Quadruple rrm experience - Transcriptomic study of a quadruple rrm mutant which belong to a new family of AGO hook proteins
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ABSTRACT: rs10-01_rrm - quadruple rrm experience - What is the role of the RRM protein family in plants? Plants were grown on soil in controlled environment under LD (16 h light/8 h dark) and the rosette leaves (8-leaf stage seedlings) were collected for RNA preparation. Keywords: normal vs transgenic comparison
Project description:rs10-01_rrm - quadruple rrm experience - What is the role of the RRM protein family in plants? Plants were grown on soil in controlled environment under LD (16 h light/8 h dark) and the rosette leaves (8-leaf stage seedlings) were collected for RNA preparation. Keywords: normal vs transgenic comparison 2 dye-swap - CATMA arrays
Project description:Cotton fiber is actually unicellular trichome, therefor its length is really hard to be modified but very meaningful to fiber quality and yield. We have reported the function of the second RRM domain of Oryza sativa FCA in rice cell size regulation. Data shows it is highly conserved across dicotyledonous and monocotyledonous plants. Here we provide evidence showing that the second RRM domain of Brassica napus FCA worked in Gossypium hirsutum, leading to the enlargement of multiple types of cells, such as pollen, cotyledon petiole and cotton fiber. In the transgenic cotton, the length of unicellular cotton fiber increased by about 10% and fiber yield per plant also showed a dramatic increase, ranging from 35% to 66%, over the control. Thus, this RRM domain may be an ancient and common cell size regulator and has great economic value on cotton industry. FCA encodes a strong promoter of the transition to flowering in Arabidopsis thaliana, which contains two RRM (RNA recognition motif) domain and a WW protein interaction domain (Macknight et al., 1997). We have previously found that cell size and yield of rice (Oryza sativa) can be increased by ectopic expression of the first RRM domain of OsFCA (Hong et al., 2007). The second RRM domain of OsFCA can also increase cell size (Attia et al., 2005), suggesting OsFCA-RRMs each play a role in homeostatic cell size regulation. We designate them as Oryza sativa cell size RRM 1 (Os-csRRM1) and Oryza sativa cell size RRM 2 (Os-csRRM2), respectively. Both of them exhibit a high degree of evolutionary conservation in plant. For Os-csRRM2, significant homology was observed in Triticum aestivum (90% identity), Hordeum vulgare (90% identity), Lolium perenne (82% identity), Zea mays (81% identity),Ricinus communis (76% identity), Vitis vinifera (68% identity), Arabidopsis thaliana (68% identity) and Brassica napus (64% identity) (Fig. 1). The high degree of conservation suggests that this RRM domain might have similar function in different plants. Indeed, we observed that overexpression of Bn-csRRM2 also increased the cell size of B. napus (unpublished data). As cotton fiber length is a key factor in cotton yield and quality, we investigated whether this attribute could be enhanced by constitutive expression of Bn-csRRM2. Transgenic and wild-type cotton were grown in same condition. The leaves of 25 day and 45 day plants were harvested for microarray analysis. RNA samples were isolated from 3 biological replications using TRIzol (Invitrogen) as described by the manufacturer. Microarray analyses were carried out using Agilent Cotton Gene Expression Microarray (G2519F-022523). Microarrays were scanned on Agilent Technologies Scanner G2505C and data points were extracted using Agilent Feature Extraction software (Version 10.7.1.1). Comparisons were made between transgenic samples and their corresponding wild-type samples.
Project description:Transcriptional profiling of Arabidopsis dark-induced senescence comparing wild type (Col-0) with pif quadruple (pif1/3/4/5) mutant. After synchronized germination, the plants were grown under continuous white light for 7 days and transferred to darkness for 2 days to induce senescence. Goal was to determine the effect of PIFs on transcriptomic regulation during dark-induced senescence. Two-condition experiment, wild type vs. pif quadruple mutant. Biological replicates: 3 wild type replicates, 3 mutant replicates.
