The apoplastic enzyme, ascorbate oxidase (AO), is a blue copper oxidase that catalyses oxidation of ascorbate (AsA) to monodehydroascorbate (MDHA). In Arabidopsis thaliana, AO is encoded by three genes (At4g39830, At5g21105 and At5g21100) designated AO1, AO2, and AO3 respectively. Since AsA is the most abundant antioxidant in the apoplast and AO is active in this compartment, the regulation of apoplastic AsA redox status by AO and its role in development and environmental perturbations has become a subject of interest. Phylogenetic analysis showed that AO is present in higher plants, pteridophytes, mosses and green algae. Amino acid sequence analysis showed that AO2 and AO3 shared higher sequence identity than AO1. In silico analyses found that AO1 had a distinct expression pattern and subcellular localisation compared to AO2 and AO3, suggesting AO1 might be involved in alternative functions. Consistent with previous studies, AO activity was high in actively growing tissue of wild-type (WT) A. thaliana, supporting a possible role of AO in cell expansion. ao1, ao3 and ao1ao3 T-DNA insertion mutants were characterised. ao1 had similar level of AO activity to WT, while ao3 and ao1ao3 had 10-20% of WT AO activity. Compared with WT, these T-DNA insertion mutants did not show any phenotypic differences under unstressed or stressed (high light and drought) growth conditions. An artificial microRNA construct (amiR-AO) to silence all three AO genes was developed. Also, an overexpression plasmid (35S::AO3) harbouring AO3 gene was constructed. These constructs were used to transform A. thaliana. AO activity was undetectable in the amiR-AO line, while the 35S::AO3 line had 3-fold higher AO activity than the WT. Under unstressed normal growth conditions, the amiR-AO line had bigger rosette size, whereas the 35S::AO3 line exhibited early flowering and smaller number of rosette leaves. The amiR-AO line accumulated more anthocyanin and AsA than WT when acclimated to high light, whereas the 35S::AO3 line accumulated less anthocyanin than WT. In response to drought, the amiR-AO line did not show phenotypic differences compared to WT, while the 35::AO3 line had higher rate of leaf water loss and appeared to have greater sensitivity to drought. These results suggest that AO perturbation could, to some extent, affect the growth and stress response of A. thaliana although the effect is small.

No description available.

Cold hardy Japanese bunching onion (Allium fistulosum L.) was used as a novel model system to examine the role of the apoplast in intracellular freezing avoidance. Since intracellular freezing avoidance is critical to both sensitive and resistant plants, non-acclimated and cold acclimated onion tissue was compared. The large 250 (length) x 50 (width) x 90 µm (thickness) intact single cell epidermal layer system allowed direct observation of the freezing process during freezing and thawing on a single cell basis in live intact tissues in non-acclimated (23/18°C) and acclimated (2 weeks at 12/4°C) plants. Under acclimation treatment, freezing resistance increased from an LT50 of -12°C at zero time to -27°C after two weeks exposure. Ice nucleation was always initiated in the apoplastic space in both non-acclimated and acclimated cells and then penetrated into the intracellular space. We provide direct evidence that lethal intracellular freezing damage was avoided in cold acclimated and CaCl2 treated cells through visible blockage of ice propagation from the apoplastic space to the intracellular region. Subsequent investigation of the apoplastic space revealed cold acclimation increased: a) the insoluble crude cell wall and pectin (galacturonic acid) content, b) total cell wall protein quantity, c) pectin methylesterase activity in the apoplastic space particularly in epidermal cells compared to non-epidermal cells, and d) the levels of un-methylated pectin. The increasing freezing resistance of Japanese bunching onion more depends on the efficiency of PME not only the increase in the amount of apoplast compositions. Collectively, these changes likely led to the observed significant reduction in cell wall permeability. In this way, freezing stress resistance may potentially be increased through avoidance of intracellular freezing by altering key factors related to apoplast permeability and blocking ice propagation across the cell wall.

