bluuebunny/crossref_metadata_2025 huggingface dataset:
Chlorophyllase and peroxidase activity during degreening of maturing canola (Brassica napus) and mustard (Brassica juncea) seed
Authors: Johnson‐Flanagan Anne M. , Spencer Mary S.
Published: March 2003

Chlorophyllase and peroxidase activities were measured in relation to seed maturation and degreening in canola (Brassica napus cvs Westar and Alto) and mustard (Brassica juncea cvs Cutlass and Lethbridge 22A). Samples of seed collected at the same moisture content were pooled, then divided and used for each assay. During maturation the green pigment (chlorophyll and related pigments) content of canola seed decreased linearly and was lower than that measured in mustard at all moisture contents studied, except for the highest and lowest moisture contents. Chlorophyllides and pheophorbides were detected in canola and were essentially absent in mustard. This difference in accumulation of dephytylated pigments infers differences in the pigment degradation pathways in Brassica species. Interspecific differences in the enzymology of degreening were found. Green pigment degradation was associated with increased chlorophyllase activity and low peroxidase activity in canola and low Chlorophyllase and high perosidase activity in mustard. The possible role of ethylene in seed degreening is discussed.
Antioxidant compound responses to chilling stress in differentially sensitive inbred maize lines
Authors: Hodges D. Mark , Andrews Christopher J. , Johnson Douglas A. , Hamilton Robert I.
Published: March 2003

Chilling temperatures increase the amounts of potentially lethal toxic oxygen compounds present within plants. These toxic oxygen compounds can be scavenged by antioxidant compounds such as ascorbate and β‐carotene. Three developmental stages (first, third and fifth leaf) of four inbred lines of maize (Zea mays L.) exhibiting differential sensitivity to chilling were examined in order to determine if the chilling‐sensitive line had lower concentrations of antioxidant compounds than did the tolerant lines. Plants were exposed to one of three treatments: (1) control (25°C constant), (2) control treatment plus a short‐term chilling exposure of 11°C one day prior to harvesting, and (3) long‐term (11°C constant) chilling exposure. Total ascorbate, total glutathione, β‐carotene, α‐tocopherol and chlorophyll contents were quantified, and ratios of dehydroascorbate/ascorbate and reduced/oxidized glutathione were determined. Lower concentrations of β‐carotene were found in the chilling‐sensitive relative to those in the chilling‐tolerant lines for the first‐leaf stage under both short‐ and long‐term chilling treatments. Concentrations of total ascorbate and glutathione and β‐carotene in the chilling‐sensitive line increased as the chilling treatment progressed and as the plants developed until they ultimately became either significantly higher or no different relative to the tolerant lines. Results suggest that this sensitive line became less sensitive to chilling‐induced oxidative stress with development.
White light‐induced sugar distribution controls growth and osmotic properties in the coleoptile and the first leaf in Zea mays seedlings
Authors: Parvez Mohammad Masud , Wakabayashi Kazuyuki , Hoson Takayuki , Kamisaka Seiichiro
Published: March 2003

The correlation of white light‐induced changes in osmotic concentration in the coleoptile and the first leaf and the growth rate of these organs in maize seedlings, was examined in relation to sugar distribution and invertase activity. One hour irradiation with white light decreased the osmotic concentration in basal zones of the coleoptile and increased it in the apical zones of the first leaf. The change in the osmotic concentration was positively correlated with the growth rate of both organs. The amount of total osmotic solutes in each zone was highly correlated with that of soluble sugars. Light decreased the activity of wall‐bound invertase in the coleoptile, but increased it in the first leaf. A high correlation existed between the content of soluble sugars and invertase activity in both organs. During 1 h incubation in the light, ca 2 µmol of soluble sugars per seedling was lost from the coleoptile and gained in the first leaf. Light promoted sugar exudation from the excised coleoptile, but the amount of soluble sugar exuded represented 5% of sugar loss from the coleoptile in intact seedlings.

These results indicate that in maize seedlings white light controls the growth rate of the coleoptile and the first leaf through the osmotic concentration. Light may have an osmoregulatory function in the control of sugar distribution between the coleoptile and the first leaf by regulating the activity of wall‐bound invertase.
Changes in transcript levels of chloroplast psbA and psbD genes during water stress in wheat leaves
Authors: He Jun‐Xian , Wen Jiang‐Qi , Chong Kang , Liang Hou‐Guo
Published: March 2003

In order to examine whether the decrease in gene expression of chloroplast DNA‐encoded polypetides contributes to the inhibition of photosystem II (PSII) function during water stress, changes in transcript and template levels of chloroplast psbA and psbD genes (encoding the D1 and D2 reaction center proteins of PSII, respectively) were investigated in spring wheat leaves (Triticum aestivum L. cv. Longchun No. 10) using northern, Southern and dot blot analyses. The results of northern hybridization indicated that stressing wheat seedlings in polyethylene glycol (PEG) solutions with an osmotic potential of −0.5 MPa for 0, 24, 48 and 72 h, caused marked declines in the steady state levels of the psbA and psbD transcripts but did not alter their transcript processing patterns. RNA dot blot analysis further demonstrated that over the whole range of water stress investigated, the transcript levels of the two genes declined by 2‐ and 3‐fold, respectively, relative to the same amount of total RNA. As total RNA decreased 3‐fold during the process of stress, the transcript levels of psbA and psbD genes actually declined by 6‐ and 9‐fold, respectively. These results suggest that water stress affects the expression of the psbA and psbD genes, possibly at the transcriptional level. Southern and DNA dot blot analyses consistently showed that water stress did not affect the template levels of either psbA or psbD genes, suggesting that the decreased abundance of psbA and psbD transcripts under water stress is not due to limited gene templates but likely a result of lowered gene transcriptional activity and/or changed mRNA stability.
Is tissue culture a viable system with which to examine environmental and hormonal regulation of cold acclimation in woody plants?
Authors: Baldwin Brian D. , Bandara Manjula S. , Tanino Karen K.
Published: March 2003

