A subclass of HSP70s regulate development and abiotic stress responses in Arabidopsis thaliana

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1 Journal of Plant Research A subclass of HSP70s regulate development and abiotic stress responses in Arabidopsis thaliana Linna Leng 1 Qianqian Liang 1 Jianjun Jiang 1 Chi Zhang 1 Yuhan Hao 1 Xuelu Wang 2 Wei Su 1,* 1 State Key Laboratory of Genetic Engineering and Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai , China 2 College of Life Science and Technology, Huazhong Agricultural University, Wuhan , China These authors contributed equally to this work *Correspondence: weisu@fudan.edu.cn Supplementary materials Fig. S1 Fig. S2 Fig. S3 Fig. S4 Fig. S5 Fig. S6 Fig. S7 Table S1 Table S2 Materials and methods Confocal microscopy

2 24 Supplementary figures!!"#$%&'&()#!"#$%&*&()#!"#$%&+&()#!"#$%&,&()#!"#$%&-&()# " # $ Fig. S1 Subcellular localizations of HSP70-1 to HSP70-5 in Nicotiana benthamiana pavement leaves. Microscope images of Nicotiana benthamiana leaves transient expressing HSP70-1-GFP to HSP70-5-GFP were stained with DAPI. (a d) The image of DAPI channel (a), GFP channel (b), bright field (c), Merge (d) showed that the HSP70-1 to HSP70-5 proteins were located both in cytosol and nucleus. Bar = 100 µm. 30 2

31!"#$%!"#$%&' ()*$%&'+&'%(,-./)&*+(,-./)&'%( 32 33 34 35 36 Fig. S2 HSP70-5 gene expression in wild type and hsp70-5 mutant. The TUB2 gene was used as the quantitative control.

3 31!"#$%!"#$%&' ()*$%&'+&'%(,-./)&*+(,-./)&'%( Fig. S2 HSP70-5 gene expression in wild type and hsp70-5 mutant. The TUB2 gene was used as the quantitative control. The amplified products of HSP70-5 are 1,941 bp (full length of HSP70-5 CDS) and that for TUB2 are 664 bp (741 1,304). PCR products amplified for 30 cycles were used to detect the expression of HSP70-5, 25 cycles and 30 cycles for TUB

38 39 40 41 42 43 Fig. S3 Genotype identification of hsp70-1/4 and hsp70-2/4/5 mutants. Genomic DNA was used as a template for genotyping PCR.

4 Fig. S3 Genotype identification of hsp70-1/4 and hsp70-2/4/5 mutants. Genomic DNA was used as a template for genotyping PCR. L, left primer; R, right primer; T, T-DNA primers (LBb1.3 for hsp70-1 to hsp70-4, LB1 for hsp70-5); M, marker. The mutant hsp70-1/4 was hsp70-1 -/- hsp70-3 +/- hsp70-4 -/- and mutant hsp70-2/4/5 was hsp70-2 -/- hsp70-3 +/- hsp70-4 -/- hsp70-5 -/- 44 4

5 45 & &'()*+,-.,/012,.-+,340562,6-,*56*+,)-46758,2608*, $" $! #" #! "! 9-4:! %%%!"#$%&'()* %%%!"#$%&+()(,* " # &'()*+,-.,/012,.-+,340562,6-,*56*+,.4-<*+758,2608*, ;! $! #!! 9-4:!!"#$%&'()* %%% %%%!"#$%&+()(,* &'()*+,-.,/012,.-+,340562,6-,*56*+,)+05=>758,2608*, ;! $! #!! 9-4:!!"#$%&'()! %%% %%%!"#$%&+()(,! $ %?>*,4*586>,-.,6>*,2747@'*2, A=(B #C$!CE!CD!C! 9-4:!!"#$%&'()* %%% %%%!"#$%&+()(,*?>*,<7/6>,-.,6>*,26*(2,A((B #C!!CE!CF!CD!C$!C! 9-4:!!"#$%&'()* %%% %%%!"#$%&+()(,* Fig. S4 Statistical analyses of developmental phenotypes of the hsp70-1/4 double mutant and hsp70-2/4/5 triple mutant. (a c) Both mutants showed developmental phenotypes of accelerating bolting (a), flowering (b) and branching (c). (d) The siliques of both mutants were shorter than Col fully expanded siliques from 6 individual plants (5 siliques from each plant) were measured. (e) The stems were thinner in both mutants than Col-0. Fifteen stem segments (5 cm long) excised from the base of the main stem were measured. Student s t-test was used to determine significance. ***P <

