Supplementary Material

SUMO proteases ULP1c and ULP1d are required for development and osmotic stress

responses in Arabidopsis thaliana

Plant Molecular Biology

Authors: Pedro Humberto Castro, Daniel Couto, Sara Freitas, Nuno Verde, Alberto P. Macho, Stéphanie Huguet, Miguel Angel Botella, Javier Ruiz-Albert, Rui Manuel Tavares, Eduardo Rodríguez Bejarano, Herlânder Azevedo*

*Corresponding author:Herlânder Azevedo

CIBIO, InBIO - Research Network in Biodiversity and Evolutionary Biology,

Universidade do Porto, Campus Agrário de Vairão, 4485-661 Vairão, Portugal

Tlf: +351 252660421

Fax: +351 252661780

Email: hazevedo@cibio.up.pt

The following Supplementary Material is available in this document:Fig. S1 In silico analysis of ULP1c and ULP1d expression patterns.Fig. S2 In silico characterization of ULP1c and ULP1d functional redundancy. Fig. S3 Characterization of ulp1c, ulp1d and ulp1c/d mutants. Fig. S4 Exposure of soil-grown ulp1c/d mutant plants to long-term drought.Fig. S5 Quantification of morphological traits of mesophyll cells and stomata in 4-week-old and 2-week-old plants.Fig. S6 Seed germination rate in medium supplemented with 400 mM mannitol.Fig. S7 Quantitative RT-PCR analysis of RD29a and KIN1.Table S1 List of primers used for genotyping T-DNA insertion lines.Table S2 List of quantitative RT-PCR (qPCR) primers.Methods S1 Histochemical GUS staining.Methods S2 PEG-infused plates.Methods S3 Quantification of real-time stomatal aperture.Methods S4 ABA inhibition of stomatal opening.Methods S5 SDS-PAGE and Western-blot analysis of SUMO-conjugate levels. Methods S6 In silico analysis.Supplementary References

Fig. S1 In silico analysis of ULP1c and ULP1d expression patterns. a, b ULP1c and ULP1d display similar expression patterns concerning plant anatomy (a) and development (b). ULP1d generally displays higher expression levels over ULP1c. Analysis was conducted in Genevestigator v4 (genevestigator.com)

Fig. S2 In silico characterization of ULP1c and ULP1d functional redundancy. a Phylogenetic reconstruction of Arabidopsis ULPs in conjunction with human SENP and yeast (S. cerevisiae) ULPs; numbers represent bootstrap values (1000 replicas). b, c Correlation, using microarray expression data, of ULP1c and ULP1d expression, during development (b) or other (c) experimental conditions. d Co-expression network (20 highest-ranking genes) of ULP1c and ULP1d. e Co-expression score of annotated members of the Arabidopsis thaliana ULP gene family. Analysis was performed in GeneMANIA (genemania.org/); connecting line thickness reflects a weighted sum of co-expression data sources. f ULP1c and ULP1d syntenic relationship

Fig. S3 Characterization of ulp1c, ulp1d and ulp1c/d mutants. a, b Morphology (a) and relative root growth rate inhibition (b) of seedlings grown in standard conditions for seven days and transferred to media supplemented with 100 mM NaCl for seven days (n=11); scale bar indicates 1 cm. c, d Morphology (c) and root growth rate (d) of 2-week-old in vitro-grown plants (n=16); scale bar indicates 1 cm. e Silique morphology following clearing in ethanol; scale bar indicates 1 mm. f Number of seeds per silique (n=9). g Morphology of soil-grown 4-week-old plants evidencing complementation of the ulp1c/d mutant; scale bar indicates 1 cm. Error bars represent SEM. Asterisk represents statistically significant differences of mutant in relation to the wild-type (unpaired t test; *, P<0.05)

Fig. S4 Exposure of soil-grown ulp1c/d mutant plants to long-term drought. a Seeds from Col, ulp1c, ulp1d and ulp1c/d genotypes were sown onto soil and watered normally for three weeks. Plants were then subjected to drought (water deprived) for two weeks. b Col, ulp1c/d and C-ulp1c/d 12-day-old soil-grown seedlings were exposed to long term drought (water deprived) and visually scored for the presence of stress symptoms after 12 days (n≥15).

Fig. S5 Quantification of morphological traits of mesophyll cells and stomata in 4-week-old and 2-week-old plants. a Quantification of the relative density of mesophyll cells in the fifth rosette leaves of 4-week-old wild-type, ulp1c/d and C-ulp1c/d plants; n=8 independent plants with 6 measurements per plant. b-d Quantification of the relative density of stomata (b), relative stomata length (c), and relative quantification of stomata length/width ratio (d), in the abaxial surface of 4-week-old wild-type, ulp1c/d and C-ulp1c/d plants; n=8 independent plants with 6 measurements per plant. e Quantification of stomata length/width ratio in 2-week-old Col, ulp1c, ulp1d, ulp1c/d and C-ulp1c/d seedlings grown in standard conditions; n≥37). f Morphology of stomata in 2-week-old seedlings; scale bar indicates 5 µm. Error bars represent SEM. Letters represent statistically significant differences between genotypes (unpaired t test)

Fig. S6 Seed germination rate inferred by the formation of green cotyledons, in medium supplemented with 400 mM mannitol (n=10).