In vitro‐grown saskatoon berry (Amelanchier alnifolia Nutt.) plantlets were exposed to various hormonal treatments, dormancy‐inducing and cold acclimation conditions to determine if this in vitro system would be viable for dormancy/hardiness studies in woody plants. Low temperature induced significant hardiness levels in plantlets to −27°C after 6 weeks at 4°C but did not approach liquid nitrogen levels of fully hardened, field‐grown buds. Control plantlets were consistently killed at −5°C throughout this period. Significant hardiness was attained under both short and long day/low temperature conditions; however, hardiness was reduced under continuous light or dark treatments. A pre‐exposure to the typical short photoperiod regime of woody plants did not significantly increase the rate of acclimation in these plantlets. The presence/absence of phytohormones in the media have a pronounced influence on the ability of plantlets to cold acclimate. Hormone‐free media increased hardiness to −10.5°C after 2 weeks in treatment. Addition of abscisic acid (ABA) increased cold hardiness levels (−12°C) while addition of benzylaminopurine (BAP) to this hormone‐free media decreased hardiness to −5.3°C. A combination of BAP and ABA treatments produced LT50 values intermediate between individual applications of either hormone. Conversely, α‐naphthaleneacetic acid (NAA) could not counteract the ABA‐induced hardiness. ABA treatments alone were not able to harden plantlets to the extent attained under low temperature acclimation conditions. Further, ABA could not maintain the hardiness levels of cold‐acclimating treatments and plantlets de‐acclimated to −9°C in BAP + ABA media. Subculturing in itself significantly elevated cold hardiness in plantlets to −9°C on BAP + NAA media within 3 days after subculture and thereafter plantlets dehardened to −5°C. While tissue culture has value in specific cases, caution should be taken when using tissue‐cultured plantlets as a system to evaluate environmental regulation of cold acclimation in woody plants, in part, due to the influence of phytohormones in the media.
Characterization of potato tuber lipoxygenase cDNAs and lipoxygenase expression in potato tubers and leaves
Authors: Fidantsef Ana L. , Bostock Richard M.
Published: March 2003

A collection of cDNAs was isolated from a λgt11 potato (Solanum tuberosum L.) tuber library by using a soybean lipoxygenase (LOX; linoleate:oxygen oxidoreductase, EC 1.13.11.12) sequence as a hybridization probe. These cDNA clones were classified by restriction patterns and sizes and two clones, plox1 and plox2, were selected for sequencing and further analysis. These clones were found to be virtually identical and contain full‐length cDNAs having a high degree of homology with other cloned LOX genes. plox1 contains a 2 806‐bp insert, with an open reading frame that encodes a protein of 861 amino acids (96.9 kDa), consistent with molecular mass estimates of the principal tuber LOX deduced from SDS‐PAGE. The deduced sequence contains iron‐binding domains that are conserved among LOXs. Restriction analysis of potato genomic DNA confirmed that potato LOX is comprised of a gene family with at least three members. The plox1 sequence hybridized to a transcript of 2.8 kb in gel blot analyses of total RNA extracted from potato tuber. Treatment of potato disks and leaves with methyl jasmonate or jasmonic acid increased LOX enzyme activity, as well as abundance of LOX mRNA, consistent with jasmonate's reported effect on LOX expression in plants. The fungal elicitor arachidonic acid (AA) strongly induced LOX enzyme activity in leaves and induced LOX mRNA transcripts in potato leaves and tuber disks. Infection of potato leaves with Phytophthora infestans induced LOX activity as well as LOX mRNA accumulation in both incompatible and compatible interaction types. LOX expression was compared with that of a wound‐inducible proteinase inhibitor (PINII) and a pathogenesis‐related protein (P4). Jasmonates induced PINII but not P4 transcript accumulation in potato leaves. In contrast, AA induced P4 but not PINII transcript accumulation in potato leaves. Infection with P. infestans resulted in a gene expression pattern similar to that observed with AA.
Freezing tolerance, cold acclimation and oxidative stress in potato. Paraquat tolerance is related to acclimation but is a poor indicator of freezing tolerance
Authors: Seppänen M. M. , Majaharju M. , Somersalo S. , Pehu E.
Published: March 2003

