Impact of the plastidial stringent response in plant ... · PDF fileImpact of the plastidial stringent response in plant growth and ... The fatty acid composition of shoots from plants

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.167

NATURE PLANTS | www.nature.com/natureplants 1

Impact of the plastidial stringent response in plant growth and stress

responses

Mikika Maekawa1, Rina Honoki1, Yuta Ihara1, Ryoichi Sato1, Akira Oikawa2,3, Yuri Kanno3,

Hiroyuki Ohta1,6, Mitsunori Seo3, Kazuki Saito3,4 & Shinji Masuda5,6,*

1Graduate School of Bioscience & Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan 2Faculty of Agriculture, Yamagata University, Tsuruoka 997-8555, Japan 3RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan 4Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan 5Center for Biological Resources & Informatics, Tokyo Institute of Technology, Yokohama 226-8501, Japan 6Earth-Life Science Institute, Tokyo Institute of Technology, Tokyo 152-8551, Japan *e-mail: [email protected] (S.M.).

http://dx.doi.org/10.1038/nplants.2015.167

2 NATURE PLANTS | www.nature.com/natureplants

SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.167

Supplementary Methods Transient expression of GFP fusion proteins. To construct GFP fusions of RSH2

and RSH3, full-length cDNAs of RSH2 and RSH3 were amplified from RT-PCR

products with gene-specific primers (Supplementary Table 2). The PCR products were

first cloned into the pDONRzeo vector using BP reactions (Invitrogen). The inserted

fragments were then cloned into pGWB5 (kindly provided by Dr. Nakagawa of Shimane

University) using LR reactions (Invitrogen). The obtained constructs were introduced

into Agrobacterium by electroporation. The Agrobacterium strains were grown in LB

medium until late-log phase and then collected by centrifugation at room temperature.

The harvested cells were suspended in ~15 ml of an infiltration buffer containing 10 mM

MES-KOH (pH 5.5), 10 mM MgSO4 and 2% (w/v) sucrose to an adjusted OD600 of ~0.7.

The suspensions were injected through pores into leaves of N. benthamiana using

syringes. Plants were incubated for 3 days under continuous light (40 µmol photons m-2

s-1) at 23°C. GFP or chlorophyll fluorescence was observed with a confocal laser

scanning microscopy (LSM780, ZEISS).

Starch Quantification. Shoot tissues were homogenized in 2 ml of 80% ethanol

followed by incubation at 80°C for 1 h. After centrifugation (15,000 × g, 10 min), the

supernatant and pellet were collected as glucose and starch fractions, respectively.

Distilled water (0.6 ml) was added to the pellet and mixed well. The mixture was

incubated at 95°C for 1 h, and then combined with the same volume of 35 U/ml

amyloglucosidase (Sigma-Aldrich). The mixture was incubated at 55°C for 1 h to

hydrolyze starch into glucose. The glucose concentration was determined using a

glucose assay kit (Glucose-CII-test Wako, Wako Pure Chemical Industries) according to

the manufacturer’s protocol.

Histology and electron microscopy. For electron microscopy, rosette leaves were

harvested from 28-day-old plants and fixed on ice with 0.1 M cacodylate buffer (pH 7.2)

containing 2.5% (w/v) glutaraldehyde and left overnight in the dark. The samples were

washed several times with the same buffer, and post-fixed with 1% osmium tetroxide

(w/v) for 2 h on ice. Then, the samples were dehydrated using a graded acetone series

and embedded in Spurr’s low-viscosity resin48. Thin sections were stained with uranyl

acetate and lead citrate, and observed under a JEOL 1200 EX transmission electron




microscope (JEOL). Transverse sections were also prepared from the resin, followed by

staining with Toluidine Blue and analysis using a Nikon ECLIPSE 80i microscope

(Nikon). Leaf length, width and area, as well as petiole size, were measured using

Image J software (http://www.rsb.info.nih.gov/ij). To visualize palisade cells, the seventh

leaf of each line grown for 21 days was fixed in a buffer containing 3.5%

paraformaldehyde, 5% acetic acid and 50% ethanol overnight, and then incubated with

a buffer composed of 80 g chloral hydrate, 30 ml water and 10 ml glycerol. The cleared

leaves were photographed using a confocal microscope (TCS SE; Leica).

