments. The present results show that the Γ‐subunit mRNA levels of F0F1‐ATPase are up‐regulated during biological stress conditions and elicitor treatments in potato and correlate with the increase of respiration rate and metabolic activity of mitochondria observed during infection processes in plants.
Kinetic characterization of succinate‐dependent plasma membrane‐bound nitrate reductase in tobacco roots 1
Authors: Wienkoop Stefanie , Ullrich Wolfram R , Stöhr Christine
Published: March 2003

Plasma membrane‐bound nitrate reductase (PM‐NR) of tobacco (Nicotiana tabacum L. cv. Samsun) roots reduces nitrate with NADH and/or succinate as electron donor. The present paper reports on significant differences of the succinate‐dependent activity (succ‐PM‐NR) to known NADH‐dependent soluble and plasma membrane‐bound NR. The experiments were performed with plasma membrane vesicles containing the hydrophobic PM‐NR. In a temperature course, succ‐PM‐NR activity attained the highest rates of total activity (succinate‐nitrate) at 50°C, NADH‐dependent PM‐NR (NADH‐PM‐NR) only at 30°C. The temperature responses of partial reactions with domains of NR diverged, but indicated that the heme domain was the most sensitive to high temperatures and could limit succ‐PM‐NR. In contrast to NADH, succinate did not supply electrons to ferricyanide reduction. The pH optima of the overall reaction with succinate were 5.6 and 8.0 at 30°C, pH 7.0 at 50°C, with a much higher rate in the latter case. The affinities of succ‐PM‐NR for both substrates (succinate and nitrate) were highest at pH 5.6 and higher at 50°C than at 30°C. Malonate showed competitive inhibition with succinate, but not with NADH as substrate. We assume that structural differences in the flavin domain of at least one of the subunits of root PM‐NR may be responsible for the succinate‐dependent in addition to the NADH‐dependent activity, but a more detailed analysis is necessary.
Proton pumping pyrophosphatase from higher plant mitochondria
Authors: Vianello Angelo , Macrì Francesco
Published: March 2003

In higher plant cells, there are some enzymes capable of utilizing pyrophosphate (PPi) as an energy donor. Among these, membrane‐bound proton pumping pyrophosphatases (H+‐PPiase) have been identified. In addition to the well‐known vacuolar H+‐PPiase (V‐PPiase), there is evidence for the presence of a mitochondrial H+‐PPiase. This enzyme is localized on the inner surface of the inner membrane and catalyzes the specific hydrolysis of PPi, coupled to proton transport, with a H+/PPi stoichiometry of ca 2. This activity is Mg2+‐requiring, is stimulated by monovalent cations, and is inhibited by Ca2+, F− and diphosphonates. The H+‐PPiase contains a catalytic head which is constituted by a 35‐kDa protein which is loosely bound to the inner membrane. This protein exhibits a PPiase activity, stimulated by phospholipids, with characteristics very similar to the membrane‐bound enzyme. The mitochondrial PPiase is distinct from the V‐PPiase, because an antibody raised against the 35‐kDa protein does not react with tonoplast membranes. The mitochondrial H+‐PPiase seems to have an F‐type structure, similar to the F‐ATP synthase and the membrane‐bound PPiases from mammalian and yeast mitochondria. It is suggested that, beside synthesizing PPi, this enzyme may act as a buffer for the electrochemical proton gradient, by hydrolyzing PPi, during conditions of oxygen deprivation.
Isoenzymic forms of carbonic anhydrase in the red macroalga Porphyra leucosticta
Authors: Mercado Jesús M. , Viñegla Benjamín , Figueroa Félix L. , Niell F. Xavier
Published: March 2003

