EXPERIMENTAL PROCEDURES
Data sources and phylogenetic analyses
Genome sequences for the bryophytes P. patens, the lycophyte S. moellendorffii
and the selected microalgae were retrieved from the websites listed in Table S1. A
local blast database was constructed. Proteins from all genomes were blasted with the
Arabidopsis proteins (Table S2). Blast results were parsed for ≥25% amino acid
identity with E-values ≤1e-10. The CDD (Conserved Domain Database;
http://www.ncbi.nlm.nih.gov/cdd/), Pfam (http://pfam.sanger.ac.uk/search) and
SMART (http://smart.emblheidelberg.de/) databases were used to search for
structurally conserved domains within specific proteins. Sequences ultimately
selected are listed in Data S1. Parsed hits for all species were aligned using
CLUSTALW (Chenna et al. 2003). Gaps and ambiguously aligned sites were
removed using gBlock (Talavera and Castresana 2007). Sequences that caused
aberrant alignments and whose real identity could not be confirmed were removed
manually. ProtTest (Darriba et al. 2011) was used to select models of protein
substitution and rate heterogeneity that best fit each data set. Phylogenetic analyses
were performed with a maximum likelihood method using PhyML 3.0 (Guindon et al.
2010). Bootstrap support values were estimated using 100 pseudo-replicates. The
number of paralogs in each case was determined by counting the number of sequences
for each lineage in the ortholog clade.
Abscisic acid (ABA) analysis
Samples were analyzed for ABA using a modified version of the method
described by Turečková et al (Turečková et al. 2009). Briefly, samples were extracted
in 1 mL cold methanol/water/acetic acid (10/89/1, v/v/v) and 20 pmol of [2H6] (+)
ABA was added as an internal standard. After 1 h, the homogenates were centrifuged
and the pellets were re-extracted for another hour. The combined supernatants were
then cleaned on Oasis® HLB cartridges (60 mg, 3 ml, Waters, Milford, MA, USA).
They were then methylated, purified by ABA-specific immunoaffinity extraction
(Hradecká et al. 2007) and analysed by UHPLC-ESI (+)-MS/MS (Micromass,
Manchester, UK).
Cytokinin (CK) analysis
Samples were analyzed for their CK content as described by Novák et al (Novák
et al. 2008). In brief, 500 mg (DW) was extracted overnight in Bieleski’s extraction
solution. Stable isotope-labeled CK internal standards (IS) were added (3 pmol of
each compound per sample in the case of the CK bases, ribosides and O-glucosides,
and 5 pmol of each CK nucleotide). The following IS were used: [13C5]tZ, [2H5]tZR,
[2H5]tZ9G, [2H5]tZOG, [2H5]tZROG, [2H5]tZRMP, [13C5]cZ, [2H3]DHZ, [2H3]DHZR,
[2H3]DHZ9G, [2H7]DHZOG, [2H3]DHZRMP, [2H6]iP, [2H6]iPR, [2H6]iP9G,
[2H6]iPRMP, [2H7]BA, [2H7]BAR, [2H7]BA9G, [2H7]BARMP, [15N4]mT and
[15N4]oT. The supernatant was purified using SCX cartridges (1g per 6ml Bond Elut,
Agilent Technologies, CA, USA) and a weak anion-exchange column (DEAE A-25,
GE Healthcare, Sweden) on C18 (200mg per 6ml Bond Elut Plexa, Agilent
Technologies, CA, USA). The nucleotide fraction was then enzymatically treated
(alkaline phosphatase from E. coli - 10U per 5ml of sample) and purified on Plexa
C18 (200mg per 6ml). Finally, immunoaffinity chromatography using monoclonal
antibodies raised in a mouse was performed to obtain pure extracts for
UHPLC-(+)ESI-MS/MS analysis of CKs.