Potato is a species commonly cultivated in temperate areas where the growing season may be interrupted by frosts, resulting in loss of yield. Cultivated potato, Solanum tuberosum, is freezing sensitive, but it has several freezing‐tolerant wild potato relatives, one of which is S. commersonii. Our study was aimed to resolve the relationship between enhanced freezing tolerance, acclimation capacity and capacity to tolerate active oxygen species. To be able to characterize freezing tolerant ideotypes, a potato population (S1), which segregates in freezing tolerance, acclimation capacity and capacity to tolerate superoxide radicals, was produced by selfing a somatic hybrid between a freezing‐tolerant Solanum commersonii (LT50=‐4.6°C) and ‐sensitive S. tuberosum (LT50=‐3.0°C). The distribution of non‐acclimated freezing tolerance (NA‐freezing tolerance) of the S1 population varied between the parental lines and we were able to identify genotypes, having significantly high or low NA‐freezing tolerance. When a population of 25 genotypes was tested both for NA‐freezing and paraquat (PQ) tolerance, no correlation was found between these two traits (R = 0.02). However, the most NA‐freezing tolerant genotypes were also among the most PQ tolerant plants. Simultaneously, one of the NA‐freezing sensitive genotypes (2022) (LT50=‐3.0°C) was observed to be PQ tolerant. These conflicting results may reflect a significant, but not obligatory, role of superoxide scavenging mechanisms in the NA‐freezing tolerance of S. commersonii. The freezing tolerance after cold acclimation (CA‐freezing tolerance) and the acclimation capacity (AC) was measured after acclimation for 7 days at 4/2°C. Lack of correlation between NA‐freezing tolerance and AC (R =‐0.05) in the S1 population points to independent genetic control of NA‐freezing tolerance and AC in Solanum sp. Increased freezing tolerance after cold acclimation was clearly related to PQ tolerance of all S1 genotypes, especially those having good acclimation capacity. The rapid loss of improved PQ tolerance under deacclimation conditions confirmed the close relationship between the process of cold acclimation and enhanced PQ tolerance. Here, we report an increased PQ tolerance in cold‐acclimated plants compared to non‐acclimated controls. However, we concluded that high PQ tolerance is not a good indicator of actual freezing tolerance and should not be used as a selectable marker for the identification of a freezing‐tolerant genotype.
Interaction between photoperiod, photosynthesis and ethylene formation in tomato plants (Lycopersicon esculentum cv. Ailsa Craig and ACC‐oxidase antisense pTOM13)
Authors: Jensen E. B. , Veierskov B.
Published: March 2003

Wild‐type and ACC‐oxidase antisense tomato plants (Lycopersicon esculentum Mill. cv. Ailsa Craig and pTOM13) were grown in environment‐controlled rooms for 21 days under photoperiods of 8, 16 or 23.5 h at an irradiance of 300 µmol m−2 s−1. Photosynthetic pigments, photosynthesis, soluble carbohydrates, starch and ethylene were measured on the last fully expanded leaf. Increasing the photoperiod from 8 to 16 h stimulated all measured growth parameters in both cultivars. However, when the photoperiod was increased to 23.5 h, foliar yellowing and deformation were observed in the wild‐type Ailsa Craig whereas no change was observed in pTOM13. It was not possible to relate these foliar changes in Ailsa Craig to destruction of the photosynthetic apparatus by excess carbohydrate levels in the leaves. Because pTOM13 was antisense to ACC‐oxidase. it is proposed that yellowing and deformation in leaves of wild‐type tomato plants grown under long photoperiods may be caused by stress ethylene induced by a long photoperiod.
Molecular characterization, organ distribution and stress‐mediated induction of two glutathione peroxidase‐encoding mRNAs in sunflower (Helianthus annuus)
Authors: Roeckel‐Drevet P. , Gagne G. , De Labrouhe D. Tourvieille , Dufaure J.‐P. , Nicolas P. , Drevet J. R.
Published: March 2003

Using a reverse transcription PCR (RT‐PCR) amplification strategy, with degenerate primers localized in highly conserved domains of known glutathione peroxidase (GPX) proteins, GPX‐encoding cDNA fragments were generated from sunflower (Helianthus annuus L.) hypocotyl RNA. Along with the sequences of two distinct GPX‐encoding cDNAs (GPXha‐1 and GPXha‐2) and the alignment of their conceptually translated products with to date cloned plant GPXs, we report here that the two corresponding mRNAs are qualitatively and quantitatively differentially expressed in various organs of H. annuus. Using northern blotting we have shown that more sunflower GPX‐like transcripts accumulate in plants which have been subjected to either wounding or infection by a biotrophic pathogen (Plasmopara halstedii) suggesting that the corresponding genes are inducible under various stress conditions.
Characterization, induction by wounding and salicylic acid, and activity against Botrytis cinerea of chitinases and β‐1,3‐glucanases of ripening grape berries
Authors: Derckel Jean‐Paul , Audran Jean‐Claude , Haye Bernard , Lambert Bernard , Legendre Laurent
Published: March 2003

In order to better understand the defense strategy of grape berries (Vitis vinifera L. cv. Pinot noir) as they mature, the activities of the defense‐related proteins, chitinase (CHV, EC 3.2.1.14) and β‐1,3‐glucanase (laminarinase, EC 3.2.1.39) were first estimated in berries at different maturation stages. Chitinase levels rose proportionally to the berry reducing sugar content, an indicator of the berry ripening degree, up to values 10 times higher than the ones seen in resting grapevine leaves. This rise in activity was due to the accumulation of two isoforms, CHV 5 and CHV 11. One more chitinase isoform, CHV 12, appeared in senescent berries. Conversely, no glucanase activity could be detected in berries at any maturation stage. Accumulation of chitinases and (β‐1,3‐glucanases could be stimulated by wounding the berry peduncle. Adding salicylic acid to the wounded berries only potentiated the wounding effect on the berry chitinase activity. The most active chitinase isoform, CHV 5, was purified to homogeneity. It represented about 40% of the total extractable protein content of a ripe berry. Its molecular mass was estimated to be 31 kDa. The peptide sequencing of four of its tryptic fragments revealed strong homologies to several class IV chitinases. Finally, it was shown to inhibit the germination of conidia of Botrytis cinerea by 50% at a concentration of 7.5 µg ml−1.
Analysis of intercellular washing fluids of potato tubers and detection of increased proteolytic activity upon fungal infection
Authors: Olivieri Florencia , Godoy Andrea V. , Escande Alberto , Casalongué Claudia A.
Published: March 2003