Different isoenzymes of carbonic anhydrase (CA; EC 4.2.1.1) have been separated using thalli of the red macroalga Porphyra leucosticta Thuret in Le Jolis. Homogenates of the thallus were centrifuged in order to separate soluble and membrane proteins. The fraction containing membrane proteins was subdivided by centrifuging into two fractions: green and nongreen membrane proteins. CA activity was detected in all the fractions. Because external CA (measured on intact thallus) represented 15% of total activity, it was concluded that most of the CA (ca. 80%) was soluble and internal. Direct evidence regarding the different function of external and internal CA was obtained by determining the effects on photosynthesis of two specific CA inhibitors with different capacity for entering cell. It was concluded that internal CA was necessary to ‘trap’ the CO2 entering the cell and thus maintain a favorable CO2 gradient that permits its diffusive entry. Changes in the O2 evolution rate at inorganic carbon (Ci) concentration saturating for photosynthesis and on the photosynthetic conductance for Ci were found when external CA was inhibited. Based on these changes and the significant CA activity (ca. 9% of the total activity) found in nongreen membrane fraction, the presence of external CA associated with plasma membrane was postulated. The presence of CA associated with chloroplast membrane was also suggested.
Copper affects the enzymes of the ascorbate‐glutathione cycle and its related metabolites in the roots of Phaseolus vulgaris
Authors: Gupta Manisha , Cuypers Ann , Vangronsveld Jaco , Clijsters Herman
Published: March 2003

The involvement of the ascorbate‐glutathione cycle in the defence against Cu‐induced oxidative stress was studied in the roots of Phaseolus vulgaris L. cv. Limburgse vroege. All the enzymes of this cycle [ascorbate peroxidase (APOD), EC 1.11.1.11; monodehydroascorbate reductase (MDHAR), EC 1.6.5.4; dehydroascorbate reductase (DHAR), EC 1.8.5.1; glutathione reductase (GR), EC 1.6.4.2] were increased, and the total ascorbate and glutathione pools rose after a 15 μM root Cu treatment. In the first hours after the start of the experiment, the accumulation of dehydroascorbate (DHA), formed as a result of a Cu‐mediated direct oxidation of ascorbate (AA), was limited by a non‐enzymatic reduction using glutathione (GSH) as the reductant. At 24 h, the enzyme capacities of both DHAR and GR were increased to maintain the redox status of the AA and GSH pools. After 72 h of Cu application, the DHAR capacity was inhibited and MDHAR was responsible for maintaining the AA pool in its reduced form. Although the GR capacity was enhanced after 72 h in the treated plants, the GSSG/GSH ratio was increased. This could be due to direct participation of GSH in the detoxification of Cu through reduction and complexation.
Simulated weightlessness and hyper‐g results in opposite effects on the regeneration of the cortical microtubule array in protoplasts from Brassica napus hypocotyls
Authors: Skagen Else B. , Iversen Tor‐Henning
Published: March 2003

Enzymatic digestion of the cell wall of Brassica napus hypocotyls gave a heterogeneous suspension of protoplasts with the cortical microtubules (CMTs) randomly organised or CMTs organised in parallel. The effect of variable g‐influences has been tested on CMT organisation. In contrast to the 1 g‐protoplasts, which reorganised the CMTs into parallel arrays during the 96 h test period, the frequency of randomly‐oriented CMTs in the protoplasts exposed to simulated weightlessness (0 g) on a 2‐D clinostat increased significantly during the same period. The opposite effect was obtained when the protoplasts were exposed to hyper‐g (7 or 10 g), where the reorganisation of the CMTs into parallel arrays was accelerated compared to the 1 and 0 g‐protoplasts. These results indicate that a unidirectional gravity force is a necessity for the reorganisation of CMTs in protoplasts to parallel arrays and that CMTs act as responding elements that are able to sense different levels of gravity. Besides the inability of the protoplasts to reorganise the CMTs into parallel arrays, the quantity of CMTs in the individual protoplast decreased during 4 days of simulated weightlessness, both compared to the CMTs quantity in the protoplasts immediately after isolation and compared to the 1 g‐ and hyper‐g‐protoplasts after 24 and 48 h of g‐exposure. The size of the protoplasts was also affected by the g‐exposure. Protoplasts exposed to simulated 0 g increased significantly after 24 and 48 h, whereas the 1 g‐ and 10 g‐protoplasts maintained the same size during the 48 h test period.
Biochemical and phenotypical characterization of transgenic tomato plants overexpressing a basic peroxidase
Authors: El Mansouri Iman , Mercado José A. , Santiago‐Dómenech Nieves , Pliego‐Alfaro Fernando , Valpuesta Victoriano , Quesada Miguel A.
Published: March 2003