6 54! hsp70-1/4 hsp70-2/4/5 Col-0 " # $ % Col-0 hsp70-1/4 hsp70-2/4/5 & Col-0 hsp70-1/4 hsp70-2/4/5 55 6

7 Fig. S5 phenotypic images of the hsp70-1/4 double mutant and hsp70-2/4/5 triple mutant. (a) Both mutants began bolting, but the Col-0 did not. (b) The image was captured when the Col-0 began bolting. (c) Both mutants began flowering and branching, but the Col-0 did not. (d) The image was captured when the Col-0 began flowering and branching. The images were taken in (a) 17d, (b) 20 d, (c) 22 d, (d) 25 d of the plants. (e) The siliques were shorter in hsp70-1/4 mutants and hsp70-2/4/5 mutants than Col-0. (f) The stems were thinner in hsp70-1/4 mutants and hsp70-2/4/5 mutants than Col-0. 7

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9 Fig. S6 The single HSP70 overexpression lines showed similar sensitivities to salt, osmotic stress, and high glucose stress as well as exogenous ABA treatment as compared with the wide type. (a, b) The images of primary roots of the five HSP70 overexpression lines and the wild type in primary root growth inhibition assays. (c-f) Statistical data for relative primary root lengths showed in (a) and (b). The relative primary root lengths of plants treated with 100 mm NaCl (c), 300 mm mannitol (d), 3 % glucose (e), and 10 µm ABA (f) compared with those of plants grown on normal 1/2 MS plates or mock. Values are means ± SE from three independent experiments (n > 15). Student s t-test was used to determine significance. *P < 0.05; ***P <

10 73 74 Supplementary table Table S1 Sequencing of the qrt-pcr products of 5 individual HSP70s genes Gene Name Locus Sequencing of the q-rt PCR amplified products Alignment site cdna HSP70-1 At5g02500 GACCTCTCTCTTTCTCCTATCTCTATCTCTTTT ACTTGCTTTTTTTTGATCTGTTAAGACTTTTTA TGTTGGGCTTTTTTAAAGAAGCCCATTTTGTG GTGTTTTTTGGTTAGTACTATTTTGAACAATG GTTGGTTCTATACCAGTTTAGCTACGATGACG GATAAAATT HSP70-2 At5g02490 GTGTTCTCTTAGTTATTTTGTCTTTTTATTTGA ACTCTCTCAATTTAGTGTTGGATTTTATGTTCT ACATTTTCTTTGATCTAAGTACTTTCTCTCTAT TGTTTGGTCATTGGTGACCCCTTTCTCTTAAC AATGCTTTAGTGGTCACTTGGTAGAAAGCTTT GTTATGAAAATTTCTTTACATGTCAGATTCAC ATAAG HSP70-3 At3g09440 TAACTTTCTCTCTTACTCTCTTACTCTCAGTCT TTATGTGTTTGTATTTCAACATTTTCCTGTTTT GTCCCCTAGTTTTTTTTTTCTTTTTCTTTCTTGT ATTGACTCTATTTTGAGGGCTCGCTTGTTTCG ATGAGCTCCTTATTTTTTTTAATCTATAACAG GAATGTTTTGA HSP70-4 At3g12580 GAGATTCTAGTTGGTTTCTTGTTCTTAGTTTTA TCTTTCTATGTCACTCTGAAACTGGTGTGTGA TCATTTTGATGCTTTAAGAATTTAGCTTTAC HSP70-5 At1g16030 AGCGTGTTTAGTGTGTTAAGACGAAAGAGTG GCTTTGGACCATTAGCGAGTCTTTTCTTTGTA TTGTGTCAAATAGTGTTGTGTACTCATAGGTG TTTTGCTAGTGAACGATAGTTGTATGCTTTAC ATATTTCAGCTGTTTCAGTTGTT