We present the first procedure for extracting intercellular fluids of potato (Solanum tuberosum L. cv. Spunta) tubers. Intercellular washing fluids were isolated from healthy and Fusarium‐infected potato tissue. The electrophoretic pattern using SDS‐PAGE indicated differences between the fluids from the two tissues. A significant extracellular proteolytic activity was accumulated during the infection with Fusarium solani f. sp. eumartii. A major proteolytic band with an apparent molecular mass of 70 kDa and another of approximately 30 kDa were detected after separation of intercellular fluids by casein gel electrophoresis. Proteolytic activity was principally inhibited by diisopropylfluorophosphate, which is indicative of the involvement of serine protease(s). In vitro degradation assay indicated that specific potato proteins from healthy tubers were hydrolyzed by fluid proteases from infected tubers. The biological role of such activity in potato‐Fusarium interaction is still unknown. Our results suggest that the intercellular serine protease has a fungal origin.
Superoxide‐producing NAD(P)H oxidases in plasma membrane vesicles from elicitor responsive bean plants
Authors: Van Gestelen Peter , Asard Han , Horemans Nele , Caubergs Roland J.
Published: March 2003

Higher plants produce active oxygen species (AOS) that regulate their defence responses against pathogenic elicitation. Etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse vroege) were used to measure the in vivo‐induced AOS production and to search for plasma membrane bound NAD(P)H‐dependent oxidases producing AOS. Immersed bean plants showed a substantial production of H2O2, as determined by the peroxidase (EC 1.11.1.7)‐dependent oxidation of 3,5‐dichloro‐2‐hydroxybenzenesulfonic acid (DHBS). Addition of the elicitor polygalacturonase (PGase, EC 3.2.1.15) from Aspergillus japonicus or the phosphatase inhibitor, cantharidin, resulted in a transient increase of AOS synthesis. Plasma membrane vesicles, purified from etiolated bean seedlings, showed an NAD(P)H‐dependent superoxide (O2−) production that was highly stimulated with naphthoquinones. Protein solubilisation and anion exchange chromatography resolved a basal and three naphthoquinone‐stimulated NAD(P)H‐dependent O2− oxidase fractions. The natural phenol, apigenin, was also a strong inducer of the naphthoquinone‐dependent enzymes, when it was used in the presence of peroxidase. Although, the relation of these different in vitro‐determined plasma membrane NAD(P)H‐dependent O2− oxidases to the in vivo elicitation of H 2O2 has not been elucidated so far.
Respiratory metabolism during cold storage of apple fruit. II. Alternative oxidase is induced at the climacteric
Authors: Duque Paula , Arrabaça João D.
Published: March 2003

Changes in mitochondrial respiration were investigated during cold storage of apple fruit (Malus domestica Borkh. cv. Reinette du Canada). Postharvest calcium treatment of the apples had no effect on the aspects of mitochondrial metabolism studied, suggesting that the role of calcium in fruit ripening is not related to respiration. A transient increase in mitochondrial oxidative capacity occurred during storage around day 80 after harvest. This climacteric burst was associated with an enhanced capacity of the alternative pathway, but not of the cytochrome pathway. The major factor determining this rise in alternative pathway capacity at the climacteric was the transient increase in the amount of alternative oxidase (AOX) protein. Antibodies raised against the Sauromatum guttatum enzyme recognized a reduced form product of ampproximately 37 kDa and a 67‐kDa band corresponding to the oxidized form of the protein. During cold storage of the apple fruit, alternative pathway capacity correlated with the levels of the AOX and was ampparently not dependent on major changes in the oxidation state of the enzyme. This is the first work to report that the respiratory climacteric and the induction of AOX are tightly and temporally linked.
Alteration of pectic polysaccharides in cell walls, extracellular polysaccharides, and glycan‐hydrolytic enzymes of growth‐restricted carrot cells under calcium deficiency
Authors: Konno Haruyoshi , Nakashima Susumu , Maitani Toshihiko , Katoh Kenji
Published: March 2003

Suspension‐cultured carrot (Daucus carota L. cv. Kintoki) cells were grown in calcium (Ca2+)‐deficient and normal liquid media. Cell growth was limited by the Ca2+ deficiency. Similar amounts of pectic fractions were extracted from the walls of control and Ca2+‐deprived cells, but the fractions from the walls of Ca2+‐deprived cells showed a substantial decrease in galacturonic acid content. However, after 15 days of culture, Ca2+‐deprived cells released galacturonic acid‐rich extracellular polysaccharides at twice the rate of control cells. The polysaccharides consisted of a mixture of several polymers containing predominantly arabinose, galactose and galacturonic acid. Ca2+‐deprived cells also secreted three times more extracellular proteins, containing many glycan‐hydrolytic enzymes, into the medium than did normal cells. SDS‐PAGE analysis revealed several distinct changes in the polypeptide pattern in the medium of control and Ca2+‐deprived cells. Activities of α‐galactosidase, β‐glucosidase and exo‐polygalacturonase increased considerably during Ca2+ deficiency, whereas α‐
l‐arabinofuranosidase and β‐galactosidase activities were much reduced.
Metabolism of l[U‐14C]‐arginine and	l[U‐14C]‐ornithine in maturing and vernalised embryos and megagametophytes of Picea abies
Authors: Santanen Ajra , Simola Liisa Kaarina
Published: March 2003

The metabolism of the polyamine precursors arginine and ornithine was studied in maturing and vernalised seeds of Picea abies (L.) Karst. (Norway spruce) in feeding experiments. Incorporation of radioactivity from these