Tomato plants (Lycopersicon esculentum Mill. cv. Pera) were transformed via Agrobacterium tumefaciens with the binary vector pKYLX71 containing a tomato basic peroxidase (EC 1.11.1.7) gene, tpx1, under the control of the cauliflower mosaic virus (CaMV35S) promoter. Transgenic plants showed a 2–5‐fold increase in the activity of the peroxidase ionically bound to the cell wall, whereas soluble peroxidase activity remained similar or even lower than wild‐type plants. Isoelectric focusing showed the presence of a new isoperoxidase of pI ca 9 in the ionically bound extract. Western blot also showed the presence of a new band at 41 kDa that was absent in the wild‐type extract. A 40–220% increment of lignin content of the leaf was found in transgenic plants. Shoot phenotype of transgenic plants was similar to wild type, although under stress, the plants appeared wilted and the new leaves had a reduced area and were thicker than wild‐type or older transgenic leaves. The root system was underdeveloped in transgenic plants, but the rooting ability of the stem was not affected by the overexpression of peroxidase. Finally, the morphogenetic response of cotyledon and hypocotyl explants from transgenic plants was evaluated. In the case of cotyledons, the percentage of explants with shoot was not different from wild‐type plants. For hypocotyl, one of the transgenic lines showed a 30% reduction in the percentage of shoot organogenesis. The results are discussed in relation to the role of tpx1 in lignin synthesis.
In situ hybridization study of the rice phloem thioredoxin h mRNA accumulation – possible involvement in the differentiation of vascular tissues
Authors: Ishiwatari Y. , Nemoto K. , Fujiwara T. , Chino M. , Hayashi H.
Published: March 2003

Thioredoxin (TRX) h was identified as a major protein in rice (Oryza sativa L. cv. Kantou) phloem sap. Accumulation of TRX h mRNA in fully expanded rice leaves was detected only in the companion cells. To clarify the role of TRX h in phloem development, we performed in situ hybridization of TRX h mRNA in rice plants during the course of vascular development. TRX h mRNA was detected in all cell types at an early stage of development and became gradually confined to the companion cells in later stages. The possible roles of TRX h in vascular development are discussed.
Allocation of carbon to growth and secondary metabolites in birch seedlings under UV‐B radiation and CO2exposure
Authors: Lavola Anu , Julkunen‐Tiitto Riitta , de la Rosa Tania M. , Lehto Tarja , Aphalo Pedro J.
Published: March 2003