11 77 Table S2 The sequences of primers used in this study Purpose Name Primer sequence Hsp70-1-GFP Hsp70-1(BamHI)-F CGGGATCCATGTCGGGTAAAGGAGAAGG Hsp70-1(XbaI)-R GCTCTAGAGTCGACCTCCTCGATCTTAGG Hsp70-2-GFP Hsp70-2(KpnI)-F GGGGTACCATGGCTGGTAAAGGAGAAGGTCC Hsp70-2(XbaI)-R GCTCTAGATTAGTCGACTTCCTCGATCTTGGG Hsp70-3-GFP Hsp70-3(BamHI)-F CGGGATCCATGGCTGGTAAAGGAGAAGGTC Hsp70-3(XbaI)-R GCTCTAGAGTCGACTTCCTCAATCTTGGG Hsp70-4-GFP Hsp70-4(KpnI)-F GGGGTACCATGGCGGGTAAAGGTGAAGGT Hsp70-4(XbaI)-R GCTCTAGAATCAACTTCTTCAATCTTTGGGC Hsp70-5-GFP Hsp70-5(KpnI)-F GGGGTACCATGGCGACGAAATCAGAGAAAG Hsp70-5(XbaI)-R GCTCTAGAATCCACCTCTTCGATCTTGGG ProHsp70-1:GUS phsp70-1(psti)-f AACTGCAGAGTTATCGGGTATTTGAGAAAAAA phsp70-1(bamhi)-r CGGGATCCTTTTATCGGAAGATTTGGAAACTA ProHsp70-2:GUS phsp70-2(psti)-f AACTGCAG GCGGTGCTGGACCTAAGATC phsp70-2(bamhi)-r CGGGATCCTATCACACGAAGATAGAAAAAGCTA ProHsp70-3:GUS phsp70-3(psti)-f AACTGCAGGATCAAGATTGTGGTCAGGTAAGA phsp70-3(bamhi)-r CGGGATCCTGTTAACGCTACTCAGGATTAAGC ProHsp70-4:GUS phsp70-4(psti)-f AACTGCAGTACGAAGCCACTTGAGTGATGAT phsp70-4(bamhi)-r CGGGATCCTATTAGAGATCAGAATTGTTCGCC ProHsp70-5:GUS phsp70-5(kpni)-f GGGGTACCCATCTTTTAAGTCATGGACACTG phsp70-5(saci)-r CGCGAGCTTGTTGCTAAAAAAAAGCTTCAG Genotyping Hsp70-1-F AAGGAGAAGGACCAGCTATCG Hsp70-1-R TCTTCGCTCTCTCACAGGAAG Genotyping Hsp70-2-F GGTACGACGTACTCTTGCGTC Hsp70-2-R CTCAATCTCCTGAGGGCTCTC Genotyping Hsp70-3-F GTCCACTTTGCATGCTTCTTC Hsp70-3-R ATCATCGCTATGGATCTACCG Genotyping Hsp70-4-F CCAAATACGAAGCCACTTGAG Hsp70-4-R TACCGAAGACGGTGTTGGTAG Genotyping Hsp70-5-F TCCTAACGAACATGTTCTCCG Hsp70-5-R AGGTCCAGGATCTTCTGCTTC Genotyping SALKLBb1.3 ATTTTGCCGATTTCGGAAC Genotyping SAIL LB1 GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC qrt-pcr Hsp70-1-F TTGTTGGACATTGACCTCTC Hsp70-1-R GGCAAACTTTTAATTTTATCCG qrt-pcr Hsp70-2-F GTGTTCTCTTAGTTATTTTGTC 11

