Project description:Flowering of Arabidopsis thaliana is accelerated by several environmental cues, including exposure to long days. The photoperiod-dependent promotion of flowering involves the transcriptional induction of FLOWERING LOCUS T (FT) in the phloem of the leaf. FT encodes a mobile protein that is transported from the leaves to the shoot apical meristem, where it forms part of a regulatory complex that induces flowering. Whether FT also has biological functions in leaves of wild-type plants remains unclear. In order to address this issue, we first studied the leaf transcriptomic changes associated with its over expression in the companion cells of the phloem. To this end, transgenic A. thaliana plants that misexpress FT from the pGAS1 promoter in a ft-10 tsf-1 double mutant background were employed (pGAS1:FT ft-10 tsf-1). In these transgenic plants, the use of the pGAS1 promoter ensures that the FT transgene is expressed in phloem companion cells of the minor veins, recreating the spatial pattern of expression described for the native gene. In this studuy, the transcriptome of leaves of pGAS1:FT ft-10 tsf-1 transgenic plants was compared to that of ft-10 tsf-1 and Col-0 plants using Tiling Arrays.
Project description:This sudy focuses on the identification of transcripts in the shoot phloem of the model plant Arabidopsis thaliana. Transcripts expressed in the phloem tissue (parenchyma cell, companion cell, sieve element) were excised by laser microdissection pressure catapulting (LMPC). These were compared with transcripts isolated from leaf phloem exudates by EDTA-chelation technique. Optimization of sample harvest resulted in RNA of high quality from both sources. Modifications of the RNA amplification procedure obtained RNA of sufficient yield and quality for microarray experiments. Microarrays (Affymetrix, ATH1) hybridized with RNA derived from phloem tissue by LMPC or phloem sap allowed us to differentiate between phloem located and mobile transcript species. The datasets provide a search criterion for phloem-based signals and will facilitate reverse genetic studies and forward genetic screens for phloem and long distance RNA signaling mutants. Keywords: profiles of mobile and stationary Arabidopsis phloem transcripts
Project description:ABSTRACT:The SODIUM POTASSIUM ROOT DEFECTIVE 1 (NaKR1) encodes a soluble metal binding protein that is specifically expressed in companion cells of the phloem. The nakr1-1 mutant phenotype includes high Na+, K+, Rb+ and starch accumulation in leaves, short roots, late flowering and decreased long-distance transport of sucrose. Using traditional and DNA microarray-based mapping, a 7 bp deletion was found in an exon of NaKR1 that caused a premature stop. The mutant phenotypes were complemented by the native gene and by GFP and GUS fusions driven by the native promoter. NAKR1-GFP was mobile in the phloem, it moved into sieve elements and into a novel symplasmic domain of the root meristem. Grafting experiments revealed that the high Na+ accumulation was due primarily to loss of NaKR1 function in the leaves. This supports a role for the phloem in recirculating Na+ to the roots to limit Na+ accumulation in leaves. The onset of root phenotypes coincided with NaKR1 expression after germination. Short root length was primarily due to a decrease in cell division rate in the root meristem indicating a role for NaKR1 expression in the phloem in root meristem maintenance. 3 hybridizations each of mutant NAKR1 and wildtype for deletion identification.
Project description:ABSTRACT:The SODIUM POTASSIUM ROOT DEFECTIVE 1 (NaKR1) encodes a soluble metal binding protein that is specifically expressed in companion cells of the phloem. The nakr1-1 mutant phenotype includes high Na+, K+, Rb+ and starch accumulation in leaves, short roots, late flowering and decreased long-distance transport of sucrose. Using traditional and DNA microarray-based mapping, a 7 bp deletion was found in an exon of NaKR1 that caused a premature stop. The mutant phenotypes were complemented by the native gene and by GFP and GUS fusions driven by the native promoter. NAKR1-GFP was mobile in the phloem, it moved into sieve elements and into a novel symplasmic domain of the root meristem. Grafting experiments revealed that the high Na+ accumulation was due primarily to loss of NaKR1 function in the leaves. This supports a role for the phloem in recirculating Na+ to the roots to limit Na+ accumulation in leaves. The onset of root phenotypes coincided with NaKR1 expression after germination. Short root length was primarily due to a decrease in cell division rate in the root meristem indicating a role for NaKR1 expression in the phloem in root meristem maintenance.
