Project description:Aim To identify genes specifically involved in the storage reserve mobilisation programme in Arabidopsis. Background During germination and early post-germinative growth in Arabidopsis seed storage reserves are broken down to provide energy and nutrients for the developing seedling. Genes encoding enzymes involved in the mobilisation of both storage lipid and protein are expressed strongly 1-2 days following imbibition and then fall to very low levels. The regulatory mechanisms controlling expression of these genes are poorly understood. Although germination and reserve mobilisation occur at the same time we have obtained evidence using Abscisic Acid (ABA) treated seeds that the events are regulated by two separate programmes. Arabidopsis seeds treated with 10mM ABA still express genes involved in the mobilisation of storage reserves and break down storage lipid even though germination is blocked. ABA treated seeds therefore provide a powerful system for the identification of genes involved specifically in the reserve mobilisation programme. Microarray analysis will allow us to gain a global undertanding of the processes involved in storage reserve mobilisation and should also result in the identification of regulatory genes involved in this process. Experimental Set-up All seeds are Col-4 imbibed on plates containing 1/2 MS media for 4 days in the dark. All plant material will be grown in controlled environment growth rooms under defined light and temperature regimes. There are two parts to the experimental design. 1. Time course to identify genes involved in both seed storage reserve and germination programmes Comparison of mRNAs from seeds immediately after imbibition (storage reserve and germination programme genes being induced) with 2 day (storage reserve genes maximally expressed) and 7 day old seedlings (storage reserve and germination programme genes off, photoautotrophic genes on) will allow the identification of genes exclusively presentor present at enhanced levels at the peak of reserve mobilisation (day 2). 2. ABA treatment experiment to identify genes involved in the storage reserve programme ABA treatment blocks germination but does not block reserve mobilisation. Microarray analysis will be performed on RNA isolated from 2 day old seed imbibed in the presence of 10mM ABA. Comparison of the results of this experiment with those of the time course experiment should allow us to distinguish between genes involved in the two major programmes that we have uncovered. Experiment Overall Design: 4 samples

Project description:Aim To identify genes specifically involved in the storage reserve mobilisation programme in Arabidopsis. Background During germination and early post-germinative growth in Arabidopsis seed storage reserves are broken down to provide energy and nutrients for the developing seedling. Genes encoding enzymes involved in the mobilisation of both storage lipid and protein are expressed strongly 1-2 days following imbibition and then fall to very low levels. The regulatory mechanisms controlling expression of these genes are poorly understood. Although germination and reserve mobilisation occur at the same time we have obtained evidence using Abscisic Acid (ABA) treated seeds that the events are regulated by two separate programmes. Arabidopsis seeds treated with 10mM ABA still express genes involved in the mobilisation of storage reserves and break down storage lipid even though germination is blocked. ABA treated seeds therefore provide a powerful system for the identification of genes involved specifically in the reserve mobilisation programme. Microarray analysis will allow us to gain a global undertanding of the processes involved in storage reserve mobilisation and should also result in the identification of regulatory genes involved in this process. Experimental Set-up All seeds are Col-4 imbibed on plates containing 1/2 MS media for 4 days in the dark. All plant material will be grown in controlled environment growth rooms under defined light and temperature regimes. There are two parts to the experimental design. 1. Time course to identify genes involved in both seed storage reserve and germination programmes Comparison of mRNAs from seeds immediately after imbibition (storage reserve and germination programme genes being induced) with 2 day (storage reserve genes maximally expressed) and 7 day old seedlings (storage reserve and germination programme genes off, photoautotrophic genes on) will allow the identification of genes exclusively presentor present at enhanced levels at the peak of reserve mobilisation (day 2). 2. ABA treatment experiment to identify genes involved in the storage reserve programme ABA treatment blocks germination but does not block reserve mobilisation. Microarray analysis will be performed on RNA isolated from 2 day old seed imbibed in the presence of 10mM ABA. Comparison of the results of this experiment with those of the time course experiment should allow us to distinguish between genes involved in the two major programmes that we have uncovered. Keywords: development_or_differentiation_design