Fig. S7 Quantitative RT-PCR analysis of RD29a and KIN1 expression levels in the wild-type (Col), ulp1c/d, siz1-2 and siz1-2 ulp1c/d backgrounds, in seedlings grown in vitro in standard or mannitol-supplemented medium

Table S1 List of primers used for genotyping T-DNA insertion lines.

Primer name Primer sequence (5' to 3') Description

ULP1c RP GACATACCTTCCTGCAGCTTG Genotyping of ulp1c-1 (SALK_050441)ULP1c LP CTCTGCAATTGCATCATTCTG

ULP1d RP GGCTGAGCTTCTTCTTCATCC Genotyping of ulp1d-1 (SALK_029340)ULP1d LP TTCAGATGTTTTACCGCAAGG

SIZ1-2 RP CACGACAGATGAAGCATTGTG Genotyping of siz1-2 (SALK_065397)SIZ1-2 LP GAGCTGAAGCATCTGGTTTTG

LBb1.3 ATTTTGCCGATTTCGGAAC Genotyping SALK T-DNA insertion lines

Table S2 List of quantitative RT-PCR (qPCR) primers. Primers were designed to ensure specific amplification within the Arabidopsis transcriptome, generate 100-250 bp PCR amplification products, and have 50-60% GC content and ~60ºC Tm. When possible, one of the primers was designed to span an exon junction.

Gene(AGI code)

Primer name Primer sequence (5' to 3') Tm GC(%)

Productsize (bp)

ULP1c ULP1c qRT F1 TGCGAGCGAGTACAGCCTCA 57.9 60.0 232(At1g10570) ULP1c qRT R1 AATCTTGGCAGCGACCGCCA 59.3 60.0

ULP1d ULP1d qRT F1 GGGAAAGCTGAGCACAGTGCA 57.6 57.1 200(At1g60220) ULP1d qRT R1 TCCCAAGACCACTCCCTAGGAGT 57.6 56.5

QQS QQS qRT F1 TTCTCCACAGCGACCAGTTG 60.3 55.0 210(At3g30720) QQS qRT R1 GTAGAACTGAAGCCCGACCC 60.1 60.0

RD20 RD20 qRT Fw1 ACACCGAAGGAAGGTATGTCCCAG 57.9 54.2 142(At2g33380) RD20 qRT Rv1 AGCCATCCAAAAGGATCGATTGCC 57.8 50.0

GOLS1 GOLS1 qRT Fw1 GGTTCACTACTGTGCAGCGGGTTC 60.0 58.3 237(At2g47180) GOLS1 qRT Rv1 GACGGTGCGGTCACGTAGTT 57.1 60.0

CIPK11 CIPK11 qRT Fw1 TTGCTTGTGGTGGTGGAGGCAC 59.8 59.1 127(At2g30360) CIPK11 qRT Rv1 TAGCCGCGTTTGTTGACGACG 58.4 57.1

RPT2 RPT2 qRT F1 GTGCTAAGGCTTGCAACGAG 59.8 55.0 207(At2g30520) RPT2 qRT R1 TGCCTGAATGGTCTCTGACG 59.8 55.0

CalB CalB qRT Fw1 ACGACGGTGGATCCATTCCGGA 59.7 59.1 167(At2g45670) CalB qRT Rv1 TCTTTCCAGCCAGCAAGTGCCA 59.0 54.6

RD29a RD29a RT Fw1 ACCAGCAGCACCCAGAAGAAGTTG 59.3 54.2 200(At5g52310) RD29a RT Rv1 GCCTGGTGCATCGATCACTTCAGG 59.9 58.3

KIN1 KIN1 RT Fw1 AGAATGCCTTCCAAGCCGGTCAG 59.2 56.5 122(At5g15960) KIN1 RT Rv1 ACTCTTTCCCGCCTGTTGTGCTC 59.4 56.5

ACT2 ActinF CTAAGCTCTCAAGATCAAAGGCTTA 52.7 40.0 211(At3g18780) ActinR ACTAAAACGCAAAACGAAAGCGGT

T57.2 40.0

CalB - Calcineurin B subunit-related; Tm - Melting temperature; bp - Base pair

Methods S1 Histochemical GUS staining. Plants were vaccum infiltrated with a GUS staining solution, containing 100 mM sodium-phosphate buffer (pH 7.0), 20% (v/v) methanol, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide and 0.3% (v/v) Triton X-100. Blue-coloration of whole plants in different developmental stages was recorded with a bright field microscope (Leica DM 5000) or a magnifying glass (Wild Heerbrugg) coupled to a CCD color camera (Leica DFC 320). GUS stained tissues and plants shown in this paper represent the typical results of at least three independent lines for each construct.

