Project description:The loss of rooting capability following the transition from the juvenile to the mature phase is a known phenomenon in woody plant development. Eucalyptus grandis was used here as a model system to study the differences in gene expression between juvenile and mature cuttings. RNA was prepared from the base of the two types of cuttings before root induction and hybridized to a DNA microarray of E. grandis. In juvenile cuttings, 363 transcripts were specifically upregulated, enriched in enzymes of oxidation/reduction processes. In contrast, in mature cuttings, 245 transcripts were specifically upregulated, enriched in transcription factors involved in the regulation of secondary metabolites. A gene encoding for nitrate reductase (NIA), an enzyme that is known to be involved in nitric oxide (NO) production, was among the genes that were upregulated in juvenile cuttings. Concomitantly, a transient burst of NO upon excision was higher in juvenile cuttings than in mature ones. Treatment with a NO donor improved rooting of both juvenile and mature cuttings. A single NIA gene was found in the newly released E. grandis genome sequence, the cDNA of which was isolated, overexpressed in Arabidopsis plants and shown to increase NO production. Therefore, higher levels of NIA in E. grandis juvenile cuttings might lead to their better ability to produce NO and form adventitious roots. The Arabidopsis transgenic plants did not exhibit significantly increased lateral or adventitious roots, suggesting that spatial and temporal rather than a constitutive increase in NO is favorable for root formation. E. grandis seedlings were grown from seeds to the age of 6-7 months. The seedlings were pruned either at 10-15 cm or at 150-200 cm above the ground. The newly developed shoots were used as cuttings. To induce rooting, auxin (6000 ppm of K-IBA), was applied by quick dipping (20 sec). Stem cuttings taken from low-pruned seedlings were found to be easy-to root, exhibiting 45% rooting after 14 days and 60% after 35 days. These cuttings were defined as juvenile (RNA from two biological replicates was extracted). In contrast, stem cuttings taken from high pruned seedlings, barely rooted (5%) after 35 days, and were defined as mature (RNA from two biological replicates was extracted). The two biological replicates was used to dye swap experimental design.
Project description:The loss of rooting capability following the transition from the juvenile to the mature phase is a known phenomenon in woody plant development. Eucalyptus grandis was used here as a model system to study the differences in gene expression between juvenile and mature cuttings. RNA was prepared from the base of the two types of cuttings before root induction and hybridized to a DNA microarray of E. grandis. In juvenile cuttings, 363 transcripts were specifically upregulated, enriched in enzymes of oxidation/reduction processes. In contrast, in mature cuttings, 245 transcripts were specifically upregulated, enriched in transcription factors involved in the regulation of secondary metabolites. A gene encoding for nitrate reductase (NIA), an enzyme that is known to be involved in nitric oxide (NO) production, was among the genes that were upregulated in juvenile cuttings. Concomitantly, a transient burst of NO upon excision was higher in juvenile cuttings than in mature ones. Treatment with a NO donor improved rooting of both juvenile and mature cuttings. A single NIA gene was found in the newly released E. grandis genome sequence, the cDNA of which was isolated, overexpressed in Arabidopsis plants and shown to increase NO production. Therefore, higher levels of NIA in E. grandis juvenile cuttings might lead to their better ability to produce NO and form adventitious roots. The Arabidopsis transgenic plants did not exhibit significantly increased lateral or adventitious roots, suggesting that spatial and temporal rather than a constitutive increase in NO is favorable for root formation.
Project description:Rooting capability is one of the economically traits lost during the juvenile to mature phase change in woody plants. A comprehensive microarray analysis was performed to compare the profiles of gene expression in juvenile and mature cuttings from E. grandis, either auxin treated or untreated on days, 0, 1, 3, 6, 9 and 12 post excision. At the end of the experiment, root primordia were observed only in auxin treated juvenile cuttings. Clustering the expression profiles revealed that the time after excision contributed to expression differences more than the age or auxin. Maximum differences contributed by the age and auxin occurred on day 6 which correlated with the kinetics of root primordia formation. These included genes related to the microtubules (MTs) system. Therefore, expression of 42 transcripts annotated as tubulin or MTs associated proteins (MAPs) was validated in the same RNA samples. The results suggest developmental and auxin regulation of MTs. To determine the relevance to adventitious root (AR) formation, subtle perturbations to MTs were performed with trifluralin during induction. While juvenile cuttings were not affected, improved rooting was obtained in mature cuttings. Taken together it suggests that specific MTs remodeling is required for AR formation in E. grandis.
Project description:Background: The change from juvenile to mature phase in woody plants is often accompanied by a gradual loss of rooting ability, as well as by reduced microRNA (miR) 156 and increased miR172 expression. Results: We characterized the population of miRNAs of Eucalyptus grandis and compared by Northern blot the gradual reduction in miR156 and increase in miR172 expression during development to the loss of rooting ability. Forty known and eight novel miRNAs were discovered and their predicted targets are listed. The expression pattern of nine miRNAs was determined during adventitious root formation in juvenile and mature cuttings. While the expression levels of miR156 and miR172 were inverse in juvenile and mature tissues, no mutual relationship was found between high miR156 expression and rooting ability, or high miR172 expression and loss of rooting ability. This is shown both in E. grandis and also in E. brachyphylla, in which explants that underwent rejuvenation in tissue culture conditions were also examined. Conclusions: It is suggested that in these Eucalyptus species, there is no correlation between the switch of miR156 with miR172 expression in the stems and the loss of rooting ability.