Project description:Arabidopsis seed germination is coordinated with the strong induction of metabolic pathways required for the mobilisation and utilization of seed storage reserves. These are essential to support the seedling before the establishment of photoauxotrophic growth. The activity of genes encoding enzymes required for lipid mobilisation is regulated largely at the level of transcription, but our knowledge of how this regulation occurs is extremely limited. After germination the rate of lipid reserve mobilisation is determined by the carbohydrate status of the seedling and by the osmotic potential of the growth substrate. The plant response to both of these requires the action of the hormone abscisic acid (ABA). We have shown that this regulation is tissue specific (Penfield et al., 2004 Plant Cell 16, 2705-2718), and that although lipid breakdown in the embryo is inhibited by ABA, lipid breakdown in the endosperm tissues is not. Furthermore, in many species the action of the endosperm is central to the processes controlling seed germination, yet very little is known about gene expression in this tissue, or of the function of the endosperm in mature Arabidopsis seeds. Mature Arabidopsis seeds can be dissected into embryo and endosperm/seed coat fractions, with the latter fraction containing RNA only from the endosperm as the seed coat cells undergo programmed cell death during the latter stages of seed development. In this experiment we divide seeds into embryo and endosperm tissues and transcript profile both shortly after germination, or when germination and lipid reserve mobilisation are inhibited by ABA or by the gibberellin biosynthesis inhibitor paclobutrazol. In this way we can discover the endosperm transcriptome and uncover candidate regulators of lipid mobilisation by searching for genes showing differential expression between embryo and endosperm before and after ABA treatment. Experimenter name = Steven Penfield Experimenter phone = 01904 328759 Experimenter fax = 01904 328762 Experimenter department = Department of Biology Experimenter institute = Centre for Novel Agricultural Products Experimenter address = University of York Experimenter address = PO BOX 373 Experimenter address = York Experimenter zip/postal_code = YO10 5YW Experimenter country = UK Keywords: organism_part_comparison_design

Project description:Arabidopsis seed germination is coordinated with the strong induction of metabolic pathways required for the mobilisation and utilization of seed storage reserves. These are essential to support the seedling before the establishment of photoauxotrophic growth. The activity of genes encoding enzymes required for lipid mobilisation is regulated largely at the level of transcription, but our knowledge of how this regulation occurs is extremely limited. After germination the rate of lipid reserve mobilisation is determined by the carbohydrate status of the seedling and by the osmotic potential of the growth substrate. The plant response to both of these requires the action of the hormone abscisic acid (ABA). We have shown that this regulation is tissue specific (Penfield et al., 2004 Plant Cell 16, 2705-2718), and that although lipid breakdown in the embryo is inhibited by ABA, lipid breakdown in the endosperm tissues is not. Furthermore, in many species the action of the endosperm is central to the processes controlling seed germination, yet very little is known about gene expression in this tissue, or of the function of the endosperm in mature Arabidopsis seeds. Mature Arabidopsis seeds can be dissected into embryo and endosperm/seed coat fractions, with the latter fraction containing RNA only from the endosperm as the seed coat cells undergo programmed cell death during the latter stages of seed development. In this experiment we divide seeds into embryo and endosperm tissues and transcript profile both shortly after germination, or when germination and lipid reserve mobilisation are inhibited by ABA or by the gibberellin biosynthesis inhibitor paclobutrazol. In this way we can discover the endosperm transcriptome and uncover candidate regulators of lipid mobilisation by searching for genes showing differential expression between embryo and endosperm before and after ABA treatment. Experimenter name = Steven Penfield; Experimenter phone = 01904 328759; Experimenter fax = 01904 328762; Experimenter department = Department of Biology; Experimenter institute = Centre for Novel Agricultural Products; Experimenter address = University of York; Experimenter address = PO BOX 373; Experimenter address = York; Experimenter zip/postal_code = YO10 5YW; Experimenter country = UK Experiment Overall Design: 18 samples were used in this experiment

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Transcription profiling of Arabidopsis ABA treated seeds to identify genes involved in the induction of storage reserve mobilisation

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