Universiẗat, Diss., 2002--Würzburg.

No description available.

In der vorliegenden Arbeit wurden die für den radialen Stofftransport durch die Wurzel Höherer Pflanzen wichtigen apoplastischen Barrieren der Wurzeln von sieben Pflanzenarten (Vicia faba L.; Typha glauca Godr.; Ricinus communis L.; Quercus petraea (Matt.) Liebl.; Fagus sylvatica L.; Picea abies (L.) Karst.; Zea mays L.) mikroskopisch charakterisiert und chemisch analysiert. Nach enyzmatischer Isolation der Gewebe wurde die Biopolymerzusammensetzung von Suberin und Lignin der isolierten Zellwände nach Depolymerisierung durch Umesterungsreaktion (Abbau von Suberin) oder Thioacidolyse (Abbau von Lignin) mittels Gaschromatographie und Massenspektroskopie aufgeklärt. Außerdem wurde Sprossknollenperiderm der Kartoffel (Solanum tuberosum L.) verschiedener post harvest Luftfeuchtebedingungen, sowie neugeformtes Wundperiderm chemisch-analytisch und auf die Permeabilität für Wasser hin untersucht. Zusätzlich zu den mikroskopischen und chemischen Analysen wurden die hydraulischen Leitfähigkeiten von Maiswurzeln verschiedener Kulturbedingungen und die Aufnahme von Rubidium-Ionen über die Maiswurzeln untersucht. Dabei wurde die Auswirkung von Salzstress (100mM NaCl), und eine Applikation des Phytohormons Abscisinsäure (10µM ABA) bei der Aufzucht der Pflanzen auf apoplastische Barrieren untersucht. Auch die Rubidiumaufnahme von bei Nitratmangel (0.00 M NO3-) aufgewachsenen Rizinuspflanzen wurde ermittelt und mit der chemischen Zusammensetzung der apoplastischen Barrieren korreliert. Die Ergebnisse dieser Arbeit zeigen, dass: -monocotyle Pflanzen wesentlich höhere Aromatenanteile im Suberin apoplastischer Barrieren besitzen als dicotyle Pflanzen; -bei der Bewertung des Suberingehaltes apoplastischer Barrieren histochemische Methoden unzureichend sind; -die Flächenbelegung mit Suberin auch innerhalb gleicher Entwicklungsstadien bei verschiedenen Pflanzen stark unterschiedlich sein kann; -der Verknüpfungsgrad der Monomeren im Suberin stark unterschiedlich sein kann; -Suberin keine 100%ige Barriere für Wasser und Ionen darstellt; -Suberin auch eine Barriere gegen unkontrollierte Gasdiffusion darstellen kann; -der Stofftransport (z.B. Rb-Ionen) durch zusätzliche Suberinmengen verlangsamt werden kann, geringere Suberinmengen den Stofffluss aber nicht signifikant erhöhen wie bei Nitratmangelpflanzen gezeigt wurde; -eine direkte Ableitung der Funktion für den Wasser und Stofftransport aus dem Suberingehalt nicht ohne eine Extraktanalyse der Gewebe möglich ist, und in jedem Fall die Notwendigkeit besteht eine Flächenbelegung mit Suberin oder Wachsen zu ermitteln; -die Variabilität von Pflanzen verschiedenen Genotyps und die Entwicklung vieler verschiedener Anpassungsstrategien zum Schutz vor Stress eine Abschätzung funktioneller Aspekte aus monokausaler Sichtweise (z.B.: Suberingehalt) unmöglich macht. Um der Vielfältigkeit pflanzlicher Strategien gerecht zu werden, ist daher die Integration vieler unterschiedlicher Untersuchungsmethoden in interdisziplinärer Arbeitsweise notwendig. / The aim of this thesis was to examine the chemical composition of suberised apoplastic barriers in roots of higher plants and to relate the obtained results to the function of these barrier tissues for the diffusive transport of ions and water. For the analysis of the chemical composition (mainly suberin and also lignin), the roots were digested enzymatically, and the remaining material was separated in two fractions: one fraction consisting of rhizodermal and hypodermal cell walls (RHCW) and the second one of endodermal cell walls (ECW). Xylem vessels were not analised. Suberin content of the isolated cell walls was determined after transesterification and GC/FID and GC/MS analysis