In plants, the allocation of carbon to secondary metabolites has been shown to be determined by both the availability of resources (e.g., CO2concentration) and by specific stress factors (e.g., ultraviolet [UV]‐radiation). It has been suggested that, in combination, CO2and UV‐B radiation may differentially affect plant growth and morphogenic parameters, and elevated CO2may ameliorate the effects of UV‐B radiation. In the present work, the effects of increased atmospheric CO2concentration and UV‐B radiation on growth and the accumulation of different types of secondary metabolites were studied in silver birch (Betula pendulaRoth). Seedlings were exposed to 350 and 700 μmol mol−1of CO2in a greenhouse. At both CO2levels, additional UV‐B was either present (8.16 kJ m−2day−1of biologically effective UV‐B irradiance) or absent. The time course of accumulation of individual secondary compounds and the shifts in allocation of carbon between biomass and the secondary metabolites (phenolic acids, flavonoids, condensed tannins) were studied during a 1‐month‐long exposure. Additionally, the activities of enzymes (l‐phenylalanine ammonia‐lyase [PAL], EC 4.3.1.5; peroxidase, EC 1.11.1.7; polyphenol oxidase, EC 1.10.3.1) were determined for leaves. UV‐B radiation significantly increased biomass, PAL activity, and the accumulation of phenolic acids and flavonoids in seedlings. Elevated CO2concentration increased the activities of all the enzymes studied and the accumulation of condensed tannins in leaves, especially with UV‐B radiation. Because the observed UV‐B induction of flavonoids was smaller under a high CO2concentration, it was suggested that the excess of carbon in the atmosphere may moderate the effect of UV‐B by increasing the metabolic activity of leaves (high enzyme activities) and by changing the allocation of internal carbon between different primary and secondary metabolites in the plant. Our results demonstrate the significant increase in the allocation of carbon to secondary metabolites without any large change in growth due to the elevation of CO2concentration and UV‐B radiation. There also was a stronger impact of CO2than UV‐B on the phenolic metabolism of birch seedlings.
Purification and characterisation of two ACC oxidases expressed differentially during leaf ontogeny in white clover
Authors: Gong Deming , McManus Michael T.
Published: March 2003

Two isoforms of ACC oxidase (ACO) (EC 1.4.3), expressed differentially during leaf ontogeny in white clover (Trifolium repens L.), have been identified and purified to homogeneity. One isoform, designated MGI, was purified from mature green leaf tissue while the second isoform, designated SEII, was purified from senescent leaf tissue. The isolation and purification of these isoforms were achieved using a combination of hydrophobic interaction chromatography, anion exchange chromatography, chromatofocusing and gel filtration column chromatography. The Mr of both MGI and SEII was determined to be 37.5 kDa by gel filtration, and 37 kDa (MGI), 35 kDa (SEII) by SDS‐PAGE, indicating that both isoforms are active as monomers. During purification, both isoforms were recognised by a polyclonal antibody directed against a recombinant polypeptide derived from a white clover ACO gene expressed in mature green leaf tissue, TR‐ACO2. In addition to molecular mass, differences between the two isoforms were observed in terms of pH optima, isoelectric point (pI), Km for ACC, optimal requirements for the co‐substrate ascorbate, and NaHCO3 and Fe2+ as co‐factors. The identification of distinct ACC oxidases from the same tissue at different developmental stages shows that the now widely observed transcriptional regulation of the ACO gene family in higher plants is also expressed in terms of differential regulation of enzyme isoforms.
Ultraviolet‐B radiation induces complex alterations in stomatal behaviour
Authors: Jansen Marcel A. K. , Van Den Noort Ria E.
Published: March 2003

Both visible and UV wavelengths play an important role in controlling stomatal aperture. We have analysed effects of UV‐B radiation on stomatal aperture inVicia faba, and found them to be complex. Depending on the metabolic state of the guard cell, high fluences of UV‐B either stimulate stomatal openingorstomatal closing. Neither of these responses is readily reversed, i.e. once stomata have been exposed to UV‐B, they are unable to re‐adjust their aperture in response to environmental stimuli like changes in light, humidity or ABA. This lack of responsiveness is unlikely to be due to widespread cellular damage, as UV‐induced stomatal closure is largely reverted in response to the H+‐ATPase activator fusicoccin. It is speculated that UV‐B impacts upstream from the plasmalemma based enzyme complexes which facilitate the solute fluxes leading to stomatal opening. Our data may help accommodate seemingly contradictory reports on the effects of UV‐B on stomatal aperture and/or conductance.
Endogenous hormone levels in explants and in embryogenic and non‐embryogenic cultures of carrot
Authors: Jiménez Víctor M. , Bangerth Fritz
Published: March 2003