Project description:Cotton fiber is actually unicellular trichome, therefor its length is really hard to be modified but very meaningful to fiber quality and yield. We have reported the function of the second RRM domain of Oryza sativa FCA in rice cell size regulation. Data shows it is highly conserved across dicotyledonous and monocotyledonous plants. Here we provide evidence showing that the second RRM domain of Brassica napus FCA worked in Gossypium hirsutum, leading to the enlargement of multiple types of cells, such as pollen, cotyledon petiole and cotton fiber. In the transgenic cotton, the length of unicellular cotton fiber increased by about 10% and fiber yield per plant also showed a dramatic increase, ranging from 35% to 66%, over the control. Thus, this RRM domain may be an ancient and common cell size regulator and has great economic value on cotton industry.
Project description:Transcriptional profiling of Arabidopsis dark-induced senescence comparing wild type (Col-0) with pif quadruple (pif1/3/4/5) mutant. After synchronized germination, the plants were grown under continuous white light for 7 days and transferred to darkness for 2 days to induce senescence. Goal was to determine the effect of PIFs on transcriptomic regulation during dark-induced senescence.
Project description:Small RNA-dependent pairing of AGO/PIWI-containing effector complexes to chromatin-bound nascent transcripts has been proposed as a universal mechanism for guiding RNA-mediated TGS in eukaryotes. Likewise, Pol V-dependent transcripts have been implicated in the targeting of AGO4 to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we show that the AGO hook platforms of PolV and SPT5L, another component of the transcriptional complex, are functionally redundant yet essential for RdDM at a genome-wide level. Synthesis of Pol V transcripts is uncoupled from AGO4 recruitment in AGO hook-minus plants, challenging the prevailing RNA-based mechanism of AGO4 targeting to chromatin in RdDM. Transcription is essential to lock the PolV transcription complex into a stable and productive DNA-bound state potentiating interactions of AGO4 with DNA. Consistent with this idea, laser UV-assisted crosslinking shows specific AGO4-DNA interaction at RdDM loci, suggesting a revised model for Pol V-mediated DNA methylation in plants, which explains the exquisite specificity of methylation.
Project description:Total RNA was isolated with RNAiso regent (Takara) from 12-day-old seedlings of the areb1 areb2 abf3 abf1 quadruple mutant and WT plants grown on GM agar plates with or without treatment of 50 uM ABA, dehydration stress, or 250 mM NaCl for 6 h.
Project description:Essential metals such as iron are required for healthy plant growth. Fe is an important cofactor and catalytic element in many biological processes. Fe and other metals can also be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalisation. The NAS genes are supposed to play an important role in Fe homeostasis. They are coding for enzymes called nicotianaminesynthase (NAS), which synthesize nicotianamine (NA) by a one-step condensation reaction of three molecules S-adenosyl-methionine. NA acts as a chelator for Fe, Cu, Ni and Zn and might be involved in the transport and allocation of Fe throughout the plant. We generated quadruple T-DNA insertion mutant nas plants to investigate NA function as described in Klatte et al., 2009, Plant Physiol. The nas4x-1 plants show an interveinal leaf chlorosis when turning from vegetative to reproductive stage, which intensifies when growing under Fe deficiency conditions. nas4x-1 plants have strongly reduced NA contents and show an elevated Fe deficiency response in roots. By performing microarray experiments we want to reveal global changes on transcriptional level in roots and leaves of nas4x-1 mutant compared to wild type plants grown under Fe supply and Fe deficiency conditions, respectively. The loss of NAS genes has a strong impact on the regulation of other metal homeostasis genes and allows to draw conclusions about nicotianamine function in metal homeostasis of A.thaliana. For this study, four-week old nas4x-1 mutant and wild type plants were exposed for 7 days to plant medium with and without Fe supply. These conditions have been established previously and have resulted in a reproducibly strong interveinal leaf chlorosis of nas4x-1 plants compared to wild type, especially upon Fe deficiency conditions. The experiment was repeated three times in consecutive weeks to obtain three independent biological repetitions. Rosette leaves and roots of five week-old plants were harvested, RNA was isolated and microarray hybridization was performed. 24 Total samples were analyzed. We generated the following pairwise comparisons: WT + Fe vs. – Fe, nas4x-1 + Fe vs. – Fe, + Fe WT vs. + Fe nas4x-1, - Fe WT vs. - Fe nas4x-1, roots and leaves