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Peer Review Report for 10.1105/tpc.16.00728
OsFTIP1-Mediated Regulation of Florigen Transport in Rice Is Negatively Regulated by a Ubiquitinlike Domain Kinase OsUbDKγ4
Shiyong Song, Ying Chen, Lu Liu, Yanwen Wang, Shengjie Bao, Xuan Zhou, Zhi Wei Norman Teo, Chuanzao Mao,
Yinbo Gan, and Hao Yu
Plant Cell. Advance Publication March 2, 2017; doi: 10.1105/tpc.16.00728
Corresponding author: Hao Yu ([email protected])
Review timeline:
TPC2016-00728-RA
Submission received:
Sep. 16, 2016
1st Decision:
Oct. 19, 2016 request revision
TPC2016-00728-RAR1 1st Revision received:
Jan. 4, 2017
2nd Decision:
Feb. 7, 2017 accept with minor revisions
TPC2016-00728-RAR2 2nd Revision received:
Feb. 9, 2017
3rd Decision:
Feb. 10, 2017 acceptance pending, sent to science editor
Final acceptance:
Mar. 1, 2017
Advance publication:
Mar. 2, 2017
REPORT: (The report shows the major requests for revision and author responses. Minor comments for revision and
miscellaneous correspondence are not included. The original format may not be reflected in this compilation, but the
reviewer comments and author responses are not edited, except to correct minor typographical or spelling errors that
could be a source of ambiguity.)
TPC2016-00728-RA 1st Editorial decision – request revision
Oct. 19, 2016
We have received reviews of your manuscript entitled "OsFTIP1 Regulation of Florigen Transport in Rice Is Mediated
by a Ubiquitin-like Domain Kinase OsUbDKγ4." Thank you for submitting your best work to The Plant Cell. The
editorial board agrees that the work you describe is substantive, falls within the scope of the journal, and may
become acceptable for publication pending revision, and potential re-review. We ask you to pay attention to the
following points emphasized by the reviewers in preparing your revision.
As requested by reviewer 1 include more statistical analysis of flowering-time data of the comparison of single and
double mutants; also include data testing whether OsFTP1 interacts with both RFT and Hd3a and discuss these as
requested. Also reviewers 1 and 3 requested more controls and repetition for immunoblots and TEM analysis.
Reviewer 2 requested that you include additional citations and discuss differences with your previously published
work in Arabidopsis. Reviewer 3 stressed the need for higher resolution confocal images and additional controls for
these.
The figures are very well done, but there are a few minor comments on the supplemental materials dealing with fonts
in one figure and in the tables. Please download and view in Acrobat in comment mode.
Please contact us if there are ambiguous comments or if you wish to discuss the revision.
Given the nature of the comments, we are offering you 90 days to complete the revision. If a revision is not returned
within this time frame, and if you have not been granted an extension, we will withdraw the manuscript, which will
leave you free to submit the work elsewhere. If you need an extension, we encourage you to contact us at any point
before submitting your revision.
---------------------------------------------------------------------------- Reviewer comments:
[Reviewer comments shown below along with author responses]
TPC2016-00728-RAR1 1st Revision received
Editor comments [shown in decision letter above] and author responses:
Jan. 4, 2017
Peer Review Report for 10.1105/tpc.16.00728
RESPONSE: We would like to thank the Editors and three reviewers for the time committed in reviewing this
manuscript, and for all the detailed suggestions on improving this manuscript. We have revised the manuscript to
fully address the reviewers’ concerns and criticisms as follows.
Reviewer comments and author responses:
Reviewer #1:
FLOWERING LOCUS T (FT) encodes a mobile protein (called "florigen") that promotes flowering in Arabidopsis.
Recent reports demonstrated that the movement of FT from the companion cells to the sieve elements and from the
sieve elements to the SAM is assisted by at least two other proteins, FTIP1 and NaKR1, respectively. In rice, two
orthologs of the Arabidopsis FT, Hd3a and RFT1, mediate flowering time under short days and long days,
respectively. The mechanisms involved in the transport of these proteins remain unknown, even though similarities
with the Arabidopsis model are predicted. In this paper, Song and colleagues performed an elegant genetic screen of
genome-edited mutants to identify components of the florigen transport machinery in rice. They isolated a rice
ortholog of the Arabidopsis FTIP1 gene (OsFTIP1) and generated new mutant alleles of hd3a and rft1. OsFTIP1 and
RFT1 genes were expressed in the companion cells and their respective mutants flowered late under long days. The
authors also showed that OsFTIP1 interacts with RFT1. Their microscopic and biochemical data suggest that the
movement of RFT1 from the companion cells to the SAM is assisted by OsFTIP1. Moreover, the authors identified an
Unbiquitin-like Domain kinase (OsUbDKy4) that interacts with OsFTIP1 and regulates its protein abundance.