Dynamics of phytohormone metabolism transcripts under nitrogen-deleted and
nitrogen-replete conditions via mRNA-Seq based time-series transcriptomes
Members of Nannochloropsis sp. are heterokonts and are found widely in the
marine environment as well as in fresh and brackish waters. These algae are of
industrial interest due to their ability to grow rapidly, synthesize large amounts of TAG
and high-value polyunsaturated FA (e.g. eicosapentaenoic acid; EPA), and tolerate
broad environmental and culture conditions (Radakovits et al. 2012, Vieler et al. 2012,
Wang et al. 2012, Wang et al. 2014). Nannochloropsis oceanica IMET1, an industrial
strain of EPA and lipid production (Wang, et al. 2014), was selected as a model here
for probing hormone metabolism in microalgae. It was inoculated into modified f/2
liquid medium, which was prepared with 35 g l-1 sea salt, 1 g l-1 NaNO3, 67 mg l-1
NaH2PO4*H2O, 3.65 mg l-1 FeCl3*6H2O, 4.37 mg l-1 Na2EDTA*2H2O, trace metal
mix (0.0196 mg l-1 CuSO4*5H2O, 0.0126 mg l-1 NaMoO4*2H2O, 0.044 mg l-1
ZnSO4*7H2O, 0.01mg l-1 CoCl2, and 0.36 mg l-1 MnCl2*4H2O), and vitamin mix (2.5
µg l-1 VB12, 2.5 µg l-1 biotin, and 0.5 µg l-1 thiamine HCl). The cells were grown in
liquid cultures under continuous light (approximately 50 µmol photons m-2 s-1) at 25˚C
and aerated by bubbling with a mixture of 1.5% CO2 in air. Mid-logarithmic phase algal
cells were collected and washed three times with axenic seawater. Equal numbers of
cells were re-inoculated in nitrogen replete medium (N-replete condition, or N+) and
nitrogen-deprived medium (N-depleted condition, or N-) with 50 µmol m-2 s-1 light
intensity. Cell aliquots were collected for RNA isolation after being transferred to the
designated conditions for 3, 4, 6, 12, 24, and 48 h. Three biological replicates of algal
cultures were established under each of the above N+ and N- conditions, respectively.
Total algal RNA under the above conditions was extracted using Trizol reagents
(Invitrogen). For mRNA-Seq, the poly (A)-containing mRNA molecules were purified
using Sera-mag Magnetic Oligo (dT) Beads (Thermo Scientific) and were fragmented
into 200- to 300-bp fragments by incubation in RNA Fragmentation Reagent (Ambion)
according to the manufacturer’s instructions. The fragmented mRNA was then purified
away from the fragmentation buffer using Agencourt® RNAClean beads (Beckman
Coulter). The purified, fragmented mRNA was converted into double-stranded cDNA
using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) by priming
with random hexamers. Strand nonspecific transcriptome libraries were prepared using
the NEBNext® mRNA Library Prep Reagent Set (New England Biolabs) and
sequenced for 2×90bp runs (paired-end, PE) using Illumina HiSeq2000.
Additional details on the methods and analysis of this time-series transcriptome
dataset were provided in (Li et al. 2014), which was published during the review of
this submission.
Physiological responses of N. oceanica to exogenous ABA and CKs
Cells were synchronized by alternating light/dark (12 h/12h) cycles with a final
extended dark period (Umen and Goodenough 2001, Zachleder 1994). The following
three experiments were performed separately: (i) Synchronous cells (with an initial
concentration of 1×106 ml-1) were cultured in flask supplemented with
6-benzyladenine (BA, an artificial CK), ABA or an equivalent amount of HCl or
DMSO under 50 μmol photons m-2s-1 light. Cell aliquots were collected for DW
determination after five days or for cell cycle analysis by flow cytometry as described
at indicated time (Marie et al. 2001). Cell division in randomly selected fields was
investigated microscopically at 100× magnification. (ii) Synchronous cells were
inoculated in fresh medium and cultured to a concentration of 5×107 ml-1, and then
were collected and cultured in photobioreactors suspended in N+ or N- medium
respectively. BA was applied to cells under N-. Cell aliquots were diluted and cell
numbers were determined under a microscope using a counting chamber. DW were
determined after 144 h. (iii) Cells at 5×107 ml-1 were harvested and re-suspended into
fresh N+ medium containing 0.5 μM or 5 μM ABA under high light (150 or 300 µmol
photons m-2 s-1) or into N- medium containing 0.5 μM or 5 μM ABA under darkness
or 50 µmol photons m-2 s-1 light. The Fv/Fm values were determined using Imaging
PAM.
Analysis of DNA content and cell cycle of N. oceanica by flow cytometry
The DNA content and cell cycle were determined using flow cytometry
(FACSAria, BD Biosciences) as described by (Marie et al. 2001) with some
modifications. (i) Cell aliquots were collected and washed with PBS solution (pH
7.4~7.6) once. (ii) Samples were fixed by adding 2.5% glutaraldehyde (v/v), frozen in
liquid nitrogen and stored at −80°C for delayed analysis. Frozen samples were thawed
at 37°C. (iii) 10 μl RNase A (10 mg/mL, DNase and protease-free, Thermo Scientific)
was added for every 1 ml of sample. Incubation was at 37°C for 30 min. (iv) 10 μl
SYBR Green I working solution and 30 μl 1 M potassium citrate was added and the
reactions incubated for 15 min at room temperature in the dark. (v) 10 μl of a 1 × 105
beads/ml suspension of 0.95-μm fluorescent microspheres per 1 ml of sample was
added. (vi) The sample was analyzed for 4 min at a low running rate (<50 μl/min), and
10,000 cells were analyzed for each sample. Data were collected and cell phase
analyzed with MODFIT.
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