Project description:Comparison of Arabidopsis T87 cells transformed with an empty vector vs transcription factor (TF)-overexpressed lines of T87 cells.
Project description:We investigated genome-wide changes in mRNA translation in Arabidopsis thaliana T87 suspension cell cultures which thought to be one of the host materials for bioreactor. Global translational repression was observed in cells of 8 day after inoculation that is thought to be stressful condition by the nutrient deficiency and hypoxia. This suggested the negative effect of the global translational repression on transgene expression. On the other hand, previous study using heat stress showed that some mRNAs were actively translated under such stressful condition, suggesting the existence of mRNA that were actively translated in cells of 8 day after inoculations. To identify mRNAs that escape global translational repression on 8 day and its cis-elements would be the 1st step to make the system for higher transgene expression by the escaping global translational repression. To this end, we subjected polysomal RNA and non-polysomal RNA from sucrose gradient fractionated cell lysates to the co-hybridization on Agilent Arabidopsis 4 Oligo Microarrays. The ratio of signal intensities (polysomal RNA: total RNA) was used as an indicator of the translation state for each transcript.
Project description:We investigated genome-wide changes in mRNA translation in Arabidopsis thaliana T87 suspension cell cultures which thought to be one of the host materials for bioreactor. Global translational repression was observed in cells of 8 day after inoculation that is thought to be stressful condition by the nutrient deficiency and hypoxia. This suggested the negative effect of the global translational repression on transgene expression. On the other hand, previous study using heat stress showed that some mRNAs were actively translated under such stressful condition, suggesting the existence of mRNA that were actively translated in cells of 8 day after inoculations. To identify mRNAs that escape global translational repression on 8 day and its cis-elements would be the 1st step to make the system for higher transgene expression by the escaping global translational repression. To this end, we subjected polysomal RNA and non-polysomal RNA from sucrose gradient fractionated cell lysates to the co-hybridization on Agilent Arabidopsis 4 Oligo Microarrays. The ratio of signal intensities (polysomal RNA: total RNA) was used as an indicator of the translation state for each transcript. Experiment using two-fractionated mRNA, Polysomal mRNA vs. total mRNA. Biological replicates: 1
Project description:Xylem vessels function in the long-distance conduction of water in land plants. The NAC transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). We previously isolated seiv (suppressor of ectopic xylem vessel cell differentiation induced by VND7) mutants, which are suppressor mutants of VND7-inducible xylem vessel cell differentiation. Here, we report that the responsible genes for seiv3, seiv4, seiv6, and seiv9 are protein ubiquitination-related genes encoding PLANT U-BOX46 (PUB46), uncharacterized F-BOX protein, PUB36, and UBIQUITIN-SPECIFIC PROTEASE1, respectively. We also found the decreased expression of genes downstream of VND7 and abnormal xylem transport activity in the seiv mutants. Upon VND7 induction, ubiquitinated levels from 492 and 180 protein groups were up- and down-regulated, respectively. Proteins for cell wall biosynthesis and protein transport were ubiquitinated by the VND7 induction, whereas such active protein ubiquitination was not observed in the seiv mutants. We detected the ubiquitination of three lysine residues in VND7: K94, K105, and K260. Substituting K94 with arginine significantly decreased the transactivation activity of VND7, suggesting that the ubiquitination of K94 is crucial for regulating VND7 activity. Our findings highlight the crucial roles of target protein ubiquitination in regulating xylem vessel activity.