C‐labelled amino acids into liberated CO2, amino acids, polyamines, proteins and cell wall fractions, as well as polyamine levels were determined in embryos and megagametophytes. Ornithine and especially arginine decarboxylation was more active in the embryo than in the megagametophytic cells, and vernalisation increased arginine metabolism more than it increased ornithine metabolism. Both precursors were metabolised to each other, to other amino acids, and to polyamines. The only polyamine in which radioactivity incorporated was free putrescine, showing either a slow synthesis or a high degradation rate of spermidine and spermine in maturing spruce seeds. The putrescine level was approximately 10 times higher in the embryo than in the megagametophytic tissues, whereas spermidine and spermine levels were almost the same in both tissues. The label from arginine and ornithine was also incorporated into proteins as amino acids and post‐translationally as polyamines. Higher radioactivity was seen in the small ≤14‐kDa polypeptides. Protein hydrolysates of the embryo and the megagametophytic tissues contained spermidine and spermine and their degradation product 1,3‐diaminopropane (DAP), suggesting that polyamines may play a role in the accumulation of seed storage protein and in the maturation of spruce seeds.
Glutathione transferases involved in herbicide detoxification in the leaves of Setaria faberi (giant foxtail)
Authors: Hatton Pamela J. , Cummins Ian , Cole David J. , Edwards Robert
Published: March 2003

Setaria faberi (giant foxtail) is a major grass weed of maize in North America and can prove difficult to control using selective herbicides. In grasses, tolerance to the chloro‐s‐triazine and chloroacetanilide classes of selective herbicides is associated with their rapid detoxification by glutathione conjugation catalysed by glutathione transferases (GSTs, EC 2.5.1.18). We were therefore interested in characterising the GSTs in S. faberi and comparing them with the corresponding enzymes in maize. Four S. faberi GST isoenzymes (SfaGSTs 1 to 4) with activities toward the triazine herbicide atrazine, the chloroacetanilide herbicides metolachlor and alachlor and the diphenyl ether herbicide fluorodifen were purified from the foliage of young plants. These GSTs detoxified herbicides with similar efficiencies to those determined for GST isoenzymes from maize, but their levels of expression in the leaves were 20‐fold lower than those reported in the crop. All SfaGSTs were composed of two subunits and the 28 kDa subunit of the isoenzyme SfaGST1 reacted strongly to an antiserum raised to the maize theta‐type GSTZm GSTI‐II. SfaGST1 also appeared to be very similar in substrate specificity to the major maize GST ZmGSTI‐I. The similarity of SfaGST1 and ZmGSTI subunits was confirmed by RT‐PCR using primers specific to ZmGSTI, with a 370 bp DNA amplification product from S. faberi showing 88% identity at the nucleotide level to the corresponding sequence of ZmGSTI. However, SfaGSTs also differed significantly from ZmGSTs. Unlike maize, only one isoenzyme, SfaGST2, detoxified chloroacetanilides. Also, SfaGST3 and SfaGST4 resembled tau‐type GSTs from maize in showing high activities toward fluorodifen, but these SfaGSTs were not recognised by an antiserum raised to the maize tau‐type GST ZmGSTV‐VI. SfaGST4 also differed from the ZmGSTs described to date in showing high activities toward atrazine. Our results demonstrate that while some GSTs are conserved in grass crops and weeds, others are quite different.
Effect of microgravity on the cell cycle in the lentil root
Authors: Yu F. , Driss‐Ecole D. , Rembur J. , Legué V. , Perbal G.
Published: March 2003

Characteristics of the cell cycle in cortical regions (0–0.6 mm from the root‐cap junction) of the primary root of lentil (Lens culinaris L.) during germination in the vertical position on earth were determined by iododeoxyuridine labelling and image analysis. All cells were in the G1 phase at the beginning of germination and the duration of the first cell cycle was about 25 h. At 29 h, around 14% of the cortical nuclei were still in the G2 or M phases of the first cell cycle, whereas 53 and 33% of the nuclei were respectively in the G1 or S phase of the second cell cycle. In parallel, the cell cycle was analysed in root tips of lentil seedlings grown in space during the IML 2 mission (1994), (1) on the 1‐g centrifuge for 29 h, (2) on the 1‐g centrifuge for 25 h and placed in microgravity for 4 h, (3) in microgravity for 29 h, (4) in microgravity for 25 h and placed on the 1‐g centrifuge for 4 h. The densitometric analysis of nuclear DNA content showed that in microgravity there were less cells in DNA synthesis and more cells in G1 than in the controls on the 1‐g centrifuge (flight and ground). The comparison of the sample grown continuously on the 1‐g centrifuge in space and of the sample grown first in 1‐g and then in microgravity indicated that 4 h of microgravity modified cell cycle, increasing the percentage of cells in the G1 phase. On the contrary, the transfer from microgravity to the 1‐g centrifuge (for 4 h) did not provoke any significant change in the distribution of the nuclear DNA content. Thus the effect of microgravity could not be reversed by a 4 h centrifugation. As the duration of the first cell cycle in the lentil root meristem is about 25 h, the results obtained are in agreement with the hypothesis that the first cell cycle and/or the second G1 phase was lengthened in absence of gravity. The difference observed in the distribution of the nuclear DNA content in the two controls could be due to the fact that the 1g control on board was subjected to a period of 15 min of microgravity for photography 25 h after the hydration of the seeds, which indicated an effect of short exposure to weightlessness. The mitotic index of cortical cells was greater on the 1‐g centrifuge in space than in any other sample (flight and ground) which could show an effect of the centrifugation on the mitosis.
Polymorphic glutamate dehydrogenase in lilac vitroplants as revealed by combined preparative IEF and native PAGE: Effect of ammonium deprivation, darkness and atmospheric CO2 enrichment upon isomerization
Authors: Refouvelet Eric , Daguin Florence
Published: March 2003