Carrot (Daucus carota L. F1 hybrid Starca) excised hypocotyls were cultured on Murashige and Skoog medium with and without 2,4‐dichlorophenoxy acetic acid (2,4‐D) to determine the effect of this plant growth regulator on their further development and their endogenous hormone levels. Culture in the absence of 2,4‐D stimulated root development at one end of the hypocotyl segments and increased the endogenous levels of free indole‐3‐acetic acid (IAA), zeatin/zeatin riboside and N6(Δ2‐isopentenyl) adenine/N6(Δ2‐isopentenyl) adenosine, as determined by radio‐immunoassay. On the other hand, the presence of 2,4‐D in the culture medium promoted callus induction and proliferation, together with abscisic acid (ABA) accumulation, in the hypocotyl segments during the first weeks of culture. When the callus segments generated in the hypocotyl sections cultured in the presence of 2,4‐D were cultivated further, the development of two callus types was observed, one composed of preglobular and globular embryos and the other translucent, watery and lacking any sign of organisation. The embryos of the first type germinated when callus segments were transferred to regeneration conditions, while no change was observed when the second type was induced to regenerate. Higher levels of free IAA and ABA were obtained in the embryogenic calli when compared to the non‐embryogenic, while no differences were observed among callus types in the other hormones evaluated. The possible role of the different plant hormones during induction of somatic embryogenesis is discussed.
Rice α‐mannosidase digesting the high mannose glycopeptide of glutelin
Authors: Kishimoto Tadashi , Hori Hidetaka , Takano Daisuke , Nakano Yoshiyasu , Watanabe Mayumi , Mitsui Toshiaki
Published: March 2003

α‐Mannosidase (EC 3.2.1.24) from rice dry seeds was purified to homogeneity. Optimum pH and Km for pNP‐α‐Man hydrolysis were pH 4.3–4.5 and 1.04 mM, respectively. The enzyme digested mannobioses such as Manα‐1,2Man, Manα‐1,6Man, Manα‐1,3Man but Manα‐1,4Man. Zn2+ ion was required for the activity, whereas EDTA and swainsonine inhibited the activity by 80 and 96%, respectively. The rice storage protein, glutelin was prepared and its basic subunits were shown to have high mannose‐type sugar chains by two‐dimensional mapping using NH2‐P and C18 silica columns. They were Man9GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2 and Man5GlcNAc2. All these oligosaccharides were digested by the purified α‐mannosidase, and Man‐GlcNAc2 and mannose were formed. Glycopeptides, having these high mannose‐type sugar chains, could also be digested by the α‐mannosidase. Subunits were prepared from glutelin basic subunit and the richest subunit among them, subunit 2 (isoform 2), was digested by the α‐mannosidase. Isoform 2 was digested by V8 protease only partially and slowly. However, isoform 2, pre‐treated with the α‐mannosidase, was rapidly and completely digested by V8 protease.
Brassinosteroid‐induced exaggerated growth in hydroponically grown Arabidopsis plants
Authors: Arteca Jeannette M. , Arteca Richard N.
Published: March 2003

The effects of root application of brassinolide (BL) on the growth and development of Arabidopsis plants (Arabidopsis thaliana ecotype Columbia [L.] Heynh) were evaluated. Initially, all leaves were evaluated on plants 18, 22, 26 and 29 days old. The younger leaves were found to exhibit maximal petiole elongation and upward leaf bending in response to BL treatment. Therefore, based on these results leaves 6, 7 and 8 on 22–24‐day‐old plants were selected for all subsequent studies. Elongation along the length of the petiole in response to BL treatment was uniform with the exception of an approximately 4 mm region next to the leaf where upward curvature was observed. Both BL and 24‐epibrassinolide (24‐epiBL) were evaluated, with BL being more effective at lower concentrations than 24‐epiBL. The exaggerated growth induced by 0.1 μM BL was not observed in plants treated with 1 000‐fold higher concentrations of GA3, IAA, NAA or 2,4‐D (100 μM). In addition, no exaggerated growth effects were observed when plants were treated with 200 ppm ethylene or 1 mM ACC. All treatments with BL, NAA, 2,4‐D, IAA or ACC promoted ethylene and ACC production in wild type Arabidopsis plants, but only BL triggered exaggerated plant growth. BL also promoted exaggerated growth and elevated levels of ACC and ethylene in the ethylene insensitive mutant etr1‐3, showing that the effect of BR on growth is independent of ethylene. This work provides evidence that BR‐induced exaggerated growth of Arabidopsis plants is independent of gibberellins, auxins and ethylene.
Cloning and characterization of leaf senescence up‐regulated genes in sweet potato
Authors: Huang Yih‐Jong , To Kin‐Ying , Yap Mee‐Ngan , Chiang Wen‐Joan , Suen Der‐Fen , Chen Shu‐Chen Grace
Published: March 2003

