Study : Spatiotemporal analysis of shade-induced transcriptional reprogramming in Arabidopsis reveals patterns underlying organ-specific growth

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Name

Spatiotemporal analysis of shade-induced transcriptional reprogramming in Arabidopsis reveals patterns underlying organ-specific growth

Identifier

dXJuOkVWQS9zdHVkeS9QUkpOQTMyMDg3OA==

Source

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Link to this study on EBI European Nucleotide Archive

Description

In response to neighbor proximity plants increase growth of specific organs (e.g. hypocotyl) to enhance access to sunlight. Shade enhances the activity of Phytochrome Interacting Factors (PIFs) by releasing those bHLH transcription factors from phytochrome B-mediated inhibition. PIFs promote elongation by inducing auxin production in cotyledons. In order to elucidate spatiotemporal aspects of the neighbor proximity response, we analyzed changes in transcript abundance at different time points during a shade treatment in dissected cotyledons and hypocotyls. We concentrated our analysis on these two organs because the former is considered as the primary shade-sensing organ while elongation is rapidly triggered in the hypocotyl. We conclude that PIFs initiate transcriptional reprogramming in both organs within 15 minutes comprising regulated expression of several early auxin response genes. This suggests that hypocotyl growth is elicited by both local and distal auxin signals. With time the transcriptional response diverges increasingly between organs. We identify genes whose differential expression may underlie organ-specific elongation. Finally, we uncover a growth promotion gene expression signature shared between responses to different environments and organs. Overall design: Arabidopsis thaliana Col-0 was grown for 5 days in a 16 h : 8 h, light : dark photoperiod at 21°C on one horizontal plate of c. 140 mm in diameter per sample. Each plate contained 25 ml ½ MS medium covered with a nylon mesh. On day 6 at ZT2, seedlings were either kept in high R/FR or transferred to low R/FR. After 0, 15, 45, 90 or 180 min after the onset of low R/FR samples of both light qualities were imbibed within 75 sec in ice-cold 100 % (v/v) acetone and two times subjected to about 600 mbar below atmospheric pressure for 5 min while remaining on ice. The dissection of acetone-fixed seedlings was carried out in 70 % (v/v) 4°C-cold ethanol under a binocular lens. Cotyledon and hypocotyl material of 50 seedlings per sample were collected in 100 % (v/v) ethanol. For each time point and light condition duplicates were prepared. Plant materials were manually ground using pestles and total RNA was extracted using the RNeasy Kit (QIAGen) with on-column DNA digestion, according to the manufacturer’s instructions. Finally RNA samples were precipitated using 3M NAOH (pH 5.2) and 100 % (v/v) ethanol. The precipitate was visualize with glycogen and washed with 80 % (v/v) ice-cold ethanol. Stranded libraries were prepared using 400 ng high quality RNA using the TruSeq protocol according to th manufacturer’s instructions (Illumina). This included RNA purification steps using AMPure XP beads and cDNA preparation using a mix of random and polyA primer. RNA-seq libraries were subsequently sequenced with a HISEQ 2500 (Illumina) in the Genome Technology Facility (GTF).

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Genotype

Accession number	Name	Taxon