The activity and polymorphism of glutamate dehydrogenase (GDH) were studied in basal callus of lilac (Syringa vulgaris L.) vitroplants. Native PAGE alone revealed seven bands staggered at regular intervals. Preparative liquid‐vein IEF allowed the separation of six to ten GDH fractions with charges ranging between 5.18 and 7.08. Analysis of these GDH fractions in native PAGE indicated that up to seven GDH bands can be detected for each fraction. This suggests the existence of seven isoforms of the enzyme with subunits presenting different isoelectric points. Dark‐ and ammonium‐controlled forms were found to be the more acidic and faster migrating ones in native PAGE. The results support for the first time that atmospheric CO2 enrichment increases GDH activity dramatically and modifies isomerization of the enzyme.
Leaf phenolics of three willow clones differing in resistance to Melampsora rust infection
Authors: Hakulinen Johanna , Sorjonen Sanja , Julkunen‐Tiitto Riitta
Published: March 2003

Leaf phenolic composition in three Salix myrsinifolia Salisb. clones (V8, V45, and V43), inoculated with Melampsora rust, was analyzed to detect local rust‐induced alterations during different stages of infection (2, 7, and 21 days after inoculation [DAI]). Phenolic levels and percentage of uredial area varied significantly between clones. In the most resistant clone,V8, the levels of some phenolic compounds were lower in rust‐infected plants than in control plants at the initial stages of rust infection, suggesting a rapid response of phenolic metabolism to rust attack. Moreover, the clone V8 contained the highest constitutive (+)‐catechin level. In clone V45, rust infection caused the most pronounced increase in the levels of individual phenolics at 7 DAI. This increase may have been effective in retarding the subsequent spread and development of rust. In the most susceptible clone V43, rust‐induced phenolic responses were less pronounced and delayed. The results suggest that in specific willow‐rust interactions, constitutive levels of phenolics, as well as induced phenolic responses, may contribute to the expression of rust resistance. In general, rust‐induced alterations in willow phenolic levels are highly specific to genotype and compound and vary depending on the stage of rust development.
Differential expression of DAHP synthase and chorismate mutase in various organs of Brassica juncea and the effect of external factors on enzyme activity
Authors: Sharma Ranjana , Jain Mukesh , Bhatnagar Raj K. , Bhalla‐Sarin Neera
Published: March 2003

The multibranched shikimic acid pathway was discovered as the biosynthetic route to the aromatic amino acids phenylalanine, tyrosine and tryptophan and a host of other secondary metabolites. An extensive body of work is available on the characterization of various enzymes of this pathway in order to understand the underlying mechanisms of aromatic amino acid biosynthesis and secondary metabolism in higher plants. In the present investigation, selective assays, based on feedback regulation patterns and divalent cation requirements, were used to monitor the isozyme profiles of two of the key regulatory enzymes of this pathway. 3‐Deoxy‐ d‐arabino heptulosonate‐7‐phosphate synthase (DAHP synthase/DS) (EC 4.1.2.15) and chorismate mutase (CM) (EC 5.4.99.4) have been characterized from different vegetative and reproductive organs of Brassica juncea cv. Pusa Bold. An attempt has also been made to investigate the effect of external factors, such as light and wounding on the regulation of these enzymes. The results reveal differential expression of DAHP synthase and CM in various organs of Brassica and an adaptability of plants to various stresses by up or down regulation of these enzymes.
Purification and properties of a second fructan exohydrolase from the roots of Cichorium intybus
Authors: De Roover Joke , Van Laere André , De Winter Marie , Timmermans Johan W. , Van den Ende Wim
Published: March 2003

A 1‐FEH II (1‐fructan exohydrolase, EC 3.2.1.80) was purified from forced chicory roots (Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A (Con A) affinity chromatography and anion and cation exchange chromatography. This protocol produced a 70‐fold purification and a specific activity of 52 nkat mg−1 protein. The apparent size of the enzyme was 60 kDa as estimated by gel filtration and 64 kDa on SDS‐PAGE. Optimal activity was found between pH 5.0 and 5.5. The temperature optimum was around 35°C. No product other than fructose could be detected with inulin as the substrate. The purified enzyme exhibited hyperbolic saturation kinetics with an apparent Km of 58 mM for 1‐kestose (Kes) and 64 mM for 1,1‐nystose (Nys). The purified 1‐FEH II hydrolyzed the β(2?1) linkages in inulin, Kes and Nys at rates at least 5 times faster than the β(2?6) linkages in levan oligosaccharides and levanbiose. Fructose did not affect the 1‐FEH II activity but sucrose (Suc) was a strong inhibitor of this 1‐FEH II (Ki=5.9 mM). The enzyme was partially inhibited by Na‐EDTA and CaCl2 (1 mM).
PP7, a gene encoding a novel protein Ser/Thr phosphatase, is expressed primarily in a subset of guard cells in Arabidopsis thaliana
Authors: Andreeva Alexandra V. , Kearns Anne , Hawes Chris R. , Evans David E. , Kutuzov Mikhail A.
Published: March 2003