of the monomers. Prior to the chemical analysis, the anatomical structures of the roots were thoroughly examined with light microscopy using histochemical dyes and UV-light as well as scanning electron microscopy. Analysis were performed on the roots of several species: Vicia faba L., Typha glauca Godr., Quercus petraea (Matt.) Liebl., Fagus sylvatica L., Picea abies (L.) Karst., Rhicinus communis L. and Zea mays L. To reveal functional aspects for the transport of water through suberised barriers, potato tuber (Solanum tuberosum L.) skin was used as a model system because it is easy to obtain and consists to more than 20% of suberin. Additionally it is impregnated with aliphatic wax like materials. Experiments with freshly harvested potatoes showed, that suberin and wax content increased in the first days after cutting the tubers from the stele, but no additional cell layers were build. As the content of aliphatic components increased in the peridermal cell walls, the water permeability decreased. Removal of the waxes increased the water permeability of potato periderm to more than a 100 fold from 5.4∙10-11 m s-1 to 8.0∙10-9 m s-1 for the extracted ones. Surprisingly, there was no significant difference in the water permeability of periderms with different suberin content. 28 day old periderms had approx. twice as much as the suberin from freshly harvested ones, but the difference in permeability was smaller than 0.4 units. These results are showing, that suberin is not a water tight barrier as it is discussed in common text books, but to a certain extend it controls the water flux. Suberin amounts and permeabilities where used to estimate hypothetical transport data for root tissues, but the calculated values were only roughly in the range of measured root hydraulic permeabilities obtained from pressure probe experiments. Pressure probe experiments with roots of 8 day old maize seedlings showed, that a higher suberin content in the exodermis significantly reduces the radial water flow through the roots. Even the ion flux in maize roots is reduced, when barrier tissues contained more suberin. These results were obtained in experiments with rubidium-ions as a tracer for potassium-ions in maize seedlings. Apoplastic root barrier tissues, especially the endodermis were strongly enhanced, when the plants were grown under 100 mM NaCl or with 10 µM of the stress-hormone abscisic acid in the hydroponic growth-solution. On the other hand, it was shown that plants of the same species but with different genotypes responded each in a different way to stress situations. For example, whilest maize genotype Pioneer 3906 was able to cope with high salt concentrations, without altering apoplastic barriers, the genotype Across 8023 showed strong enhancement with high suberin amounts of the endodermal cell walls at the same salt concentrations. Lack of nutrients (e.g.: nitrate) in castor bean hydroponic culture leads to low suberin content in the endodermis but the uptake ability for rubidium-ions was not affected at all. Analysis of broad bean root nodules showed the relevance of high suberin amounts as a barrier for water and solutes as well as for gases. Suberin content in nodules were much higher than in the root apoplastic barriers, for that it would concluded, that the uncontrolled diffusion of oxygen into the infected zone of the nodule was inhibited by large amounts of suberin in the nodule endodermis. Another hint to this idea was the analysis of Typha glauca roots and rhizomes, which had more suberin in the exodermis than in the endodermis (in maize it was vice versa), because the submerged tissues are full of air-filled intercellular spaces and the function of higher suberin amounts in the exodermis may be to prevent oxygen loss. The results of this thesis are showing that suberin is a variable material with several functions, not only as a transport barrier for water, ions and gases which controls to a certain extend the fluxes in and out of the apoplast, but also as a barrier against pathogens with high contents of aromatic materials.