Therefore, the authors propose a model where OsUbDKy4 regulates OsFTIP1 to mediate the movement of RFT1
from the companion cells to the SAM.
The amount of genetic materials produced in this paper by using CRISPR/Cas9 technology is very impressive. The
authors generated new mutant alleles of OsFTIP1, Hd3a, RFT1 and OsUbDKγ4 in Nipponbare cultivar background.
These materials will be very useful for further studies and add an extra value to this study. They also present a series
of genetic, biochemical and microscopic experiments to elucidate how OsFTIP1 and OsUbDKy4 regulate RFT1
movement from the companion cells to the SAM under long day conditions.
Overall, the paper is potentially very interesting for a broad spectrum of readers. Furthermore, it is very well written
and comprehensive. However, I am not completely convinced that the authors interpreted correctly the results from
some of the genetic experiments. In addition, I think that new controls and data quantification should be added to
some of the presented experiments. Finally, I believe that new experiments should be included to clarify the
proposed model. In more detail:
Point 1. A main message of this paper is that OsFTIP1 contributes to the transport of RFT1 from the companion cells
to the SAM. Therefore, these two genes should act in the same genetic pathway. However, the genetic experiments
showed in this paper do not fully corroborate this idea. In Figure 4A, the flowering time of different Osftip1 and rft1
mutant combinations is shown. On average, WT plants flowered after 115 d, Osftip1-1 after 135 d, rft1-1 after 150 d
and the double mutant Osftip1-1 rft1-1 after 165 d. In other words, compared to WT plants, Osftip1-1 delays flowering
by 20 d, rft1-1 by 35 and the double mutant Osftip1-1 rft1-1 by around 50. The interpretation of the authors of this
result is (page 10, line 245): "Interestingly, as Osftip1-1 rft1-1 flowered later than the respective single mutants
(Figure 4A), both proteins may either interact to have a synergistic effect on rice flowering, or act with other unknown
co-regulator(s) involved in the control of rice flowering time". By observing the flowering time data presented in Figure
4A, it seems more realistic to me that the effect of the two mutations is additive. Indeed, the delay in flowering (in
terms of days to flower) shown by the double mutant (50 days) is very similar to the expected delay resulting from the
combination of the two single mutations (20 + 35 days). If this is the case, RFT1 and OsFTIP1 would act in two
independent genetic pathways. Moreover, in Figure 4B, the authors showed the flowering time of the transgenic
plants gRFT1-FLAG (which overexpresses RFT1) compared to the double mutants gRFT1-FLAG Osftip1 (-1 and -2).
In this experiment, Osftip1 mutations delay the flowering of gRFT1-FLAG by 20 d. The interpretation of the authors of
this result is (page 10, line 260): "Both Osftip1-1 and Osftip1-2 suppressed the early flowering phenotype of gRFT1FLAG, while supply of OsFTIP1 activity in Osftip1-1 gOsFTIP1 restored the promotive effect of gRFT1-FLAG on
flowering (Figures 4B), indicating that RFT1 effect on rice flowering is partially dependent on OsFTIP1". This is also
controversial, as the delay caused by Osftip1 mutant alleles in the gRFT1-FLAG background is similar to that
Peer Review Report for 10.1105/tpc.16.00728
observed in the WT background (20 d). In the same line, gRFT1-FLAG causes the same acceleration of flowering in
WT and Osftip1 plants (20 d) (Fig 4A and Supplemental Fig 7B). Therefore, the genetic experiments shown in the
paper might support an independent effect of OsFTIP1 and RFT1 mutations on flowering time in rice. Obviously, this
would argue against the proposed model.
The authors should clarify whether RFT1 and OsFTIP1 act in the same genetic pathway. For this purpose, the
authors might analyze the effect on flowering time of the single, double mutants and their genetic interactions by
using statistical tools (i.e. Two-Way ANOVA).