Recently, we have identified a cDNA encoding a novel protein Ser/Thr phosphatase (EC 3.1.3.16) from Arabidopsis thaliana (L.) Heynh. (ecotype Columbia), designated as PP7, which is only distantly related to the known phosphatases of the PPP family. We examined PP7 expression in A. thaliana using Northern blot analysis, reverse transcriptase‐polymerase chain reaction (RT‐PCR) and whole‐mount in situ hybridisation. Although the PP7 transcript could be detected in all organs analysed by the first two methods, whole‐mount in situ hybridisation revealed specific expression in a subset of stomata in A. thaliana seedlings. Possible implications of this expression pattern are discussed.
Kinetics of ethylene biosynthesis and its effects during maturation of white spruce somatic embryos
Authors: El Meskaoui Abdelmalek , Desjardins Yves , Tremblay Francine M.
Published: March 2003

The objective of the current investigation was to study the role of ethylene in the maturation of white spruce (Picea glauca [Moench.] Voss) somatic embryos. This was carried out by examining the effects of (1) 1‐aminocyclopropane‐1‐carboxylic acid (ACC), a direct precursor of ethylene in plant tissue, (2) silver nitrate (AgNO3), an inhibitor of ethylene action, (3) α‐aminooxyamino acid (AOA), a potent inhibitor of ethylene biosynthesis, and (4) enrichment with ethylene. Ethylene biosynthesis was biphasic and gradually increased during embryo development, whereas endogenous ACC and N‐malonylaminocyclopropane‐1‐carboxylic acid (mACC) decreased. Addition of ACC or AOA to the culture medium increased or decreased, respectively, ethylene biosynthesis by altering endogenous ACC levels during the culture period. In contrast to AOA and AgNO3, ACC and ethylene enrichment significantly decreased the production of mature somatic embryos and increased the browning of the cultures. However, the structure of the shoot apex in mature cotyledonary stage embryos formed under ethylene enrichment was similar to that in control systems. This shows that a reduction in ethylene is beneficial to maturation of white spruce somatic embryos. This is further substantiated by the finding that the inhibitory effects of AOA were partially reversed by the addition of ethylene. The possible effects of the interaction between ethylene and polyamines on somatic embryo development are also discussed.
Stomatal development and patterning in Arabidopsis leaves
Authors: Serna Laura , Fenoll Carmen
Published: March 2003

The functional unit for gas exchange between plants and the atmosphere is the stomatal complex, an epidermal structure composed of two guard cells, which delimit a stomatal pore, and their subsidiary cells. In the present work, we define the basic structural unit formed in Arabidopsis thaliana during leaf development, the anisocytic stomatal complex. We perform a cell lineage analysis by transposon excision founding that at least a small percentage of stomatal complexes are unequivocally non‐clonal. We also describe the three‐dimensional pattern of stomata in the Arabidopsis leaf. In the epidermal plane, subsidiary cells of most stomatal complexes contact the subsidiary cells of immediately adjacent complexes. This minimal distance between stomatal complexes allows each stoma to be circled by a full complement of subsidiary cells, with which guard cells can exchange water and ions in order to open or to close the pore. In the radial plane, stomata (and their precursors, the meristemoids) are located at the junctions of several mesophyll cells. This meristemoid patterning may be a consequence of signals that operate along the radial axis of the leaf, which establish meristemoid differentiation precisely at these places. Since stomatal development is basipetal, these radially propagated signals may be transmitted in the axial direction, thus guiding stomatal development through the basal end of the leaf.
Polyamine content and chilling susceptibility are affected by seasonal changes in temperature and by conditioning temperature in cold‐stored ‘Fortune’ mandarin fruit
Authors: Gonzalez‐Aguilar G. A. , Zacarias L. , Perez‐Amador M. A. , Carbonell J. , Lafuente M. T.
Published: March 2003

‘Fortune’ mandarins are prone to develop pitting and necrosis upon exposure to low temperatures. We have examined the effect of field temperature during fruit maturation and the effect of conditioning temperatures (from 2 to 37°C) prior to cold storage on the content of polyamines (PAs) and on chilling susceptibility in order to understand the role of PAs in maturation and chilling tolerance of this citrus cultivar. Chilling susceptibility and the content of PAs were more affected by seasonal changes in field temperature than by the stage of fruit maturity. The highest putrescine (Put) and spermidine (Spd) content was found in fruits exposed to the lowest field temperatures. These fruits were in turn more susceptible to develop chilling injury (CI) after storage at 2°C. Spermine (Spm), however, decreased in attached fruit with time of exposure to temperatures below 12°C. Temperature pretreatments for 3 days above 20°C of fruits detached from the tree reduced CI, the more so the higher the conditioning temperature. Put and Spd increased with temperature conditioning in detached fruits, differing from the response of fruits attached to the tree. No direct relationship between induced levels of these PAs and the tolerance to CI was found. Levels of Put and Spd increased at temperatures (22, 30 and 37°C) which increased the tolerance and also at temperatures (6 and 12°C) which accelerated the appearance of chilling symptoms. In contrast, a significant increase in Spm levels was only found after conditioning at 30 or 37°C. After cold storage a general decline in PA levels occurred in all temperature‐conditioned mandarins. In most cases no significant differences among fruit exposed to effective and non‐effective pretreatments were observed. PA content increased again after transferring cold‐stored fruits to 20°C, whereas the CI index was barely affected. In conclusion, PA changes in the flavedo of ‘Fortune’ mandarins appear to be related to variations in temperature rather than to stage of maturity or tolerance to chilling.
Expression of Δ1‐pyrroline‐5‐carboxylate dehydrogenase and proline/arginine homeostasis in Solanum tuberosum
Authors: Forlani Giuseppe , Mangiagalli Anna , Pinter Claudia , Nielsen Erik
Published: March 2003