Project description:Background: The change from juvenile to mature phase in woody plants is often accompanied by a gradual loss of rooting ability, as well as by reduced microRNA (miR) 156 and increased miR172 expression. Results: We characterized the population of miRNAs of Eucalyptus grandis and compared by Northern blot the gradual reduction in miR156 and increase in miR172 expression during development to the loss of rooting ability. Forty known and eight novel miRNAs were discovered and their predicted targets are listed. The expression pattern of nine miRNAs was determined during adventitious root formation in juvenile and mature cuttings. While the expression levels of miR156 and miR172 were inverse in juvenile and mature tissues, no mutual relationship was found between high miR156 expression and rooting ability, or high miR172 expression and loss of rooting ability. This is shown both in E. grandis and also in E. brachyphylla, in which explants that underwent rejuvenation in tissue culture conditions were also examined. Conclusions: It is suggested that in these Eucalyptus species, there is no correlation between the switch of miR156 with miR172 expression in the stems and the loss of rooting ability. Examination of microRNA in seedlings of Eucalyptus grandis
Project description:In order to identify genes specifically induced during various developmental stages of Adventitious Root (AR) formation (described in Ahkami et al. 2009) in leafy cuttings of Petunia hybrida (line W115) and to describe the series of physiological processes during adventitious rooting, a microarray-based transcriptome analysis in the stem base of the cuttings was conducted. The microarray was described by Breuillin et al. (2010) and included a normalized cDNA library from different time points after taking the cuttings from mother plant. Because physiological processes and molecular changes specifically involved in AR formation were considered as of major interest, rather than those associated with wound responses, a filtration approach was chosen to eliminate primarily wound-responsive genes. This study is mainly focused on changes in transcript abundances of genes related to specific metabolic pathways or cellular events including primary metabolism, membrane transport, cell division or signalling during various phases of AR formation.
Project description:Rooting capability is one of the economically traits lost during the juvenile to mature phase change in woody plants. A comprehensive microarray analysis was performed to compare the profiles of gene expression in juvenile and mature cuttings from E. grandis, either auxin treated or untreated on days, 0, 1, 3, 6, 9 and 12 post excision. At the end of the experiment, root primordia were observed only in auxin treated juvenile cuttings. Clustering the expression profiles revealed that the time after excision contributed to expression differences more than the age or auxin. Maximum differences contributed by the age and auxin occurred on day 6 which correlated with the kinetics of root primordia formation. These included genes related to the microtubules (MTs) system. Therefore, expression of 42 transcripts annotated as tubulin or MTs associated proteins (MAPs) was validated in the same RNA samples. The results suggest developmental and auxin regulation of MTs. To determine the relevance to adventitious root (AR) formation, subtle perturbations to MTs were performed with trifluralin during induction. While juvenile cuttings were not affected, improved rooting was obtained in mature cuttings. Taken together it suggests that specific MTs remodeling is required for AR formation in E. grandis. mRNA samlpes from mature and juvenile Eucalyptus sections immediately after cutting served as contorls. The rest of the samples were mature and juvenile sections, treated of not treated with Auxin, 1, 3, 6, 9 and 12 days after the prunning. At each time point, a loop design was performed consisting of a juvenile untreated vs. mature untreated array, a matrue untreated vs. mature auxin treated array, a matrue auxin treated vs. juvenile auxin treated array, and a juvenile auxin treated vs. juvenile untreated array. additional arrays connected between these loops, and between them and the time 0 controls. Most conditions had 3 replicates, but mature-untreated-day1 had 4 replicates, and three conditions had 2 replicates each: Juvenile-auxin-treated-day1, juvenile-untreated-day6, and mature-auxin-treated-day9.
Project description:In order to identify genes specifically induced during various developmental stages of Adventitious Root (AR) formation (described in Ahkami et al. 2009) in leafy cuttings of Petunia hybrida (line W115) and to describe the series of physiological processes during adventitious rooting, a microarray-based transcriptome analysis in the stem base of the cuttings was conducted. The microarray was described by Breuillin et al. (2010) and included a normalized cDNA library from different time points after taking the cuttings from mother plant. Because physiological processes and molecular changes specifically involved in AR formation were considered as of major interest, rather than those associated with wound responses, a filtration approach was chosen to eliminate primarily wound-responsive genes. This study is mainly focused on changes in transcript abundances of genes related to specific metabolic pathways or cellular events including primary metabolism, membrane transport, cell division or signalling during various phases of AR formation. The overall design of the custom microarray used in this study is described in Breuillin et al. (2010). In brief, a database of 24,816 unigene sequences (including 4,700 ESTs from P. hybrida cutting base and all public available P. hybrida and P. axillaris sequences) was used for construction of a custom microarray.
Project description:Gene expression analysis of chrysanthemum infected with three different viruses including Cucumber mosaic virus, Tomato spotted wilt virus, and Potato virus X have been performed using the chrysanthemum 135K microarray. Mock and each virus infected chrysanthemum plants were subjected for microarray analysis.