12 Hsp70-2-R CTTATGTGAATCTGACATGTAA qrt-pcr Hsp70-3-F TAACTTTCTCTCTTACTCTCTT Hsp70-3-R CAAAACATTCCTGTTATAGATTA qrt-pcr Hsp70-4-F GCCTTTTGGCTTTTGTTTACT Hsp70-4-R AACGGTAAAGCTAAATTCTTAAA qrt-pcr Hsp70-5-F AGCGTGTTTAGTGTGTTAAGA Hsp70-5-R AACAACTGAAACAGCTGAAATA qrt-pcr FLC-F CAAATGTCAAAAATGTGAGTATCGAT FLC-R TAAGGTGGCTAATTAAGTAGTGGGAG qrt-pcr FT-F GCTACAACTGGAACAACCTTTGGCAAT FT-R CCTCTGACAATTGTAGAAAACTGCG qrt-pcr SOC1-F GAGAAAAGTGTCAAATGTATTCGAGC SOC1-R ATTTGACCAAACTTCGCTTTCATG qrt-pcr LFY-F CCAAGTATTCAGGTACGCGAAGA LFY-R AAGCCTGACGCCATGAGCCAA qrt-pcr ARR5-F GTTTTGC GTCCCGAGATGT TAGATA ARR5-R AGCTGCGAG TAGATATCA TTAGCTT qrt-pcr ARR6-F CAAATTCCGTGACTGGATCTTAG ARR6-R GGCGAGAATCATCAGTGTAGG qrt-pcr ARR15-F CTGCTTGTAAAGTGACGACTGTTG ARR15-R AGTTCATATCCTGTTAGTCCCGGC qrt-pcr DWF4-F CATTGCTCTCGCTATCTTCTTC DWF4-R GACTCTCCTAGTTCCTTCTTGG qrt-pcr CPD-F TTACCGCAAAGCCATCCAAG CPD-R TCCATCATCCGCCGCAAG qrt-pcr DET2-F ACTACGAAGACGGAAACTGG DET2-R TCCTTGAACTTGGCAATGTA qrt-pcr IAA6-F CCATAAAGTTTGAAAACCGTTGAG IAA6-R ACGACCAGTAAGGAAATACATTTG qrt-pcr GH3-1-F TTTCACTGTGGGATCTCTGTG GH3-1-R GCATTGATTTCCATCAAAGTAGAC qrt-pcr GH3-5-F TCTCCAACCAACTCTCATCAC GH3-5-R TGCAATTTCACGTTGCTTATAC qrt-pcr CBF1-F CTTGAAAAAGAATCTACCTGAAAAGA CBF1-R TCTCCGCCTTGAGGCTCGTAAT qrt-pcr CBF2-F CCTGAATTAGAAAAGAAAGATAGATAGAG CBF2-R TGTAATCACCGCCTGAGGAAA qrt-pcr CBF3-F GAGAGAAACTATTATTTCAGCAAACC 12

13 78 79 CBF3-R CTGCTCGCAAGCGTCGGAATA qrt-pcr RAB18-F TTGTAACGCAGTCGCATT RAB18-R GATGCTCATTACACACTCATG qrt-pcr RD20-F TTTGGACCTTACTCATAAACTTAGC RD20-R TTAGTCTTGTTTGCGAGAATTGGC qrt-pcr RD22-F ACCATTGAGGAGTGTGAAGCCAG RD22-R CTAGTAGCTGAACCACACAACATGAG qrt-pcr RD29B-F AAGAACG TCGTTGCCTCA RT-PCR RD29B-R TUB2-F TUB-R GCCCGTA AGCAGTAACAG CTCTGACCTCCGAAAGCTTGC TCACCTTCTTCATCCGCAGTT 13

14 Material and methods Confocal microscopy To investigate the subcellular localizations of the GFP-tagged HSP70-1, -2, -3, -4, and -5 proteins. Agrobacteria transformed by HSP70s-GFP constructs were injected into the leaves of Nicotiana benthamiana and grown for another 2 3 d. The leaves were incubated in buffer containing 2 g ml -1 DAPI for 30 min at room temperature and were washed 3 times by sterile water containing 0.1 % triton. GFP fluorescent signals (excited at 488 nm) and DAPI signals (excited at 359 nm) were analyzed with a Leica SP8 laser confocal microscope. Images of Nicotiana benthamiana leaves were captured with a 20 objective and zoomed in to show the details. 14

Supplementary Fig 1. The responses of ERF109 to different hormones and stresses. (a to k) The induced expression of ERF109 in 7-day-old Arabidopsis

Supplementary Fig 1. The responses of ERF109 to different hormones and stresses. (a to k) The induced expression of ERF109 in 7-day-old Arabidopsis seedlings expressing ERF109pro-GUS. The GUS staining