Subject: PP2016-01546D: Decision Letter From: [email protected]Date: 2016/10/13 23:11 To: [email protected]13‐Oct ‐2016 Prof. Takehito Inaba University of Miyazaki Department of Agricultural and Environmental Sciences, Faculty of Agriculture Miyazaki 8892192 Japan RE: Ubiqui Ɵn‐proteasome dependent regulaƟon of GLK1 in response to plasƟd signals in Arabidopsis Dear Dr. Takehito Inaba: We are pleased to accept your manuscript for publicaƟon in the Plant Physiology. This acceptance is conƟngent on revision based on the comments below. In parƟcular, please consider the following: You have convincingly revised the manuscript according to the referees' comments. Two points sƟll need aƩenƟon: You addressed the issue of signi ficant which should only be used in conjuncƟon with a staƟsƟcal test. This is good. But please indicate staƟsƟcal signi ficance of di fference in all figures. Second I ask you to explain GLK1 in the Ɵtle and in the one sentence summary. To submit your revised manuscript, click: hƩp://pphys.msubmit.net/cgi ‐bin/main.plex?el=A5It5Clr5A4DbC1I2A9Ōdk2aho3EXqDh91TNBBLnAQZ If you cannot return the revised manuscript within 8 weeks of receipt, please let us know. Otherwise, we will assume that you have elected not to revise the manuscript and are withdrawing it. Thank you for allowing us to review your work. We look forward to hearing from you soon. Sincerely, Karl ‐Josef Dietz Monitoring Editor, Plant Physiology ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Reviewer comments: PP2016-01546D: Decision Letter imap://mail.cc.miyazaki-u.ac.jp:993/fetch>UID>.INBOX>42406?hea... 1 / 1 2016/10/14 9:37
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GLK1 - miyazaki-u.ac.jp€¦ · Subject: PP2016-01546D: Decision Letter From: [email protected] Date: 2016/10/13 23:11 To: [email protected] 13‐Oct‐2016 Prof. Takehito
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Prof. Takehito InabaUniversity of MiyazakiDepartment of Agricultural and Environmental Sciences, Faculty of AgricultureMiyazaki 8892192Japan
RE: Ubiqui n‐proteasome dependent regula on of GLK1 in response to plas d signals in Arabidopsis
Dear Dr. Takehito Inaba:
We are pleased to accept your manuscript for publica on in the Plant Physiology. This acceptance iscon ngent on revision based on the comments below. In par cular, please consider the following:
You have convincingly revised the manuscript according to the referees' comments. Two points s ll needa en on: You addressed the issue of significant which should only be used in conjunc on with asta s cal test. This is good. But please indicate sta s cal significance of difference in all figures. Second Iask you to explain GLK1 in the tle and in the one sentence summary.
To submit your revised manuscript, click:
h p://pphys.msubmit.net/cgi‐bin/main.plex?el=A5It5Clr5A4DbC1I2A9 dk2aho3EXqDh91TNBBLnAQZ
If you cannot return the revised manuscript within 8 weeks of receipt, please let us know. Otherwise, wewill assume that you have elected not to revise the manuscript and are withdrawing it.
Thank you for allowing us to review your work. We look forward to hearing from you soon.