Methods S2 PEG-infused plates. PEG-infused MS agar plates were prepared as follows: under sterility conditions, 20 mL of fused agarised 0.5x MS media were poured into petri plates, left to cool and then covered with 30 mL of PEG or mock overlay solution; plates were covered and the media was allowed to sit for 12-15 h. PEG overlay solution (-0.7 MPa strength) consisted of 0.5x MS basal salt mixture, 1.2 g L-1 MES and 400 g L-1 PEG 8000. Excess overlay solution was poured just before seedling transfer, and immediately sealed with parafilm to avoid water loss. Control plants were transferred to mock-infused 0.5x MS agar plates.

Methods S3 Quantification of real-time stomatal aperture. For the quantification of real-time stomatal aperture, 4-week-old plants were watered the day before the experiment; in the day of the experiment, plants were kept under day-cycle lights for at least three hours; due to the sensitivity of stomata, any type of stress was avoided; abaxial epidermal peels were collected, and immediately placed afloat in buffer (25 mM MES-KOH, pH 6.15, and 10 mM KCl), within a closed Petri dish; peels were immediately observed under a microscope, and data was recorded quickly. A similar procedure was used to analyse real-time stomatal aperture in 2-week-old in vitro-grown seedlings, gown under standard conditions or on media supplemented with 100 mM NaCl or 200 mM mannitol; in the day of the experiment, plants were kept under day-cycle lights for at least three hours; seedlings were immediately placed afloat in buffer (25 mM MES-KOH, pH 6.15, and 10 mM KCl), within a closed Petri dish; cotyledon leaves were observed under a microscope, and data was recorded quickly.

Methods S4 ABA inhibition of stomatal opening. Leaves were detached from the rosette and submerged in a stomata-opening solution (50 mM KCl; 10 μM CaCl2; 0.01% Tween 20; 10 mM MES-KOH pH 6.15) under cool white light (80 µE m-2 s-1) for three hours. Subsequently, 5 µM ABA or mock solution was added to the buffer, and the samples were incubated for one hour under identical light conditions. Epidermal peels were obtained with the help of double-sided adhesive tape, and subsequently stained with a 0.2% (w/v) toluidine blue solution and observed under a light microscope (Leica DM 5000).

Methods S5 SDS-PAGE and Western-blot analysis of SUMO-conjugate levels. Equal amounts of protein were resolved by standard SDS-PAGE, in a 10% (m/v)

acrylamide resolving gel, using a Mini-Protean Cell (Bio-Rad) apparatus. For immunoblotting, proteins were transferred to a PVDF-membrane using a Mini Trans-Blot Cell (Bio-Rad). The membrane was blocked for 1 h at 23ºC in blocking solution (5% dry milk powder in PBST). The primary antibody Anti-AtSUMO1/2 (ABCAM) was added in a 1:2000 dilution and incubated for 3 h. The membrane was washed for 3x10 min with 10 mL of PBST, and incubated with the secondary antibody Anti-Rabbit (Santa Cruz Biotechnologies) in a 1:5000 dilution, in blocking solution for 1 h. The membrane was washed as previously detailed, and developed by chemiluminescence using the Immun-Star WesternC Kit (Bio-Rad) and a Chemidoc XRS System (Bio-Rad) for image acquisition. PVDF membranes were incubated for 15 min with Ponceau S solution [0.1% (w/v) Ponceau S; 5% (v/v) acetic acid] to stain total protein levels.

Methods S6 In silico analysis. Correlation graphs for gene pair expression were generated using the CoexViewer feature at ATTED-II v7.1 (atted.jp/top_draw.shtml#CoexViewer) (Obayashi et al. 2014). Co-expression analysis was performed using GeneMANIA (genemania.org) (Warde-Farley et al. 2010), with default settings for the co-expression network. Syntenic analysis was carried out using the Plant Genome Duplication Database (chibba.agtec.uga.edu). GO term functional categorization (Biological Process) was performed in VirtualPlant 1.2 (virtualplant.bio.nyu.edu/cgi-bin/vpweb). Redundancy exclusion and scatterplot analysis were performed in REVIGO (revigo.irb.hr). For phylogenetic analysis, Arabidopsis ULP homologues were obtained using Saccharomyces cerevisiae ULPs and protein DELTA-BLAST analysis at NCBI (www.ncbi.nlm.nih.gov). SeaView v4 (Gouy et al. 2010) was used to sequentially perform (1) sequence alignment of the a.a. sequences using MUSCLE, (2) tree generation using maximum likelihood (PhyML with the LG model and bootstrap analysis of 1000 replicates).

Supplementary references

Gouy M, Guindon S, Gascuel O (2010) SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27:221-224 doi:10.1093/molbev/msp259

Obayashi T, Okamura Y, Ito S, Tadaka S, Aoki Y, Shirota M, Kinoshita K (2014) ATTED-II in 2014: evaluation of gene coexpression in agriculturally important plants. Plant Cell Physiol 55:e6 doi:10.1093/pcp/pct178

Warde-Farley D et al. (2010) The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res 38:W214-220 doi:10.1093/nar/gkq537