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A expressÃo heterÃloga de genes que codificam proteÃnas relacionadas à patogÃnse (PR-proteÃnas) representa uma alternativa promissora para o desenvolvimento de plantas resistentes ao ataque de fungos. Dentre as PR-proteÃnas com grande potencial biotecnolÃgico, estÃo as quitinases, hidrolases que catalisam a degradaÃÃo da quitina, que à um polÃmero constituinte da parede celular de muitas espÃcies de fungos fitopatogÃnicos. AlÃm da funÃÃo relacionada aos mecanismos de defesa, acredita-se que essas enzimas podem desempenhar outros papÃis, como a regulaÃÃo de processos de crescimento e desenvolvimento em plantas. O objetivo deste trabalho foi estudar o papel de uma quitinase de classe I de feijÃo-caupi (VuChiI) na defesa e no desenvolvimento vegetal e para isso duas abordagens foram conduzidas. A primeira consistiu no silenciamento do gene que codifica esta proteÃna em plantas de feijÃo-caupi. Nos experimentos de silenciamento gÃnico foram utilizados meristemas apicais de plÃntulas de feijÃo-caupi, que foram entÃo bombardeados com micropartÃculas portando um vetor de silenciamento (pKANNIBAL), onde foi clonada uma construÃÃo em grampo (intron harpin) do gene de VuChiI. As plantas regeneradas em meio de cultura de Murashige e Skoog (MS) contendo o herbicida imazapyr, como agente de seleÃÃo, foram analisadas por PCR, o que resultou na confirmaÃÃo da transformaÃÃo genÃtica de cinco plantas, a partir de 1.092 meristemas apicais bombardeados. O RNA foi extraÃdo das plantas transformadas e utilizado em anÃlises de Northern blot que possibilitou a detecÃÃo de siRNAs no RNA total extraÃdo das plantas putativamente transgÃnicas. Apenas uma das plantas transformadas produziu uma vagem contendo seis sementes, das quais apenas uma germinou. As plantas transformadas apresentaram ciclo de vida de mais de 365 dias enquanto que o esperado à de 65-70 dias. Com base nos resultados sugere-se que o silenciamento da quitinase de classe I de feijÃo-caupi pode ter promovido alteraÃÃo no desenvolvimento da planta. A segunda estratÃgia de transformaÃÃo genÃtica consistiu no co-cultivo de discos foliares de tabaco com suspensÃo de Agrobacterium tumefaciens LB4404, com o intuito de expressar a proteÃna recombinante no espaÃo extracelular das plantas regeneradas. Essa estratÃgia tem como objetivo fazer com que a proteÃna entre em contato com o fungo antes mesmo que este agente patogÃnico penetre na cÃlula vegetal. Para tanto, o inserto referente à quitinase de feijÃo-caupi foi clonado no vetor binÃrio pCAMBIA1305.2 sob controle do promotor CaMV35S e fundido ao peptÃdeo sinal GRP (glycine-rich protein de Oryza sativa), que permite a secreÃÃo da proteÃna recombinante via apoplasto. Foram produzidas trÃs linhagens de plantas que tiveram a transformaÃÃo genÃtica confirmada por PCR. As sementes obtidas das plantas transformadas, geraÃÃo R1, foram semeadas em meio seletivo para a anÃlise da transmissÃo do transgene. O teste de X2 indicou que o transgene foi transmitido para a progÃnie em proporÃÃes Mendelianas. Os modelos experimentais desenvolvidos neste trabalho poderÃo contribuir para a elucidaÃÃo dos papÃis biolÃgicos da quitinase de classe I de feijÃo-caupi. / Heterologous expression of genes encoding pathogenesis related proteins (PR-proteins) represents a promising alternative for the development of plants resistant to fungi. Among the PR-proteins with