RESPONSE: This reviewer raised a valid question on the interpretation of the genetic data. As there are 12 FTIP1like MCTPs and 13 FT-like homologs so far found in the rice genome, it is highly possible that there is cross
regulation among these homologs in rice. RFT1 could be regulated by OsFTIP1 and other unknown MCTPs, while
OsFTIP1 may also mediate transport of other FT-like proteins involved in the control of flowering time. This explains
the partially additive genetic effects of Osftip1-1 rft1-1 (which shows statistically significant difference in flowering
time compared with single mutants as presented in Figure 4A). Thus, we have been cautious in interpreting our
genetic data shown in Figures 4A and 4B, and indicated that OsFTIP1 and RFT1 may act independently with other
unknown co-regulator(s) involved in the control of rice flowering time, and that the effect of RFT1 on rice flowering
is partially dependent on OsFTIP1. These descriptions do not exclude the possible independent effects of Osftip1-1
and rft1-1 on rice flowering time as suggested by this reviewer. Our overall findings from molecular, genetic,
biochemical and bioimaging experiments support that OsFTIP1 contributes to RFT1 transport from companion cells
to sieve elements in rice.
Point 2. In the Discussion; Page 15, line 432: "Here we have shown that OsFTIP1, a rice ortholog of Arabidopsis
FTIP1 (Liu et al., 2012), specifically regulates rice flowering time under LDs through modulating RFT1 transport from
companion cells to sieve elements". I have some doubts on how OsFTIP1 can be a specific regulator of flowering
time under long day conditions. Is OsFTIP1 only expressed under long days? If not, and considering that RFT1 and
Hd3a are very similar proteins, does OsFTIP1 interact with RFT1, but not with Hd3a? In my opinion, the answers to
these questions are crucial to understand the role of OsFTIP1 in the long days-RFT1 signaling pathway.
RESPONSE: As Hd3a is the closest homolog of RFT1 in rice, we indeed included this gene in our study. We first
generated hd3a mutants through CRISPR/Cas9-mediated target mutagenesis, and found that hd3a mutants in the
Nipponbare cultivar background displayed normal flowering time under LDs, and only showed the late-flowering
phenotype under SDs (new Supplemental Figures 5A, 5B, 5D, and 5E). These results are consistent with the
observations on Hd3a RNAi lines in the Norin 8 cultivar background (Komiya et al., 2009), demonstrating that Hd3a
is not the major florigen required for rice flowering under LDs. In addition to this major phenotype data presented in
the revised manuscript, we have also tested the protein interaction between Hd3a and OsFTIP1. Although in vitro
GST pull-down assay could detect weak interaction between OsFTIP1-HA and GST-Hd3a, there was no detectable in
vivo interaction between Hd3a and OsFTIP1 in gHd3a-GFP gOsFTIP1-HA plants grown under LDs. Taken together,
these results suggest that Hd3a is not a major target of OsFTIP1 under LDs.
We agree with this reviewer that it will be interesting to study why OsFTIP1 only affects rice flowering under LDs.
OsFTIP1 was ubiquitously expressed in all tissues examined (Figure 2A), and also expressed at almost comparable
levels in leaves under LDs and SDs (new Supplemental Figure 4A). This feature is similar to that of its Arabidopsis
ortholog FTIP1, indicating that their specificity in mediating florigen transport may not lie in changes in gene
expression. This reviewer has provided a reasonable suggestion in the last comment on investigation of the
residues that mediate the interaction between RFT1 and OsFTIP1. We agree that elucidation of this question, along
with several other possible mechanisms (e.g., post-translational modifications of RFT1 and OsFTIP1), will gain
helpful insights into the functional specificity of OsFTIP1 under LDs.
Relevant Reference:
Komiya, R. et al. (2009). A gene network for long-day flowering activates RFT1 encoding a mobile flowering signal in
rice. Development 136: 3443-3450.
