DOI: 10.5061/dryad.sf7m0cgb2
title: Data for: Induction of C4 genes during de-etiolation of Gynandropsis gynandra evolved through changes in cis allowing integration into ancestral C3 gene regulatory networks

description: C4 photosynthesis has evolved repeatedly and in doing so repurposed
 existing enzymes to drive a carbon pump that limits the oxygenation
 reaction of RuBisCO. C4 proteins accumulate to levels matching those of
 the photosynthetic apparatus, and to allow this gene expression must be
 modified over evolutionary time. To better understand this rewiring of
 gene expression we undertook RNA-SEQ and DNaseI-SEQ on de-etiolating
 seedlings of C4 Gynandropsis gynandra which is evolutionarily proximate to
 C3 A. thaliana. Changes in chloroplast ultrastructure and C4 gene
 expression in G. gynandra were coordinated and rapid. C3 and C4
 photosynthesis genes showed similar induction patterns, but C4 genes from
 G. gynandra were more strongly induced than orthologs from A. thaliana.
 The cistrome of G. gynandra was enriched in TGA, TCP and homeodomain
 binding sites. Furthermore, in vivo binding data in G. gynandra
 highlighted TGA and homeodomain as well as light responsive elements such
 as G- and I-box motifs as being associated with the rapid increase in
 transcripts derived from C4 genes. Although promoters of PPDK and ASP1
 from G. gynandra contained distinct light responsive elements, promoters
 from both A. thaliana and G. gynandra allowed high expression. Deletion
 analysis of the Ppa6 gene from G. gynandra showed that regions containing
 G- and I-boxes were necessary for high expression. The data support a
 model in which accumulation of transcripts derived from C4 genes in leaves
 of G. gynandra is enhanced compared with homologs in A. thaliana because a
 variety of modifications in cis allowed integration into ancestral
 transcriptional networks.
description: <em>Gynandropsis gynandra </em>seeds were sown
 directly from intact pods and germinated on moist filter papers in the
 dark at 32°C for 24 hours. Germinated seeds were then transferred to half
 strength Murashige and Skoog (MS) medium with 0.8% (w/v) agar (pH 5.8) and
 grown for three days in a growth chamber at 26°C. De-etiolation was
 induced by exposure to white light with a photon flux density (PFD) of 350
 μmol m<sup>-2</sup> s<sup>-1</sup> and photoperiod
 of 16 hours. Whole seedlings were harvested at 0.5, 2, 4 and 24 hours
 after illumination (starting at 8:00 with light cycle 6:00 to 22:00).
 Tissue was flash frozen in liquid nitrogen and stored at -80°C prior to
 processing. <strong>RNA and DNaseI
 sequencing</strong> Before processing, frozen
 samples were divided into two, the first being used for RNA-SEQ analysis
 and the second for DNaseI-SEQ. Samples were ground in a mortar and pestle
 and RNA extraction carried out with the RNeasy Plant Mini Kit (74904;
 QIAGEN) according to the manufacturer’s instructions. RNA quality and
 integrity were assessed on a Bioanalyzer High Sensitivity DNA Chip
 (Agilent Technologies). Library preparation was performed with 500 ng of
 high integrity total RNA (RNA integrity number &gt; 8) using
 the QuantSeq 3’ mRNA-SEQ Library Preparation Kit FWD for Illumina
 (Lexogen) following the manufacturer’s instructions. Library quantity and
 quality were checked using Qubit (Life Technologies) and a Bioanalyzer
 High Sensitivity DNA Chip (Agilent Technologies). Libraries were sequenced
 on NextSeq 500 (Illumina, Chesterford, UK) using single-end sequencing and
 a Mid Output 150 cycle run. To extract nuclei, tissue
 was ground in liquid nitrogen and incubated for five minutes in 15mM PIPES
 pH 6.5, 0.3 M sucrose, 1% (v/v) Triton X-100, 20 mM NaCl, 80 mM KCl, 0.1
 mM EDTA, 0.25 mM spermidine, 0.25 g Polyvinylpyrrolidone (SIGMA),
 EDTA-free proteinase inhibitors (ROCHE), filtered through two layers