great biotechnological potentials are the chitinases, a class of hydrolases which catalyze the degradation of chitin, a polymer constituent of cell wall of several species of pathogenic fungi. Besides their function related to defense mechanisms, it is believed that these enzymes may play other roles, such as regulation of growth and development processes in plants. Using two approaches, this study sought to investigate the role of a class I chitinase of cowpea (VuChiI) in defense and plant development. The first approach consisted of silencing of the gene encoding for VuChiI in cowpea. Apical meristems excised from cowpea seeds were bombarded with microparticles containing a silencing vector (pKANNIBAL), which was cloned with a construct harboring an intron harpin of the gene VuChiI. Plants regenerated in a Murashige and Skoog (MS) media containing imazapyr as a selection agent, were analyzed by PCR, resulting in confirmation of genetic transformation of five plants from the 1092 apical meristems bombarded. Northern blot analysis of total RNA extracted from the putative transgenic plants allowed for the detection of siRNAs. Upon regeneration and germination, only one of the five transformed plants produced a pod containing six seeds. Of these, only one germinated when further planted. The transformed plants exhibited a life cycle of over 365 days, as against the normal cycle of 65-70 days. Based on these results we posit that silencing of class I cowpea chitinase may have led to changes in plant development. The second strategy consisted of co-cultivation of tobacco leaf discs with Agrobacterium tumefaciens strain LB4404 suspension to express recombinant protein in the extracellular space of the regenerated plants in order to expose the protein fungus even before its penetration into the plant cell. To achieve this, a sequence of cowpea chitinase insert was cloned into the binary vector pCAMBIA1305.2 under the control of CaMV35S promoter and signal peptide fused to glycine-rich protein of Oryza sativa (GRP), which allowed the secretion of recombinant protein via apoplast. This led to the generation of three strains as confirmed by PCR. Seeds obtained from transformed R1 generation of the plants were germinated and analyzed for transmission of the transgene using X2 test, which confirmed its transmission to the progeny in a Mendelian fashion. The experimental models developed in this work may serve to further assess the biological roles of class I chitinase from cowpea.

Micorrizas arbusculares (MAs) são associações simbióticas entre os fungos do filo Glomeromycota e a maioria das plantas. Os mecanismos moleculares que controlam o processo de colonização e desenvolvimento das MAs são ainda pouco conhecidos, mas proteínas com acúmulo diferencial em MAs podem ter papel regulatório importante. O presente trabalho teve como objetivo detectar, por meio de eletroforese bi-dimensional (2D-PAGE) e espectrometria de massas, proteínas com acúmulo diferencial no fluido intercelular (FI), membrana plasmática ou tecido radicular de cana-de-açúcar colonizada por Glomus clarum. Plântulas de cana-de-açúcar micropropagadas foram inoculadas com G. clarum e cultivadas com 20 ou 200 mg de P kg-1 de substrato. Raízes micorrizadas e não-micorrizadas, 8 semanas após a inoculação, foram utilizadas para a extração de proteínas do FI, membrana plasmática e tecido radicular (solúveis totais). As proteínas foram separadas por 2D-PAGE e analisadas por espectrometria de massas. Os perfis de proteínas solúveis totais e de membrana plasmática não revelaram proteínas relacionadas à simbiose. No entanto, três proteínas do FI, uma hidrolase aspártica putativa, uma histidina quinase putativa e uma peroxidase putativa apresentaram acúmulo