C. Detection of GFP-GLK1 in chloroplasts. Total protein extracts (lane 1) and 688
chloroplast proteins (lane 2) were resolved by SDS-PAGE and probed with antibodies 689
against GLK1 (top) and Toc75 (middle). As the negative control, the membrane was 690
also probed with an anti-actin antibody (bottom). 691
692
Figure 7. Effects of norflurazon and MG-132 on GFP-GLK1 in the transformed plants 693
A. Effects of norflurazon (NF) on the expression of GFP-GLK1. After growth under 694
normal conditions for 3 days, plants were treated with 1μM NF (+) or dimethyl sulfoxide 695
(DMSO, -) under continuous light for 5 days. The mRNA levels were analyzed by 696
real-time PCR, and expression levels were normalized to that of ACTIN2. The graph 697
shows the average of four independent transgenic lines shown in Supplemental Figure 698
S3. Error bars represent 1 SE of the mean (n=4). 699
B. Accumulation of GFP-GLK1 in the glk1glk2 double mutant transformed with 700
GFP-GLK1. Plants were treated with either 1μM NF or DMSO as described for panel A. 701
Extracted proteins were then resolved by SDS-PAGE, and proteins were probed with 702
antibodies against GLK1 or actin (Supplemental Figure S3B). GLK1 protein levels were 703
41
quantified using image acquisition software and normalized to actin levels. The graph 704
shows the average of four independent transgenic lines shown in Supplemental Figure 705
S3. Error bars represent 1 SE of the mean (n=4). 706
C. Accumulation of poly-ubiquitinated GFP-GLK1 in plants treated with MG-132. 707
Arabidopsis plants expressing GFP-GLK1 were cultured in a liquid MS medium for 2 708
weeks. Plants were then treated with 50μM MG-132 (+) in a liquid culture for 12 hours. 709
As the control, plants were also treated with DMSO (-) for the same duration. Total 710
proteins were extracted from MG-132 or DMSO-treated plants, and GFP-GLK1 protein 711
was affinity purified using monoclonal antibody against GFP. The starting material (1% 712
of the total) and eluates were then resolved by SDS-PAGE, and proteins were probed 713
with monoclonal antibody against the multiubiquitin chain. Actin amounts confirmed 714
equal loading of starting material. 715
716
Figure 8. Effects of MG-132 on GLK1 accumulation in vivo 717
A. Effects of MG-132 on the accumulation of GLK1 in wild-type (WT) and gun1-101 718
plants treated with norflurazon (NF). Plants were treated with DMSO (lanes 1 and 4) for 719
4 days or with NF for 6 days. The NF-treated plants were further treated with additional 720
DMSO (lanes 2 and 5) or 50μM MG-132 (lanes 3 and 6) for 18 hours. Extracted proteins 721
were then resolved by SDS-PAGE, and proteins were probed with antibodies against 722
GLK1 or actin. Note that the exposure time to capture GLK1 signals in WT lanes was 723
longer than that in gun1-101 lanes. 724
B. Effects of MG-132 on the accumulation of GLK1 in ppi2-2 mutant overexpressing 725
GLK1 (GLK1ox ppi2-2). Note that only the plants carrying homozygous ppi2 genotype 726
(ppi2 -/-) exhibit damaged plastids and albino phenotype. The GLK1ox ppi2-2 mutant 727
42
was grown on plates for 7 days. Then, plants were transferred to liquid MS medium. The 728
heterozygous GLK1ox ppi2-2(+/-) plants (green phenotype) were grown in liquid MS 729
medium for 6 days under continuous light and then subjected to DMSO treatment for 18 730
hours. The homozygous GLK1ox ppi2-2(-/-) plants (albino phenotype) were grown in 731
liquid MS medium for 10 days under continuous light and then treated with either DMSO 732
or 50μM MG-132 for 18 hours. Extracted proteins were then resolved by SDS-PAGE, 733
and proteins were probed with antibodies against GLK1 or actin. 734
C. Quantification of GLK1 mRNA and GLK1 protein levels in NF-treated plants (NF) and 735
both NF and MG-132-treated plants (NF + MG-132) shown in A. GLK1 protein levels 736
were quantified using image acquisition software and normalized to actin levels. The 737
mRNA levels were analyzed by real-time PCR, and the expression levels were 738
normalized to that of ACTIN2. The expression levels in WT (left) or gun1-101 (right) 739
plants treated with both NF and MG-132 was set to 1. Error bars shown in mRNA data 740
represent 1 SE of the mean (n=3). 741
