Project description:Stroma extracts were isolated from 2-week-old WT plants and incubated with either PUMPKIN-specific antibodies or with the pre-immune serum. IgGs were captured with SiMAG-Protein G beads (Chemicell) and recovered RNA was used for generation of libraries with the ScriptSeq v2 RNA-seq Library Preparation Kit (Epicentre). Primary reads were aligned to the Arabidopsis chloroplast genome (accession number NC_000932.1) using CLC Genomics Workbench, the mean RPKM values of the replicates as well as the ratio of PUMPKIN vs Pre-immuneserum were calculated. Five prominent RNA targets (trnG-UCC, trnV-UAC, petB, petD and ndhA) were identified and validated via Slot Blot analyses.
Project description:Purpose: Analyses of transcriptomes of WT and pumpkin mutants, in order to compare gene expression of typically NEP or PEP-transcribed plastid genes.
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. Experiment Overall Design: In order to identify potential tomato transcripts in dodder, microarray analysis was performed on RNA from dodder and hosts. Total RNA was extracted from the tomato host and from dodder grown on tomato, Arabidopsis, tobacco, or pumpkin. The host tomato RNA was included to verify that any transcripts detected in the parasite were in fact expressed in the host. The dodder samples grown on tobacco, Arabidopsis, and pumpkin served as controls for dodder genes that may cross-hybridize with tomato array probes, with three different host species used to minimize any host-specific effects on dodder gene expression. Samples were analyzed using the Affymetrix GeneChip Tomato Array and transcripts scored for presence or absence in each sample. Considering that host transcripts present in dodder would be at low levels and diluted with dodder transcripts, a P-value of 0.06 in at least two of three biological replicates was used as the threshold for scoring a transcript as being present.
Project description:Food fraud is a common issue in the modern food industry. The undeclared use of foreign pro-teins in meat products is a major concern in this context. Oilseeds are ideal for this purpose due to their high protein content and since huge amounts of oil meals are obtained as a by-product of oil production. Therefore, a UHPLC-MS/MS method was developed for the simultaneous de-tection of chia, coconut, flaxseed, hemp, peanut, pumpkin, rapeseed, sesame, soy, and sunflower proteins in meat products. Potential tryptic peptide markers were identified by high-resolution mass spectrometry. The final twenty peptide markers selected, which are specific for one of the ten species targeted were each measured by multiple reaction monitoring. To the best of our knowledge, twelve new heat-stable marker peptides for chia, coconut, flaxseed, pumpkin, rape-seed, sesame and sunflower have not been reported previously.
