Response to reviewers with comments on Cserhati et al.

paper, “Core cell cycle regulatory

genes in rice and their expression profiles across the growth zone of the leaf”

First of all, I would like to thank the reviewers’ valuable comments, which we think allowed us to
significantly improve the quality of our manuscript. We have carefully considered all criticisms and most
of them led to significant changes to our paper. Below we provide a detailed list of our response to each
of these comments. We hope that by these improvements the manuscript is now acceptable for
publication.

Response to Reviewer #1:

1. Comment: “In Figure 5 and Figure 6, we can recognize that both segment 1 and segment 2 are
cell division zone, but segment 3 is cell expansion zone, and cell maturation zone is located from
segment 4 toward the tip of the leaf. In addition to the authors described that “cell division
activity is restricted to the basal 2 cm of the leaf and that no endoreduplication occurs in this
species (Figure 5).” in p10, it has been reported that polyploid cells are found only in the
endosperm in rice (Endo et al., Plant J. 69: 967-977. 2012). However flow histograms showed 4C
fractions in the region from segment 3 to segment 6, which represents cell expansion and
maturation zones in Figure 5. Like maize, although the rice leaf consists of both dividing and
elongating cells in cell transition zone, cells stop elongating and attain their mature cell length in
cell maturation zone. Therefore, I don’t understand why there are still present 4C fractions in
cell maturation zone if cell maturation zone truly starts at 4 cm apart from the leaf base. I would
like you to explain this in the text. It would be also required to measure epidermal cell length
profiles of cell files along the proximal-distal axis of the rice leaf. This may help to assign cell
elongation and maturation zones in the rice leaf.”

Response: This is indeed an interesting point worth discussing. The following paragraph has
been added to the first paragraph of page 11: “​The lengths of the cells along the leaf blade
follow a characteristic sigmoidal pattern from the base to the mature zone, where the transition
from division to expansion and from expansion to mature cells are located around 2 and 4 cm
from the base, respectively (Figure 5C). ​Note that a fraction of the mature cells is in the 4C state
although they no longer divide or endoreduplcate. It is not possible to tell if these 4C cells are
arrested in G2 phase, or in the G1 of the first round of endoreduplication (only 8C cells can be
shown unambiguously to be endoreduplicating)​.”

2. Comment: “Figure 6 showed expression profiles of rice cell cycle genes throughout the rice leaf
and the values represent expression fold change compared to the leaf segment with the lowest
expression. It would be easy to compare the approximate expression levels of each gene if the
authors could be shown the values of all examined genes relative to expression levels of
constitutively expressed control gene like actin 1 in Figure 6.”

Response: The problem with comparing gene expression levels in the leaf segment to actin is that actin
is not constitutively expressed during leaf maturation. This illustrates the risk of using a single gene as a
reference in QPCR studies. To circumvent this problem we used the gnorm normalization, which bases
itself on a selection the most stably expressed genes. Indeed, actin was not in the 15 most stable
expressing genes (see in the materials and methods section (RNA isolation and qPCR analysis) so
therefore we couldn’t compare the gene expression levels to that of actin (moreover geNorm also
discarded GOS2, another more commonly used “housekeeping”/reference gene from the normalization
process. To our minds this illustrates the strong contrasts in transcriptome of dividing, expanding and
mature cells a gradient basically representing a large part of the life cycle of a cell). Nevertheless, gnorm
could be performed on a set of 7 genes (UBQ2; E2F4; CYCH1; CDKC1; CDKD; KRP1; CDKC2), providing a
robust basis for the normalisation between samples. We agree with the reviewer that our relative
expression values, while informative for comparing gene expression profiles, hide information about the
expression levels of the individual cell cycle genes. Therefore, we included the maximum gene
expression level and fold change for each gene in a separate column in Figure 6. The caption for Figure 6
has also been changed accordingly.

Response to Reviewer #2:

General Comment: “The authors appear to have used full length sequences, including the variant C- and
N-termini? Do the relationships change if only the conserved core regions are used, and how do they
define the core regions. “

Response: Originally, we decided to use the full-length protein because this way we use the most
information. The trees were remade for the CDKA, CDKB, CYCD and E2F/DP/DEL/RB gene families using
edited alignments containing constant yet also variant sequences, but the tree showed no substantial
difference compared to the one where the full protein sequence was shown. However, we chose to use
the edited alignments since this is standard procedure.