Point 3. In order to demonstrate that OsFTIP1 is involved in RFT1 transport from the companion cells to the SAM, the
authors performed a number of biochemical and microscopic experiments. In the Figure 4C, a western blot
experiment is shown where the RFT1-FLAG protein abundance in the SAM of plants of different genetic backgrounds
is compared. How were the SAM of these plants isolated? The authors argued that there is less RFT1 protein
Peer Review Report for 10.1105/tpc.16.00728
accumulation in the SAM of Osftip mutant plants compared to WT. Based on a picture of a single blot, it is difficult to
judge whether there are significant differences in protein accumulation between the studied genotypes. Thus, it is
necessary to show the result of the quantification (and statistics) of various independent blots in order to state any
conclusion from this experiment. The quantification (and statistics) of the blots should be also applied to the
experiments shown in the Figures: 4C, 7B, 7C, 7D and 7E.
RESPONSE: To isolate rice SAMs, we removed all leaves surrounding the SAMs and collected SAMs of around 400
µm in height and 500 µm in diameter under the microscope. We have complemented the above information in the
section of Immunoblotting in ‘METHODS’. All Western blots are representative examples of at least three repeats
with independent biological samples. As suggested by the reviewer, we have performed densitometric analyses of
Western blots using ImageJ, and provided the quantitative data in representative blots (Figures 4C and 7B-7E) and
also the statistical data for all repeats in Supplementary Figure 12.
Point 4. The TEM experiments shown in the Figures 2D and 4E required more controls in order to determine that the
signal detected is specific for the studied proteins. First, in the two experiments, the specificity of the antibodies
employed for the immunolocalization should be tested in WT plants (without tagged proteins). An unspecific binding
of the antibodies to different sub-cellular structures could cause misinterpretation of the results. Additionally, in the
experiment shown in Figure 4E, it is necessary to rule out that free FLAG tag is being detected. For this purpose,
another FLAG fusion protein (for example GFP:FLAG) expressed from the RFT1 promoter should be used as a
control. This will also be useful to verify that the transport of RFT1-FLAG mediated by OsFTIP1 is specific.
RESPONSE: As suggested by the reviewer, we have provided the results showing the specificity of anti-HA and
anti-FLAG antibodies for detecting OsFTIP1-HA and RFT1-FLAG, respectively, in the revised manuscript (new
Supplemental Figure 8A). In addition, we have also examined whether OsFTIP1 affects the movement of free FLAG
protein in ProRFT1:FLAG and ProRFT1:FLAG Osftip1-1 plants. As shown in new Supplemental Figure 8B,
quantitative analyses of free FLAG signals detected by anti-FLAG antibody have revealed that both the number and
frequency of free FLAG signals in companion cells and sieve elements are comparable between wild-type and
Osftip1-1 backgrounds, clearly showing that OsFTIP1 does not affect movement of free FLAG protein in companion
cell-sieve element complexes. This negative control, together with the result showing the effect of OsFTIP1 on
RFT1-FLAG localization in companion cell-sieve element complexes (Figure 4E), suggests that OsFTIP1 specifically
mediates the transport of RFT1-FLAG.
Reviewer #2:
This paper addresses the molecular mechanism controlling flowering time in rice. Little is known about this process
and thus the authors not only address a question of considerable interest for cell and developmental biologists but
also for scientists interested in application. More specifically, the authors investigate the rice ortholog of Arabidopsis
FTIP1. In previous work they showed that Arabidopsis FTIP1 controls flowering time under LD, localizes to
plasmodesmata (PD), binds to FT, and is involved in transporting FT from the companion cell to the sieve element. In
this paper they go on to show that OsFTIP1 functions in a similar fashion in rice. In addition, they identify the kinase
OsUbDKγ4 as a component of LD flowering time control and show that OsUbDKγ4 is a direct negative regulator of
OsFTIP1 by marking OsFTIP1 for degradation. This is a very interesting paper providing sound conclusions that are
based on solid experimental evidence.
Point 1. The weakest results relate to the sub-cellular localization of OsFTIP1 that in part relies on analysis of GFP
fusions in the heterologous N. benthamiana system, which is well known to be prone to artefacts. Immunogold
localization is of course technically challenging and Fig. 2D is probably OK but not that clear. It is particularly
important as the authors do not even mention an important difference between OsFTIP1 and Arabidopsis FTIP1.
According to the published work of the authors, FTIP1 also localizes to PD. This does not seem to be the case for
OsFTIP1. Moreover, the first identified member of the family, QUIRKY (QKY), was reported by the Schneitz lab to
localize to PD where it interacts with a receptor kinase. Moreover, several members of the family were found in the
PD proteome. Thus, it would only be fair to discuss these obvious differences in the paper.