Genes that are expressed during leaf senescence in sweet potato (Ipomoea batatas, cv. Tainong 57) were identified by the isolation of cDNA fragments with the mRNA differential display method. Eight senescence‐associated cDNA clones for mRNAs differentially expressed during leaf senescence were obtained and characterized. Northern blot analysis indicated that all these clones represented genes that are up‐regulated during natural leaf senescence. Among them, five cDNA clones have been obtained in full length by screening a senescing leaf cDNA library or by performing rapid amplification of cDNA ends. DNA and protein database searches revealed that clones SPA15 and SPC9 encode proteins of unknown function. The other six clones SPG31, SPC20, SPG27, SPC25, SPC15 and SPC1 showed significant sequence homology to known genes encoding a cysteine proteinase, isocitrate lyase, S‐adenosylmethionine decarboxylase, cysteine proteinase inhibitor and metallothionein‐like type I protein. The gene expression patterns represented by SPG31, SPG27 and SPA15 were found to be highly specific in senescing leaves. The corresponding transcripts for SPG31, SPG27 and SPA15 were below detectable levels in other organs such as flowers, stems, roots and tubers. The possible physiological roles of these gene products in the leaf senescence process are discussed.
Differential leaf stress responses in young and senescent plants
Authors: Abarca Dolores , Martín Mercedes , Sabater Bartolomé
Published: March 2003

Responses to low temperature, mechanical wounding and salicylic acid (SA) treatments were studied in 3‐week‐old (young) and 6‐week‐old (senescent) Arabidopsis thaliana (L.) Heynh. plants by analyzing increases in Pal1 and Pr1 expression and superoxide dismutase (SOD; EC 1.15.1.1) and peroxidase (POX; EC 1.11.1.7) activities. Young plants showed higher Pal1 transcript accumulation after low temperature and wounding. In contrast, senescent plants presented higher accumulation of Pr1 transcripts after SA treatments. Similar results were obtained with the ethylene‐insensitive etr1 mutant, suggesting that these differences are not related to increased ethylene content in senescent tissues. SOD activity and inducibility were lower, whereas POX activity and inducibility were higher in senescent plants. A possible relationship between senescence‐associated changes in responses to stress and in the metabolism of active oxygen species is discussed.
Identification and characterization of a NaCl‐inducible vacuolar Na+/H+ antiporter in Beta vulgaris
Authors: Xia Tao , Apse Maris P. , Aharon Gilad S. , Blumwald Eduardo
Published: March 2003

We have cloned, by RT‐PCR and the use of degenerate oligonucleotide primers, a Na+/H+ antiporter from Beta vulgaris that is homologous to NHX1 of Arabidopsis thaliana and is a member of the family of recently cloned plant NHX‐genes. This antiporter, BvNHX1, partially complements the salt‐sensitive phenotype of a Δena1–4Δnhx1 yeast strain. Antibodies were raised against a central portion of the BvNHX1 open reading frame that was predicted from the cloned cDNA. This antiporter was found to be highly enriched in tonoplast membranes isolated from plant tissues. BvNHX1 transcript abundance increased after salt treatments, in both suspension‐cell cultures and whole plants. BvNHX1 protein abundance in the tonoplast‐enriched membranes was also elevated after salt treatments. The vacuolar Na+/H+ antiporter activity increased up to 3‐fold when the cell were exposed to 100 mM NaCl. The increase in protein abundance in response to the salt treatment, together with the salt‐induced vacuolar Na+/H+ antiporter activity in B. vulgaris suggests that BvNHX1 plays an important role in salinity tolerance.
Effects of ozone on photosynthetic CO2 exchange, chlorophyll a fluorescence and antioxidant systems in lettuce leaves
Authors: Calatayud Angeles , Ramirez Jaime W. , Iglesias Domingo J. , Barreno Eva
Published: March 2003