D. Quantification of GLK1 mRNA and GLK1 protein levels in the GLK1ox ppi2-2 (-/-) 742
mutant treated with DMSO or MG-132 shown in B. GLK1 protein levels were quantified 743
using image acquisition software and normalized to actin levels. The mRNA levels were 744
analyzed by real-time PCR, and the expression levels were normalized to that of 745
ACTIN2. The expression level in GLK1ox ppi2-2(-/-) plants treated with MG-132 was set 746
to 1. Error bars shown in mRNA data represent 1 SE of the mean (n=3). 747
748
Figure 9. Model for the regulation of GLK1 by multiple mechanisms 749
Both developmental signals and sucrose regulate the accumulation of GLK1 protein 750
through transcriptional regulation of GLK1 gene. Plastid signals derived from damaged 751
43
plastids also regulate the expression of GLK1 through the activity of GUN1. Meanwhile, 752
plastid signals regulate the accumulation of GLK1 at protein level. Although the 753
components involved in this regulation remain to be identified, ubiquitin-proteasome 754
system appears to participate in this regulation. 755
756
757
Supplemental Figure Legends 758
Supplemental Figure S1. Expression of GLK1 fusion protein in E. coli 759
A. Schematic diagram of the construct used for bacterial expression. 760
B. Purification of GLK1-His protein. The purified NusA-TEV-GLK1-His protein was 761
cleaved into NusA (arrowhead) and GLK1-His (arrow) by TEV protease (lane 1). The 762
GLK1-His protein was further purified from the protease treated fraction using 763
preparative gel electrophoresis (lane 2). 764
765
Supplemental Figure S2. Localization of GFP-GLK1 and GFP in Arabidopsis 766
A. Gel-filtration chromatography of GFP-GLK1 proteins in Arabidopsis treated with NF. 767
Total protein extracts from the GFP-GLK1-transformed glk1glk2 mutants treated with NF 768
were resolved by gel-filtration chromatography on a Sephacryl S-300 HR column. The 769
molecular masses of the standard proteins are indicated by arrowheads. Proteins in 770
each fraction were precipitated with trichloroacetic acid and analyzed by immunoblotting 771
with antibodies indicated at the left. The level of GFP-GLK1 in the complemented line 772
was high such that it was detectable even in the plants treated with NF. 773
B. Localization of GFP in Arabidopsis leaf and root cells. Leaf (upper) and root (lower) 774
tissues of transgenic plants expressing GFP in wild-type background were observed 775
44
using a confocal laser-scanning microscope LSM 700. The images were taken as the 776
control of GFP-GLK1 shown in Fig. 6B. GFP, GFP fluorescence; Chl., chlorophyll 777
auto-fluorescence; DIC, differential interference contrast image; GFP+Chl., overlap of 778
the GFP and Chl. images. 779
780
Supplemental Figure S3. Analysis of glk1glk2 mutants complemented with GFP-GLK1 781
gene 782
A. Representative phenotype of the additional glk1glk2 lines transformed with 783
GFP-GLK1 construct. Transformed plants were first grown on MS plates for 2 weeks, 784
transferred to soil and continued to grow for another 2 weeks. Wild-type and glk1glk2 785
plants were grown on MS plates without antibiotics and then transferred to soil. 786
B. Effect of norflurazon (NF) on the accumulation of GFP-GLK1 protein. After growth 787
under normal conditions for 3 days, plants were treated with 1μM NF (+) or dimethyl 788
sulfoxide (DMSO, -) under continuous light for 5 days. Extracted proteins were then 789
resolved by SDS-PAGE, and proteins were probed with antibodies against GFP or actin 790
(top panel). The lower panel shows quantification of GLK1 protein level in each sample. 791
GLK1 protein levels were quantified using image acquisition software and normalized to 792
actin levels. The GLK1 protein level in DMSO-treated plants was set to 1 in each line. 793
C. Effects of NF on the expression of GFP-GLK1. Plants were treated with either 1μM 794
NF (+) or DMSO (-) as described for panel B. The mRNA levels were analyzed by 795
real-time PCR, and expression levels were normalized to that of ACTIN2. The 796
expression level in plants treated with DMSO was set to 1. Error bars represent 1 SE of 797
the mean (n=3). 798
799
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