Specific comments:

1. Comment: ”some speculations in the discussion have been made on a very narrow evidence
base and perhaps should be removed: for example - On the other hand, Arabidopsis contains 4
B-type CDKs while only two were found in the maize and rice genomes. It is noteworthy that the
added number of A-type and B-type CDKs (five) does not change, indicating that B-type CDK
functions in Arabidopsis might be partially taken over in rice by A-type CDKs, or vice-versa.
Interestingly, whereas most A-type CDKs of other higher plants have been reported to be
constitutively expressed (Martinez et al. 1992; M…. Gene number alone is not sufficient to allow
even speculation on function, unless a particular gene class is absent”

Response: We agree with this comment. Therefor the passages speculating on the variations of
gene numbers between species were removed:
- p. 6 on CDKA and B: “suggesting a functional redistribution of those 5 core CDKs between
rice and ​Arabidopsis​”.
- pgs. 15-16: “Still, some differences were observed between the two species which might
have some functional importance, as for example, rice possesses 3 ​CDKA proteins whereas
the ​Arabidopsis genome codes for only one. Also in maize two ​CDKA genes were found
 (Rymen et al. 2007) indicating that this may be a monocot specific characteristic. On the
other hand, ​Arabidopsis contains 4 B-type CDKs while only two were found in the maize and
rice genomes. It is noteworthy that the added number of A-type and B-type CDKs (five) does
not change, indicating that B-type CDK functions in ​Arabidopsis might be partially taken over
in rice by A-type CDKs, or vice-versa. Interestingly, whereas most A-type CDKs of other
higher plants have been reported to be constitutively expressed (Martinez et al. 1992;
Magyar et al. 1997), we have found that transcript levels of rice ​CDKA2;1 accumulate
differentially along the leaf, suggesting that the activity of this rice A-type ​CDK might be, at
least partially, transcriptionally regulated (Figure 6). Both auxin and cytokinin moderately
influence expression of the ​CDKA2 or ​CDKA1 genes in rice seedlings (Guo et al. 2007).” Since
this is the only place where Martinez et al. are referenced, the Martinez reference was
removed.

2. Comment: “on the other hand the authors do not comment much on the possible evolutionary
relationships of the various groups. From the presented phylogenetic trees, it looks as if some
groups (especially of the D-cyclins) may have been defined before monocots diverged from
dicots (cyclin D5 and D6 for example, while others are not so clear. Color coding the trees might
bring this information to the fore.”

Response: This is a very nice observation from the reviewer. We have changed Figure 3
accordingly to highlight (color-code) the different groups of cyclin Ds: 2/4, 1, 3, 7, 5, and 6.
Colored boxes and labels were added to Figure 4 so that we could distinguish between them.
Indeed, both monocot and dicot species have genes which belong to all
CDK/cyclin/E2F/DEL/DP/RB gene groups, suggesting that the D-type cyclin types diverged prior
to the supposed divergence of the mono- dicot clades. We have added a paragraph to the
discussion section highlighting this observation. Thanks for pointing this out.

3. Comment; “Check reference for dates and original sources”.

Response: The references were double-checked. References for Gonzalez, 2012; ​Kuwabara and
Gruissem​, 2014; Francis, 2011 were inserted. Lin, 2014, Martinez, 1992, and van de Peer, 1997
were removed.

4. Comment: “Double check that the cyclins, CDKs and CDK-like genes have all been included.”

Response: The gene list was double-checked. There are 55 rice core cell cycle genes. KRP6 was
missing from the list in Table 1. We inserted information for KRP6 here as well as in the
Supplementary Excel file. Annotation for KRP6 was added to the tab “cc gene annotations” and
“promoter data”. The number of genes was corrected in the tab “number of genes”.
Finally, after including all comments we felt that the order of the authors no longer corresponded with
their respective input and we therefore changed the author list accordingly. We also made several minor
improvements to the text to improve it’s readability.