Lipid analysis. The fatty acid composition of shoots from plants grown for 28 days was

measured using gas chromatographic analysis after the combined digestion and fatty

acid methyl ester formation of lipids using a reported method49. Lipids were extracted

from vegetative shoots of 28-day-old plants as described50. The extracts were applied to

silica gel 60A plates (Merck) and analyzed using 2D-TLC with chloroform/methanol/7N

NH4 (15:10:1), followed by chloroform/methanol/acetic acid/water (170:20:15:3). Lipids

were visualized by spraying 60% acetone containing 0.01% (w/v) primulin under UV

light. The lipids were identified by co-chromatography with commercial standards

(Sigma-Aldrich), and their fatty acid content was determined using gas chromatographic

analysis. To allow quantification, the lipid extracts were spiked with pentadecanoic acid

(15:0), and the absolute quantity of each lipid class was calculated based on the final

recovery of 15:0. Fatty acids were extracted from vegetative shoots of 28-day-old plants,

followed by quantification using gas chromatography. 15:0 was used as the control.

Supplementary references

49. Browse, J., McCourt, P.J. & Somerville, C.R. Fatty acid composition of leaf lipids

determined after combined digestion and fatty acid methyl ester formation from fresh

tissue. Anal. Biochem. 152, 141-145 (1986).

50. Dörmann, P., Hoffmann-Benning, S., Balbo, I. & Benning, C. Isolation and

characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl

diacylglycerol. Plant Cell 7, 1801-1810 (1995).




Supplementary Table 1 | Chlorophyll contents in mutant plants

Chl a (µg mgFW-1)

Chl b (µg mgFW-1)

Chl a + Chl b (µg mgFW-1)

Chl a / Chl b

WT 8d 0.95±0.13 0.32±0.07 1.27±0.18 3.12±0.75

18d 1.13±0.22 0.41±0.09 1.54±0.30 2.85±0.21

rsh2rsh3 8d 0.86±0.22 0.29±0.13 1.15±0.30 3.38±1.30

18d 1.06±0.24 0.34±0.13 1.40±0.34 3.51±1.30

RSH3ox2 8d 0.65±0.10*a 0.24±0.08* 0.89±0.16* 2.94±1.10

18d 0.70±0.16**b 0.27±0.12** 0.97±0.27** 2.83±0.90

Values are means ± SD (n=16). Chlorophyll contents per fresh weight (FW) were measured with plants grown for 8 or 18 days. a *P<0.05 versus WT (8d) b **P<0.05 versus WT (18d)




Supplementary Table 2 | Primers used in this study Target gene(s) Sequence (5'->3') Usage T-DNA left border

TAGCATCTGAATTTCATAACCAATCTCGATACAC rsh2 mutation genotyping

CCCATTTGGACGTGAATGTAGACAC rsh3 mutation genotyping

RSH2 (At3g14050)

GAACTTTAGGATTATAAAGCAG rsh2 mutation genotyping PCR AAACAAGAATCTAAAACTAGTG

GGTCGACATGGTGGTGGCGACGACT rsh2 mutation genotyping RT-PCR AGAACAGAATCCGATCCGAAC

AAAAAGCAGGCTCAATGGTGGTGGCGACG PCR for gateway cloning AGAAAGCTGGGTAGCTTCCCCATCCGACC

CACATCATGGTCAAAAGAGACGTAG qRT-PCR CAGCAACAATAGACTCCCAATCC

RSH3 (At1g54130)

GGTCGACATGGTGGTAGCAACGACC rsh3 mutation genotyping PCR CCAGCTACAACAACAGTCGAATTA

GGTCGACATGGTGGTAGCAACGACC rsh3 mutation genotyping RT-PCR GGTCGACATGGTGGTAGCAACGACA

AAAAAGCAGGCTCAATGGTGGTAGCAACG PCR for gateway cloning AGAAAGCTGGGTAGCTTCCCCAGCAGCCAACC

CAAATTCATTGGTTCTTCATACC qRT-PCR CCTACACGAAGAGCAGATCC

AccD (AtCg00500)

GGGGTTATGGATTTTCGGTTT qRT-PCR CTTCCTTCTTGCATTCGTGCT

RpoA (AtCg00740)