RESPONSE: We agree that this reviewer raised an important point, and have incorporated the relevant discussion in
the text. We have repeatedly examined OsFTIP1-HA localization by immunogold electron microscopy in Osftip1-1
gOsFTIP1-HA. While OsFTIP1-HA signals were consistently found in phloem companion cells, we were unable to
Peer Review Report for 10.1105/tpc.16.00728
detect its localization in plasmodesmata between companion cells and sieve elements as shown for FTIP1 in
Arabidopsis. There could be the following two main reasons among various ones that contribute to this difference.
First, compared to plasmodesmata between bigger cells (e.g., xylem vascular parenchyma cells and parenchyma
cells), it was practically very difficult to identify plasmodesmata between small companion cells and sieve elements
by transmission electron microscopy in rice. On very few occasions when we could identify plasmodesmata
between companion cells and sieve elements on the sections, we did not observe OsFTIP1-HA signals. This very
small number of negative observations did not allow us to conclude OsFTIP1-HA localization in plasmodesmata
between companion cells and sieve elements because of the dynamic nature of its localization. Second, OsFTIP1HA may indeed not localize in plasmodesmata between companion cells and sieve elements in rice. This could
indicate that regulation of RFT1 transport by OsFTIP1 may also involve some rice-specific features that are different
from those in Arabidopsis.
Point 2. Fig. 4C: The authors should present a quantification of the result.
RESPONSE: As suggested by the reviewer, we have performed densitometric analyses of Western blots using
ImageJ, and provided the quantitative data in representative blots (Figures 4C and 7B-7E) and also the statistical
data for all repeats in Supplementary Figure 12.
Point 3. Fig. 5C: Overexpression in protoplasts may lead to artefacts. If possible please provide data from transgenic
rice lines (as the authors did for OsFTIP1/RFT1 interaction). At the very least don't call it „in vivo" (more like „can
interact in plant cells”).
RESPONSE: As we did not produce these transgenic rice lines, we have deleted “in vivo” as suggested by this
reviewer.
Reviewer #3:
In the manuscript titled "OsFTIP1 Regulation of Florigen Transport in Rice Is Mediated by a Ubiquitin-like Domain
Kinase OsUbDKγ4" Shiyong Song et al. describe the identification of the rice FT (RFT1) interacting protein OsFTIP
(an ortholog of the Arabidopsis FTIP1) in rice. Twelve rice FTIP-like genes were mutagenized by the CRISPR/Cas
system and tested for a delayed flowering time phenotype under long days. Two mutant alleles named Osftip1.1 and
Osftip1.2 (genomic T insertion and T deletion, respectively) and independent siRNA producing constructs revealed
that the FTIP1 rice ortholog plays an important role in inducing long-day flowering. In addition, rice rft1 and hd3a
mutant lines were established and used to show that RFT1 but not the FT-related Hd3a plays a role in long-day floral
induction. These observations are consistent with previous reports that in rice the interacting OsFTIP1 regulates
phloem transport or availability of FT to be delivered to the shoot apex where it induces the flowering program (as
reported for A. thaliana FT and FTIP1).
According to qRT-PCR, both OsFTIP1 and the newly identified OsFTIP1 interacting UbDKγ4 are expressed in roots,
seeds, leaves, and panicles. With promoter GUS constructs the authors also showed UbDKγ4 expression in phloem
tissue. The interactions of OsFTIP1 with RFT1 and with UbDKγ4 were revealed by Y2H and CoIP assays, and with
OsFTIP and RFT1 by BiFC assays (see also comments below). A very interesting and novel aspect is that the
authors presented data indicating interaction of a ubiquitin-like domain kinase γ4 (OsUbDKγ4) with rice FTIP1. Again
rice mutants were made using the CRISPR/Cas system to establish that UbDKγ4 plays a role in flower initiation as
they flower earlier. Furthermore, hd3a mutant lines were established and used to show that RFT1 but not the FTrelated Hd3a plays a role in long-day flower induction.