Polyclonal antibodies raised in mouse against purified potato Δ1‐pyrroline‐5‐carboxylate dehydrogenase (P5C‐DH, EC 1.5.1.12), which catalyses the last step in the catabolism of both proline and arginine, were used to investigate the expression of this enzyme. Distribution of P5C‐DH in potato (Solanum tuberosum L. cv. Desiree) organs was studied at different stages during plant development. Variations in enzyme level were determined in axenically grown plantlets following the addition of exogenous proline, and in cell suspension cultures under hyperosmotic stress and after its relief. Free proline and arginine levels were also quantified, and compared to those of the enzyme. Results were consistent with a developmental, but not with an environmental, control of P5C‐DH expression. The possible involvement of specific isozymes in proline and arginine oxidation is discussed.
Effects of elevated CO2, drought and temperature on the water relations and gas exchange of groundnut(Arachis hypogaea)stands grown in controlled environment glasshouses
Authors: Clifford Sean C. , Stronach Ian M. , Black Colin R. , Singleton‐Jones Paul R. , Azam‐Ali Sayed N. , Crout Neil M. J.
Published: March 2003

Stands of groundnut (Arachis hypogaeaL. cv. Kadiri‐3) were grown in controlled environment glasshouses at mean atmospheric CO2concentrations of 375 or 700 μmol mol−1and daily mean air temperatures of 28 or 32°C on irrigated or drying soil profiles. Leaf water (Ψl) and solute potential (Ψs), relative water content (RWC), stomatal conductance (gl) and net photosynthesis (Pn) were measured at midday for the youngest mature leaf throughout the growing season. Elevated CO2and temperature had no detectable effect on the water relations of irrigated plants, but higher values of RWC, Ψland Ψswere maintained for longer under elevated CO2during progressive drought. Turgor potential (Ψp) reached zero when Ψldeclined to −1.6 to −1.8 MPa in all treatments; turgor was lost sooner when droughted plants were grown under ambient CO2. A 4°C increase in mean air temperature had no effect on Ψsin droughted plants, but elicited a small increase in Ψl; midday glvalues were lower under elevated than under ambient CO2, and Ψland gldeclined below −1.5 MPa and 0.25 cm s−1, respectively, as the soil dried. Despite the low glvalues recorded for droughted plants late in the season, Pnwas maintained under elevated CO2, but declined to zero 3 weeks before final harvest under ambient CO2. Concurrent reductions in gland increases in water use efficiency under elevated CO2prolonged photosynthetic activity during drought and increased pod yields relative to plants grown under ambient CO2. The implications of future increases in atmospheric CO2for the productivity of indeterminate C3crops grown in rainfed subsistence agricultural systems in the semi‐arid tropics are discussed.
Genes with homologies to known α‐galactosidases are expressed during senescence of barley leaves
Authors: Chrost Bozena , Krupinska Karin
Published: March 2003

α‐Galactosidase activity increases during dark induced senescence as well as during natural senescence under field conditions. Two cDNA clones named pHvSF11 and pHvSF23 were isolated from a cDNA library of senescent barley flag leaves by screening with a coffee bean α‐galactosidase cDNA as probe. Sequences of the two barley cDNAs show high homology to those of known α‐galactosidases and hence seem to represent two different α‐galactosidase genes in barley.

Expression of the genes corresponding to HvSF11 and HvSF23 was analyzed on one hand in primary foliage leaves incubated in darkness to accelerate senescence and on the other hand in flag leaves from barley plants grown under field conditions. The transcript represented by the cDNA HvSF23 is detectable in fully expanded leaves and its level is slightly upregulated during dark induced senescence of primary foliage leaves as well as during natural senescence of flag leaves. In contrast, the level of the transcripts represented by the cDNA HvSF11 decreases in flag leaves during natural senescence and inversely increases in primary foliage leaves induced to senesce by darkness.

Antibodies raised against overexpressed peptide sequences deduced from the two different partial cDNA sequences react with the same proteins. In extracts from primary foliage leaves induced to senesce by prolonged darkness two proteins were immunodecorated by these antibodies. In senescent flag leaves from a barley field an additional third protein was detected.
Characterization of a basic chitinase which is secreted by cultured pumpkin cells
Authors: Arie Mami , Hikichi Kiyoshi , Takahashi Kiyoshi , Esaka Muneharu
Published: March 2003

A basic chitinase was secreted into culture medium of pumpkin cell suspension cultures. The chitinase was purified from the culture medium. A cDNA encoding the pumpkin chitinase was cloned by reverse transcription (RT)‐PCR and rapid amplification of cDNA ends (RACE) methods. The chitinase gene was strongly expressed in pumpkin callus cells, but little or not at all in mature leaf, young leaf, cotyledon, stem, hypocotyl and root of pumpkin. No chitinase mRNA was detected in intact pumpkin fruit tissues. However, chitinase was induced during callus formation from sliced pumpkin fruit tissues. Induction also occurred in the absence of 2,4‐D, a chemical causing callus formation, suggesting that it may be independent of the presence of 2,4‐D. Perhaps, induction is caused by osmotic or wounding stress. Levels of chitinase mRNA markedly increased at 4 h after transfer of pumpkin callus cells into fresh culture liquid medium. They were also high at later stages of cell suspension culture. In transgenic tobacco BY‐2 cells, into which the pumpkin chitinase cDNA was introduced, the recombinant pumpkin chitinase was expressed and secreted into the culture medium, suggesting that the signal peptide of pumpkin chitinase also functions for secretion from tobacco BY‐2 cells.