GATGCTGTATTCATGCCTGTTGA qRT-PCR AATTCCGGGAGGCTTGATG

ClpP (AtCg00670)

GCGGTATTTGGTTCATTTGTCC qRT-PCR TCCTGAACGGGCTTCTTCC

AtpB (AtCg00480)

TCGCACAACATCTCCTATCCA qRT-PCR CGACGATAAGGGGCCAAA

PsbA (AtCg00020)

AACGGCGGTCCTTATGAACTAA qRT-PCR CCAAGGACGCATACCCAGA

PsbD (AtCg00270)

TCTGCTATGCGCTATTCATGGT qRT-PCR ATAAGTTTCTTCGGCTTGAGTTGG

RbcL (AtCg00490)

GGAGTTCCACCTGAAGAAGCA qRT-PCR TAAGCCCATCGGTCCACAC

UBQ10 (At4g05320)

GATCTTTGCCGGAAAACAATTGGAGGATGGT qRT-PCR CGACTTGTCATTAGAAAGAAAGAGATAACAGG




Supplementary Figure 1 | Isolation and phenotypes of rsh2, rsh3 and rsh2rsh3 mutants. a, Location of the T-DNA insertion in RSH2 and RSH3. Solid bars represent exons and thin lines represent introns. Numbered arrows represent primers used for RT-PCR shown in (b). b, RT-PCR analysis confirming the lack of transcripts for the target genes in each mutant. Ubiquitin10 (UBQ10) gene-specific primers were used as controls. c, Images of the WT, rsh2, rsh3, and rsh2rsh3 plants grown for 8 days on MS medium. Two plants of each line are shown. d, Images of the plants grown for 18 days on MS medium. Two plants of each line are shown.




Supplementary Figure 2 | ppGpp quantification of 21-day-old plants. ppGpp levels per fresh weight (FW) were quantified using LC-MS/MS. Values are means ± S.D. (n = 3). *P < 0.05.




Supplementary Figure 3 | Leaf sizes of WT, rsh2rsh3 and RSH3ox2 mutants. a, Length of WT (Col), rsh2rsh3 and RSH3ox2 (3ox2) leaves. b, Width of WT, rsh2rsh3 and RSH3ox2 leaves. c, Length WT, rsh2rsh3 and RSH3ox2 petioles. d, Length/width ratio of WT, rsh2rsh3 and RSH3ox2 leaves. e, Leaf area of WT, rsh2rsh3 and RSH3ox2. Plants were grown for 21 days on MS medium. Values are means ± S.D. (n = 10). *P < 0.05 versus WT.




Supplementary Figure 4 | Sizes of palisade cells from leaves. Palisade cells of the seventh leaf of WT and each mutant line grown for 21 days were analyzed. No significant differences were found. Bars = 50 mm.




Supplementary Figure 5 | Fresh and dry leaf weights. Fresh and dry leaf weights of WT and mutant lines grown for 21 days were measured. No significant differences were observed between WT and mutant leaf weights. Values are means ± S.D. (n = 3).




Supplementary Figure 6 | Metabolic state changes by ppGpp accumulation. Statistically significant changes (P < 0.05, n = 3) in metabolites in RSH3ox1 (compared with those in WT) are highlighted as red (increased) or blue (decreased). Fold change values in metabolite accumulation are expressed along a color gradient. Undetected metabolites are shown in gray. GlcN6P, glucosamine-6-phosphate; GS/GOGAT, glutamine synthase/glutamate synthase; OAA, oxaloacetate; Orn, ornithine; and PEP, phosphoenolpyruvate.




Supplementary Figure 7 | Abscisic acid, cytokinin and glucose contents are not affected by ppGpp accumulation. a, Abscisic acid levels in WT, rsh2rsh3, RSH3ox1 and RSH3ox2 grown for 21 days. b, trans-zeatin levels in WT, rsh2rsh3, RSH3ox1 and RSH3ox2 grown for 21 days. c, Glucose levels in WT, rsh2rsh3, RSH3ox1 and RSH3ox2 grown for 21 days.


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Impact of the plastidial stringent response in plant ... · PDF fileImpact of the plastidial stringent response in plant growth and ... The fatty acid composition of shoots from plants