The presented data point to a negative regulatory function of the OsUbDKγ4 kinase onto FTIP, which in turn
attenuates FT presence in the sieve elements and flowering. That RFT1 presence increased by OsFTIP1 in the
sieve elements affects flowering was shown by genetic as well as by immunogold EM localization experiments on
transgenic plants. OsUbDKγ4 interaction with OsFTIP1 seems to delay flowering as mutant plants lacking or
overexpressing OsUbDKγ4 flower earlier or later. Also some, although weaker, evidence is presented indicating that
plants lacking OsUbDKγ4 activity have higher transgenic expressed OsFTIP1 protein levels. Here it seems that
OsFTIP1 presence can be increased by MG132 treatment pointing to degradation of both OsFTIP1 and RFT1 by
proteasomes. In summary, these data suggest that OsUbDKγ4 down-regulates OsFTIP1 and RFT1 protein levels
Peer Review Report for 10.1105/tpc.16.00728
and by this means delays flowering.
In general the presented data are very interesting and consistent with the proposed functions (except some protein
localization and stability data, see comments below), and are well analyzed, and novel. The genetic analysis together
with the interaction assays is an important step towards understanding the factors regulating flower timing in
monocots.
Point 1. The MG132 treatment experiments (Figure 7 B-E) together with the OsFTIP1 and RFT1 protein stability
assays are not as convincing as the genetic experiments. First, the relative detected protein bands seen on the
western blots are not measured and calculated in relation to the loading (detected Tubulin) reference. Second, the
experimental conditions such as the MG132 concentration(s) used are not mentioned in the submitted manuscript
text as well as the method text describing the "immunoblotting" is missing. Furthermore, in general it is not described
what detection system was employed in the western-blot assays presented in the various figures. Here, if a
chemiluminescence detection system was used, the detected protein bands could have been relatively easily
quantified.
RESPONSE: As suggested by the reviewer, we have performed densitometric analyses of Western blots using
ImageJ, and provided the quantitative data in representative blots (Figures 4C and 7B-7E) and also the statistical
data for all repeats in Supplementary Figure 12. In addition, we have provided the requested experimental
conditions, such as MG132 concentrations (Figures 7C and 7D legend) and the chemiluminescent detection system
for immunoblotting (METHODs), in the revised manuscript.
Point 2. Figure 4 B: Osftip1 phenotype can be compensated by RFT1 genomic construct and ftip1 RFT1-FLAG plants
flower at the same time as the wild type suggesting a dosage effect. This was confirmed with MADS14 and MADS15
RNA flower markers as they are reduced in mutant Osftip1.1 plants in the presence of additional RFT1 (gRFT1
transgenic). Here the wild-type control is missing in the qRT experiment and in case the data are available they
should be included.
RESPONSE: We have included the quantitative real-time PCR data for wild-type plants in revised Figure 4D.
Point 3. Figure 2 C and Figure 3A and E.
The colocalization of GFP-tagged OsFTIP1 with the ER-RFP marker is not clearly noticeable.
It seems that both localize around the nucleus but the RFP tagged ER at the periphery seem to lack co-localization
with GFP-tagged OsFTIP1. Here the RFP-tagged ER seems to aggregate (an indication of prolonged laser exposure)
and these aggregates do not seem to coincide with the GFP tagged OsFTIP1. Also, a relatively high green signal
resembling cytosolic strands seems to be present. Here higher quality CLSM images would be preferable to clarify
whether OsFTIP1 is actually associated with ER structures or is present in both the cytosol and the ER. Notably,
Supplemental Figure 6 showing co-localization is more convincing. However, the red channel image has a different
resolution (is pixelated) as the merged or green channel CLSM images. This should be corrected and better highresolution images should be provided in the supplement.
RESPONSE: As suggested by the reviewer, we have replaced Figures 2C, 3A, 3E, and Supplemental Figure 6 with
high-resolution images so that colocalization between various GFP and RFP signals could be clearly identified.
Colocalization of GFP-OsFTIP1 and ER-RFP was intensive as shown in new Figure 2C.
Point 4. Similarly and as mentioned above, Figure 3A does not clearly show whether this is the ER. Also, controls are
missing showing whether FT alone is also associated with the ER. In other words, is FT localized to the ER
depending on the FTIP? This should be clarified by independent transient expression assays. Regarding the BiFC
constructs a proper control would be the combination of untagged (empty) nEFP and cYFP expression and
occurrence of BiFC.