Project description:This sudy focuses on the identification of transcripts in the shoot phloem of the model plant Arabidopsis thaliana. Transcripts expressed in the phloem tissue (parenchyma cell, companion cell, sieve element) were excised by laser microdissection pressure catapulting (LMPC). These were compared with transcripts isolated from leaf phloem exudates by EDTA-chelation technique. Optimization of sample harvest resulted in RNA of high quality from both sources. Modifications of the RNA amplification procedure obtained RNA of sufficient yield and quality for microarray experiments. Microarrays (Affymetrix, ATH1) hybridized with RNA derived from phloem tissue by LMPC or phloem sap allowed us to differentiate between phloem located and mobile transcript species. The datasets provide a search criterion for phloem-based signals and will facilitate reverse genetic studies and forward genetic screens for phloem and long distance RNA signaling mutants. Experiment Overall Design: Arabidopsis plants were cultivated under short day conditions (8 h light) until flowering. Phloem tissue was isolated by Lasermicrodissection and Pressure Catapulting (LMPC) and phloem exudate by EDTA-chelation technique. In both experiments poly-A-RNA was extracted and amplified before hybridization of microarrays (Affymetrix, ATH1). For both experiments three LMPC-derived phloem tissue (LMPC-derived phloem_1-3) and three phloem exudate samples (Phloem exudate_1-3) were analysed. Precise protocols of plant growth, sample harvest, RNA extraction and amplification are provided in Deeken et al., 2008.
Project description:Transformation of undifferentiated stem cells into cells with special functions is central for organismal development. The phloem tissue mediates long-distance transport of energy metabolites along plant bodies and is characterized by an exceptional degree of cellular specialization. How the phloem-specific developmental program is implemented is, however, unknown. Here we reveal that the ubiquitously expressed PHD-finger protein OBERON3 (OBE3) and the phloem-specific SUPPRESSOR OF MAX2 1-LIKE 5 (SMXL5) protein form a central module for establishing phloem identity in Arabidopsis thaliana (Arabidopsis). By phloem-specific ATAC-seq analyses, we show that OBE3 and SMXL5 proteins establish a phloem-specific chromatin profile.
Project description:We performed small RNA-seq (sRNA-seq) study of Arabidopsis phloem sap under iron-sufficient (+Fe; control) and iron deficient (-Fe) conditions to investigate and identify sRNAs whose expression is regulated by iron deficiency in the phloem sap.
Project description:A silencing signal in plants with an RNA specificity determinant moves through plasmodesmata and the phloem. To identify the mobile RNA we grafted Arabidopsis thaliana shoots to roots that would be a recipient for the silencing signal. Using high throughput sequencing as a sensitive detection method and mutants to block small RNA (sRNA) biogenesis in either source or recipient tissue, we detected endogenous and transgene specific sRNA that moved across the graft union. Surprisingly we found that the mobile endogenous sRNAs account for a substantial proportion of the sRNA in roots and we provide evidence that 24nt mobile sRNAs direct epigenetic modifications in the genome of the recipient cells. Mobile sRNA thus represents a mechanism for transmitting the specification of epigenetic modification and could affect genome defence and responses to external stimuli that have persistent effects in plants. Keywords: Small RNA Analysis, Epigenetics 34 unique samples, 15 Biological Replicates
Project description:A silencing signal in plants with an RNA specificity determinant moves through plasmodesmata and the phloem. To identify the mobile RNA we grafted Arabidopsis thaliana shoots to roots that would be a recipient for the silencing signal. Using high throughput sequencing as a sensitive detection method and mutants to block small RNA (sRNA) biogenesis in either source or recipient tissue, we detected endogenous and transgene specific sRNA that moved across the graft union. Surprisingly we found that the mobile endogenous sRNAs account for a substantial proportion of the sRNA in roots and we provide evidence that 24nt mobile sRNAs direct epigenetic modifications in the genome of the recipient cells. Mobile sRNA thus represents a mechanism for transmitting the specification of epigenetic modification and could affect genome defence and responses to external stimuli that have persistent effects in plants. Keywords: Small RNA Analysis, Epigenetics
Project description:An intriguing new paradigm in plant biology is that systemically-mobile mRNAs play a role in coordinating development. In this process, specific mRNAs are loaded into the phloem transport stream for translocation to distant tissues, where they may impact developmental processes. However, despite its potential significance for plant growth regulation, mRNA trafficking remains poorly understood and challenging to study. Here we show that phloem-mobile mRNAs can also traffic between widely divergent species from a host to the plant parasite, lespedeza dodder (Cuscuta pentagona Engelm.). Reverse transcriptase PCR (RT-PCR) and microarray analysis were used to detect specific tomato transcripts in dodder grown on tomato (Lycopersicon esculentum Mill.) that were not present in control dodder grown on other host species. The foreign transcripts included LeGAI, which has been previously shown to be translocated in the phloem, as well as nine other transcripts not reported to be mobile. Dodders are parasitic plants that obtain resources by drawing from the phloem of a host plant, and have joint plasmodesmata with host cortical cells. Although viruses are known to move between dodder and its hosts, translocation of endogenous plant mRNA has not been reported. These results point to a potentially new level of interspecies communication, and raise questions about the ability of parasites to recognize, use, and respond to transcripts acquired from their hosts. Keywords: mRNA trafficking, parasitism, parasitic plant