induzido em raízes micorrizadas. As atividades de peroxidases nas raízes e apoplasto das raízes foram determinadas. A atividade de peroxidase apoplástica foi maior em raízes colonizadas e cultivadas em baixo teor de P, quando comparado com controles não-inoculados. Com base na seqüência parcial de aminoácidos dessa peroxidase, um fragmento de seu gene (POX1) foi amplificado e clonado a partir de cDNA de raízes de cana-de-açúcar. A sequência obtida mostrou 90% e 91% de identidade com peroxidase de milho (NCBI) e cana-de-açúcar (TIGR), respectivamente. A análise de expressão de POX1 foi feita por PCR quantitativo a partir de transcritos extraídos de raízes micorrizadas e não-micorrizadas, em condições de baixo e alto P. O acúmulo de transcritos de POX1 em raízes micorrizadas em condições de baixo P foi 6,8 vezes maior do que em raízes micorrizadas cultivadas em condições de alto P. Raízes micorrizadas e cultivadas em condições de alto P apresentaram acúmulo de transcritos 3,9 vezes menor do que em raízes nãomicorrizadas cultivadas nas mesmas condições de P. Os dados obtidos sugerem que o controle do metabolismo de espécies ativas de oxigênio é um dos fatores que contribuem para a regulação do desenvolvimento de MAs. Estudos com plantas alteradas para a expressão de POX1 são, no entanto, necessários para elucidar a essencialidade dessa peroxidase nas MAs. / Arbuscular mycorrhizae (AM) are symbiotic associations between fungi of the phylum Glomeromycota and most of the plant species. Even though the molecular mechanisms controlling the colonization process and AM development are largely unknown, proteins with differential accumulation in AM may have important regulatory roles. The aim of this work was to detect, by bi-dimensional electrophoresis (2D-PAGE) and mass spectrometry, proteins with differential accumulation in the intercellular fluid (IF), plasma membrane or radicular tissue of sugarcane colonized by Glomus clarum Micropropagated sugarcane plantlets were inoculated with G. clarum and growth under low or high P conditions, 20 or 200 mg P kg-1 substrate, respectively. Mycorrhizal and non-mycorrhizal roots, eight weeks after inoculation, were used to extract proteins from the IF, plasma membrane and root tissue (total soluble proteins). Protein separation and analyses were performed using 2D-PAGE and mass spectrometry. The total soluble and plasma membrane protein profiles did not reveled symbiosis-related proteins. However, three proteins from the IF, a putative aspartic hydrolase, a putative histidine kinase and a putative peroxidase showed induced accumulation in mycorrhizal roots. Peroxidase activities in roots and apoplastic fluid were determined, and shown to be higher in mycorrhizal roots at low P than in non-mycorrhizal control roots. Based on the partial amino acid sequence of this peroxidase, a partial cDNA sequence of its gene (POX1) was cloned from PCR-amplified cDNA from sugarcane roots. The POX1 sequence showed 90% and 91% identity to maize (NCBI) and sugarcane (TIGR) peroxidase, respectively. Expression analyses of POX1 were perfomed using quantitative PCR of reverse transcripts from mycorrhizal and non-mycorrhizal roots at low and high P conditions. The steady state level of POX1 transcripts in mycorrhizal roots at low P condition was 6.8-fold higher than in mycorrhizal roots at high P conditions. In mycorrhizal roots at high P conditions the steady state level of POX1 transcripts was 3.9-fold lower than in nonmycorrhizal control roots. These data suggest that the metabolism of reactive oxygen species may be an important factor controlling the development of AM. Studies with plants altered in POX1 expression are, however, required to elucidate the essentiality of this peroxidase in AM.