RESPONSE: To substantiate that both OsFTIP1 and RFT1 are colocalized on ER, we have provided two sets of new
data. First, we have included the ER-RFP marker in BiFC analysis of the interaction between OsFTIP1 and RFT1. As
shown in new Figure 3E, the reconstituted EYFP signals were clearly colocalized with ER-RFP. Second, as
suggested by the reviewer, we have also performed independent transient expression assays to reveal the
colocalization of ER-GFP and RFT1-RFP on ER (new Supplemental Figure 6, upper panel), confirming that RFT1
Peer Review Report for 10.1105/tpc.16.00728
localization on ER is independent of OsFTIP1. In addition, we have also included empty nEFP and cYFP expression
constructs in our BiFC analysis (new Figure 3E) as suggested by this reviewer.
Point 5. Figure 3 D legend: Information regarding the lower panel (GST and GST-RFT1 IP) is missing. I assume that
an anti-GST AB was used to confirm the presence of GST in the IP experiment.
RESPONSE: We have included the requested information in the corresponding figure legend.
Point 6. Figure 5 F: The co-localization of OsUbDKγ4 with the RFP ER marker as mentioned in the results text (Line
326 "....revealed substantial colocalization") is not evident in the presented CLSM images. The presented green
channel image specifies a cytosolic distribution of the OsUbDKγ4 fusion rather than an ER association. To confirm
the supposed ER localization of OsUbDKγ4 I would suggest to co-express a cytosolic RFP marker and to present
comparative close-up images of the cell cortex where a typical ER mesh tagged by the GFP fusion should be easily
detected.
RESPONSE: We have replaced Figure 5F with high-resolution images so that colocalization of OsUbDKγ4-GFP and
ER-RFP could be clearly identified.
Point 7. One important finding is that OsFTIP1-HA functions and present in phloem companion cells, but not in sieve
elements (Figure 2D). This points to a highly conserved mechanism for FT transport in rice compared to A. thaliana.
Such aspects (differences / similarities) in the rice FT pathway should be addressed in more detail in the discussion
text. For example it should be mentioned in the discussion that the expression pattern of OsFTIP1 seems to be
different from that observed with FTIP1. Also I find it intriguing why OsFTIP1 is expressed in the roots. Does this
point to a more pleotropic function in phloem development as observed with the FT interacting NaKR1? Another
aspect is that one could argue that the OsFTIP1 - UbDKγ4 complex regulates the presence of FT protein (levels) in
the companion cells and thus, not necessarily mediates the transport of FT into the sieve tube but controls the
availability of mobile FT. This should be discussed/mentioned.
RESPONSE: As suggested by the reviewer, we have incorporated the discussions relevant to OsFTIP1 expression
patterns in ‘Discussion’. In addition, we have summarized the relationship among OsUbDKγ4, OsFTIP1, and RFT1 in
the first paragraph of ‘Discussion’. OsUbDKγ4 interacts with OsFTIP1 and modulates the protein stability of
OsFTIP1, which in turn regulates the movement of RFT1 from the companion cells to the sieve elements.
TPC2016-00728-RAR1 2nd Editorial decision – accept with minor revision
Feb. 7, 2017
We have received reviews of your manuscript entitled "OsFTIP1 Regulation of Florigen Transport in Rice Is
Negatively Regulated by a Ubiquitin-like Domain Kinase OsUbDKγ4." On the basis of the advice received, the board
of reviewing editors would like to accept your manuscript for publication in The Plant Cell. This acceptance is
contingent on revision based on the comments of our reviewers. In particular, please consider the following:
I have now received three reviews on the revised version of your manuscript from the same reviewers who assessed
the first version. I am pleased to inform you that all reviewers found your manuscript improved and suitable in
principle for publication in The Plant Cell. Reviewer 1 found the manuscript acceptable in this form. However,
reviewer 2 and 3 asked for textual revisions. Reviewer 3 asks for correction of a label on a figure and to a figure
legend, which I am sure you can quickly do. Reviewer 2 raises again the issue of plasmodesmata localization of
OsFTIP1 and asks that your discussion of the difference with FTIP1 is discussed more clearly, and more specifically
that you mention other examples of the QKY family and their localization. I would ask you to consider both of these
issues and to revise your manuscript as required. Furthermore, your manuscript will be checked for the statistical
analysis of data. Most of the legends appear to have all of the required information. However, in the legend of Figure
4E or in the figure itself it would be important to state in how many CC and SE cells gold particles were counted in
both genotypes.