The effects of ozone exposure on lettuce leaves were investigated by means of gas exchange, modulated chlorophyll a fluorescence and antioxidants systems. High ozone concentration decreased the rate of photosynthesis at light saturation level and changes in stomatal conductance and transpiration rate. However, an increase in intercellular CO2 concentration indicated a decrease in the carboxylation efficiency. These data agreed with a reduction of non‐cyclic electron flow and lower capacity to reduce the quinone pool. Ozone affected the ascorbate pool and decreased ascorbate peroxidase activity, increased lipid peroxidation, altered membrane properties and reduced the development of non‐photochemical quenching.
Prevention dof recurrent cytomegalovirus disease in renal and liver transplant recipients: effect of oral ganciclovir
Authors: Turgeon N. , Fishman J.A. , Doran M. , Basgoz N. , Tolkoff‐Rubin N.E. , Cosimi A.B. , Rubin R.H.
Published: March 2003

Background: Although the primary treatment of symptomatic cytomegalovirus (CMV) disease in organ transplant recipients is successful in >90% of individuals, relapsing disease, particularly in those with primary infection, remains an important problem. Previously, we had observed that the rate of symptomatic recurrence was >60% in those with primary disease (seronegative for CMV prior to transplant), and approximately 20% in those who were seropositive prior to transplant. The present study was undertaken to determine whether a maintenance regimen of oral ganciclovir for 2–3 months added to the routine 14–21 days of intravenous ganciclovir would further prevent symptomatic CMV recurrence.

Methods: From May 1995 until June 1998, all kidney and liver transplant recipients with confirmed tissue‐invasive CMV disease or CMV syndrome were treated with 14–21 days of intravenous ganciclovir (5 mg/kg b.i.d. with dose adjusted for renal dysfunction) followed by 2–3 months of oral ganciclovir (2 g daily). The incidence of recurrence of CMV disease and/or viremia during and after oral therapy was then determined over a mean follow‐up of 530.6 days.

Results: Thirty‐seven patients, 19 kidney and 18 liver transplant recipients, were studied; 5 had biopsy‐proven tissue‐invasive disease (13.5) and 32 suffered a CMV syndrome (86.5). Twenty‐one of these patients (58.6) were seronegative for CMV prior to transplant and received an allograft from a seropositive donor (D+/R−). Overall, 10 patients (27.0) developed CMV recurrence. Eight of 21 patients who were D+/R− for CMV (38.1) developed recurrence as opposed to 2 of 16 patients with other serologic status (12.5) (P=0.14). Patients with recurrent CMV disease and/or viremia had a peak antigenemia assay titer during their initial CMV event of 319.2 positive cells/2 slides compared with 109.8 positive cells/2 slides for patients without recurrent CMV infection (P=0.14); the trend of having a higher peak antigenemia assay titer among patients who recurred occurred both in patients who were at risk of primary CMV infection (D+/R− for CMV) and in those who were not. Two patients developed recurrent infection with strains of CMV that were resistant to ganciclovir.

Conclusions: This new therapeutic regimen of oral ganciclovir following intravenous ganciclovir slightly reduced the overall rate of recurrent CMV disease and/or viremia, but it still did not adequately prevent CMV recurrence in patients who are at risk of primary infection prior to transplant. Of particular concern, 2 patients with primary infection treated with this regimen developed ganciclovir‐resistant recurrent disease (Note).