 of Miracloth (Millipore) and pelleted by centrifugation at 4°C for 15 min
 at 3600 g. To isolate deproteinated DNA, 100 mg of tissue from seedlings
 exposed to 24 hours light were harvested two hours into the light cycle,
 four days after germination. DNA was extracted using a QIAGEN DNeasy Plant
 Mini Kit (QIAGEN, UK) according to the manufacturer’s instructions. 2x108
 nuclei were re-suspended at 4°C in digestion buffer (15 mM Tris-HCl, 90 mM
 NaCl, 60 mM KCl, 6 mM CaCl2, 0.5 mM spermidine, 1 mM EDTA and 0.5 mM EGTA,
 pH 8.0). DNase-I (Fermentas) at 2.5 U was added to each tube and incubated
 at 37 °C for three minutes. Digestion was arrested by adding a 1:1 volume
 of stop buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1% (w/v) SDS, 100 mM EDTA,
 pH 8.0, 1 mM Spermidine, 0.3 mM Spermine, RNaseA40 µg/ml) and incubated at
 55°C for 15 minutes. 50 U of Proteinase K were then added and samples
 incubated at 55°C for 1 h. DNA was isolated by mixing with 1 ml 25:24:1
 Phenol:Chloroform:Isoamyl Alcohol (Ambion) and spun for 5 minutes at
 15,700 g followed by ethanol precipitation of the aqueous phase. Samples
 were size-selected (50-400 bp) using agarose gel electrophoresis and
 quantified fluorometrically using a Qubit 3.0 Fluorometer (Life
 technologies), and a total of 10 ng of digested DNA (200 pg l-1) used for
 library construction. Sequencing ready libraries were prepared using
 a TruSeq Nano DNA library kit according to the manufacturer’s
 instructions. Quality of libraries was determined using a Bioanalyzer High
 Sensitivity DNA Chip (Agilent Technologies) and quantified by Qubit (Life
 Technologies) and qPCR using an NGS Library Quantification Kit (KAPA
 Biosystems) prior to normalisation, and then pooled, diluted and denatured
 for paired-end sequencing using High Output 150 cycle run (2x75 bp reads).
 Sequencing was performed using NextSeq 500 (Illumina, Chesterford UK) with
 2x75 cycles of sequencing. <strong>RNA-SEQ data
 processing and quantification</strong>  Commands
 used are available on GitHub (“command_line_steps”) but an outline of
 steps was as follows. Raw single ended reads were trimmed
 using trimmomatic (version 0.36). Trimmed reads were then quantified using
 salmon (version 0.4.234) after building an index file for a
 modified G. gynandra transcriptome. The transcriptome was modified to
 create a pseudo-3’ UTR sequence of 339 bp (the mean length of identified
 3’UTRs) for G. gynandra gene models that lacked a 3’ UTR sequence which
 was essentially an extension beyond the stop codon of the open reading
 frame. Inclusion of this psuedo 3’ UTR improved mapping rates. Each sample
 was then quantified using the salmon “quant” tool. All *.sf files had the
 “NumReads” columns merged into a single file (All_read_counts.txt) to
 allow analysis with both DEseq2 and edgeR. The edgeR pipeline was run as
 the edgeR.R R script (here and on GitHub) on the All_read_counts.txt file
 to identify the significantly differentially expressed genes by comparing
 each time-point to the previous one. A low expression filter step was also
 used. We then similarly analysed the data with the DEseq2 package using
 the DEseq2.R R script (on GitHub) on the same All_read_counts.txt file.
 This also included the Principal Component Analysis shown in Fig. 2A. The
 intersection from both methods was used to identify a robust set of
 differentially regulated genes. For most further analysis of the RNA-SEQ
 data, mean TPM values for each time-point (from three biological
 replicates) was first quantile normalised and then each value divided by
 the sample mean such that a value was of 1 represented the average for
 that sample. This processing facilitated comparisons between experiments
 across species in identifying changes to transcript abundance between
 orthologs.