Micorrizas arbusculares (MAs) são associações simbióticas entre os fungos do filo Glomeromycota e a maioria das plantas. Os mecanismos moleculares que controlam o processo de colonização e desenvolvimento das MAs são ainda pouco conhecidos, mas proteínas com acúmulo diferencial em MAs podem ter papel regulatório importante. O presente trabalho teve como objetivo detectar, por meio de eletroforese bi-dimensional (2D-PAGE) e espectrometria de massas, proteínas com acúmulo diferencial no fluido intercelular (FI), membrana plasmática ou tecido radicular de cana-de-açúcar colonizada por Glomus clarum. Plântulas de cana-de-açúcar micropropagadas foram inoculadas com G. clarum e cultivadas com 20 ou 200 mg de P kg-1 de substrato. Raízes micorrizadas e não-micorrizadas, 8 semanas após a inoculação, foram utilizadas para a extração de proteínas do FI, membrana plasmática e tecido radicular (solúveis totais). As proteínas foram separadas por 2D-PAGE e analisadas por espectrometria de massas. Os perfis de proteínas solúveis totais e de membrana plasmática não revelaram proteínas relacionadas à simbiose. No entanto, três proteínas do FI, uma hidrolase aspártica putativa, uma histidina quinase putativa e uma peroxidase putativa apresentaram acúmulo induzido em raízes micorrizadas. As atividades de peroxidases nas raízes e apoplasto das raízes foram determinadas. A atividade de peroxidase apoplástica foi maior em raízes colonizadas e cultivadas em baixo teor de P, quando comparado com controles não-inoculados. Com base na seqüência parcial de aminoácidos dessa peroxidase, um fragmento de seu gene (POX1) foi amplificado e clonado a partir de cDNA de raízes de cana-de-açúcar. A sequência obtida mostrou 90% e 91% de identidade com peroxidase de milho (NCBI) e cana-de-açúcar (TIGR), respectivamente. A análise de expressão de POX1 foi feita por PCR quantitativo a partir de transcritos extraídos de raízes micorrizadas e não-micorrizadas, em condições de baixo e alto P. O acúmulo de transcritos de POX1 em raízes micorrizadas em condições de baixo P foi 6,8 vezes maior do que em raízes micorrizadas cultivadas em condições de alto P. Raízes micorrizadas e cultivadas em condições de alto P apresentaram acúmulo de transcritos 3,9 vezes menor do que em raízes nãomicorrizadas cultivadas nas mesmas condições de P. Os dados obtidos sugerem que o controle do metabolismo de espécies ativas de oxigênio é um dos fatores que contribuem para a regulação do desenvolvimento de MAs. Estudos com plantas alteradas para a expressão de POX1 são, no entanto, necessários para elucidar a essencialidade dessa peroxidase nas MAs. / Arbuscular mycorrhizae (AM) are symbiotic associations between fungi of the phylum Glomeromycota and most of the plant species. Even though the molecular mechanisms controlling the colonization process and AM development are largely unknown, proteins with differential accumulation in AM may have important regulatory roles. The aim of this work was to detect, by bi-dimensional electrophoresis (2D-PAGE) and mass spectrometry, proteins with differential accumulation in the intercellular fluid (IF), plasma membrane or radicular tissue of sugarcane colonized by Glomus clarum Micropropagated sugarcane plantlets were inoculated with G. clarum and growth under low or high P conditions, 20 or 200 mg P kg-1 substrate, respectively. Mycorrhizal and non-mycorrhizal roots, eight weeks after inoculation, were used to extract proteins from the IF, plasma membrane and root tissue (total soluble proteins). Protein separation and analyses were performed using 2D-PAGE and mass spectrometry. The total soluble and plasma membrane protein profiles did not reveled symbiosis-related proteins. However, three proteins from the IF, a putative aspartic hydrolase, a putative histidine kinase and a putative peroxidase showed induced accumulation in mycorrhizal roots. Peroxidase activities in roots and apoplastic fluid were determined, and shown to be higher in mycorrhizal roots at low P than in non-mycorrhizal control roots. Based on the partial amino acid sequence of this peroxidase, a partial cDNA sequence of its gene (POX1) was cloned from PCR-amplified cDNA from sugarcane roots. The POX1 sequence showed 90% and 91% identity to maize (NCBI) and sugarcane (TIGR) peroxidase, respectively. Expression analyses of POX1 were perfomed using quantitative PCR of reverse transcripts from mycorrhizal and non-mycorrhizal roots at low and high P conditions. The steady state level of POX1 transcripts in mycorrhizal roots at low P condition was 6.8-fold higher than in mycorrhizal roots at high P conditions. In mycorrhizal roots at high P conditions the steady state level of POX1 transcripts was 3.9-fold lower than in nonmycorrhizal control roots. These data suggest that the metabolism of reactive oxygen species may be an important factor controlling the development of AM. Studies with plants altered in POX1 expression are, however, required to elucidate the essentiality of this peroxidase in AM.

No description available.