New Insights into the Role of Alternating Temperatures and Cyanide in the ROS-Mediated Cardoon Seed Dormancy Termination

Round 1

Reviewer 1 Report

It is necessary to display the results of Tukey's statistical analysis on the data in Table 1.

The conclusion of 100 M KCN ​​with 16 hours as the best treatment does not seem to match table 1. In table 1 the best treatment seems to be 10 M with 6 hours (it is necessary to show statistical analysis in table 1. Which criteria were used to make this conclusion?

Comments for author File: Comments.pdf

Author Response

We thank Reviewer 1 for highlighting this aspect that was overlooked in the original submission. As suggested, we added the statistical analysis results in Table 1 and also in Figure 1. For the latter, statistical significance is shown at each observation step (daily), while the statistics reported in table 1 is referred to final germination percentages. In both cases, the statistical elaborations were performed by comparing the cyanide effect for alternating and constant temperatures separately using, for each temperature regime, the direct imbibition in water as the control condition. As now reported in table 1, the Tuckey test indicated that for the constant temperature regime, 10 uM for 16 hours and 100 uM for 16 hours were both the conditions determining the most significant effect on final germination. We selected 100uM for 16 hours as the best treatment because it provided the higher final germination percentage (85.56%) respect to 10 uM for 16 hours (84.44%).

Reviewer 2 Report

The work concerns the dormancy of cardoon achenes. It is generally well done and the presented results are interesting. It requires linguistic improvement and detailing on some issues. I have put most of my detailed comments and questions in the manuscript.

Authors have to specify if embryos or achenes were treted with KCN - it makes difference. In M&M section they wrote, achenes were immersed in KCN solution. If so the effect is surprising...

In same parts there are too general statesments, they need specification. The authors should distinguish between changes at the transcriptome level and changes at the proteome level and enzymatic activities. Some additional information to Material & Methods section are also required. 

I look forward to referring to each of my comments in the manuscript. Please correct the whole manuscript when the comment concerns the whole manuscript.

Comments for author File: Comments.pdf

Author Response

We thank reviewer 2 for the valuable suggestions that improved substantially the manuscript. We amended all the raised issues in the manuscript attached. 

As regards the effect of KCN pre-treatment, the experiment was carried out using achenes. They were treated with different KCN concentrations and hours of incubation previously to be transferred to the water. Wild cardoon is a variety exhibiting seed physiological dormancy for which the pericarp imposes a block to the radicle protrusion and no embryo dormancy was documented so far. Alternating temperatures terminate achenes dormancy to a high extent (e.g. >75%) by increasing the embryo growth potential by means of a reduction of ABA/GAs ratio to overcome pericarp constraint (Huarte and Benech Arnold, 2010; Huarte et al., 2014), and this, as we recently demonstrated, is associated also with the ethylene and ROS homeostasis (Huarte et al 2018 and Huarte et al, 2020). Therefore to get more insights into the physiological mechanisms triggered by cyanide we pre-treated intact achenes with cyanide at different concentrations/times and then the achenes were imbibed at a constant temperature. This was compared with the germination of intact achenes imbibed directly at alternating temperatures which effect is known. We agree with reviewer 2 about the surprising effect of cyanide in wild cardoon, even at micromolar concentrations, and for this reason, we thought our results were worthy of publication and of future investigations.