---------------------------------------------------------------------------- Reviewer comments:
[Reviewer comments shown below along with author responses]
TPC2016-000728-RAR2 2nd Revision received
Feb. 9, 2017
Peer Review Report for 10.1105/tpc.16.00728
Editor comments [shown in decision letter above] and author responses:
RESPONSE: We would like to thank the Editors and three reviewers for the time committed in reviewing this revised
manuscript, and for the suggestions on further improving this manuscript. We have revised the manuscript to fully
address all the issues raised by reviewers as follows.
Reviewer comments and author responses:
Reviewer #1:
In the revised version of the manuscript the authors have properly addressed my previous concerns. In my opinion,
the manuscript does not require any further improvement.
Reviewer #2:
Point 1. lines 186–194: The issue regarding the PD localization of Arabidopsis FTIP1 versus OsFTIP1 has been
discussed. However, it is still not clear to me whether the authors think that the failure to reveal PD localization of
OsFTIP1 relates to a true mechanistic difference between FTIP1 and OsFTIP1 or whether they think it simply a
matter of experimental issues. The little evidence that they have at present argues against a PD localization of
OsFTIP1. The authors also suggest a „dynamic nature" of OsFTIP1 sub-cellular localization. However, they do not
show any evidence for this statement.
In light of this issue, I still find it irritating that the authors completely ignore the work of others on the PD-localization
of QKY (after all the canonical member of this gene family) or the likely PD-localization of yet other Arabidopsis family
members. It provides for an unbalanced and unfair discussion regarding how members of this gene family function at
the cellular/biochemical level. In my opinion the authors should just openly discuss the PD issue and state that it
remains to be resolved in rice.
RESPONSE: As suggested by this reviewer, we have clearly stated that “unlike the localization of FTIP1 and its
homolog QUIRKY in Arabidopsis plasmodesmata (Fulton et al., 2009; Liu et al., 2012; Vaddepalli et al., 2014), we
were unable to detect OsFTIP1-HA in plasmodesmata between companion cells and sieve elements in rice.” In
addition, we have discussed the reasons why we were unable to conclude OsFTIP1-HA localization in
plasmodesmata, and stated that this remains to be resolved in rice.
Relevant References:
Fulton, L., Batoux, M., Vaddepalli, P., Yadav, R.K., Busch, W., Andersen, S.U., Jeong, S., Lohmann, J.U., and
Schneitz, K. (2009). DETORQUEO, QUIRKY, and ZERZAUST represent novel components involved in organ
development mediated by the receptor-like kinase STRUBBELIG in Arabidopsis thaliana. PLoS Genet 5, e1000355.
Liu, L., Liu, C., Hou, X.L., Xi, W.Y., Shen, L.S., Tao, Z., Wang, Y., and Yu, H. (2012). FTIP1 is an essential regulator
required for florigen transport. Plos Biol 10, e1001313.
Vaddepalli, P., Herrmann, A., Fulton, L., Oelschner, M., Hillmer, S., Stratil, T.F., Fastner, A., Hammes, U.Z., Ott, T.,
and Robinson, D.G. (2014). The C2-domain protein QUIRKY and the receptor-like kinase STRUBBELIG localize to
plasmodesmata and mediate tissue morphogenesis in Arabidopsis thaliana. Development 141, 4139-4148.
Reviewer #3:
The authors addressed all main points that were raised and the manuscript improved in such that higher quality
confocal images (ER co-localization of the constructs) and additional controls and quantitative data such as wild-type
qRT-PCR, protein detection (e.g. Suppl. Figure 12) and BiFC controls (Figure 3E) were provided.
TPC2016-00728-RAR2 3rd Editorial decision – acceptance pending
Feb. 10, 2017
We are pleased to inform you that your paper entitled "OsFTIP1 Regulation of Florigen Transport in Rice Is
Negatively Regulated by a Ubiquitin-like Domain Kinase OsUbDKγ4" has been accepted for publication in The Plant
Cell, pending a final minor editorial review by journal staff.
Final acceptance from Science Editor
Mar. 1, 2017