Upregulation of DWARF27 is associated with increased strigolactone levels under sulfur deficiency in rice

Abstract Plants produce strigolactones (SLs) in roots in response to nitrogen or phosphate deficiency. To evaluate SL levels under other mineral deficiencies in rice, we cultivated rice seedlings in hydroponic media without nitrogen, phosphorus, potassium, sulfur, calcium, magnesium, and iron. Tiller bud outgrowth was stimulated under calcium deficiency because of low SL levels. SL levels increased under sulfur deficiency, in addition to phosphate, and nitrogen deficiencies. To explore which genes are key regulators of SL production under sulfur deficiency, we analyzed the expression of SL‐related genes in sulfur‐sufficient and sulfur‐deficient conditions. An SL biosynthesis gene, DWARF27 (D27), was strongly expressed under sulfur deficiency, and its expression was decreased by sulfur supply. The levels of D10, D17, and OsMAX1 transcripts did not differ between sulfur‐sufficient and sulfur‐deficient conditions. These results suggest that the increased SL levels under sulfur deficiency are due to a high expression of D27. A combination of nitrogen, phosphorus, and sulfur deficiencies had no additive synergistic effect on SL production. Under combined phosphorus and sulfur deficiency, the expression levels of most SL biosynthesis genes were elevated. The number of tiller buds in the d27 mutant was higher than in the wild type, but lower than in other d mutants. Under sulfur deficiency, the chlorophyll content of d27 was lower than those of other d mutants. These results indicate that D27 plays an important role in adaptation to sulfur deficiency in rice.

Responses to the reviewer's comments Reviewer 1 1. Consider modifying the title to be more representative of your conclusions. I would suggest something like, ' Up-regulation of DWARF27 transcript in sulfur deficient rice is associated with increased strigolactone levels'.
The title has changed to 'Up-regulation of DWARF27 is associated with increased strigolactone levels under sulfur deficiency in rice' (See l.9-10).
2. Make sure your conclusions regarding D27 and SL levels reflect the suggestive nature of your findings and are not overstated. According to the reviewer comment, we have revised the manuscript. We have moved some data to supplemental information or have deleted. In addition, most part of hypothetical model have been modified.
3. The possible link between D27 and Aux/IAA signaling and AM fungi (see Discussion on pg 8 and 9) is interesting but does not seem to be the focus of this study. I suggest removing most of this unsupported conjecture. The proposed model (line 272-279) is especially speculative without the Aux/IAA genes and no data on these genes presented in this study. I strongly recommend removing or heavily editing this material in the discussion.
According to the reviewer's comment, the Aux/IAA-related description has been deleted.
4. Figure 1 (Effect of Nutrient deficiency on rice tiller number) could be moved to the supplement. This is not the main point of the study and, while important, does not add much to the main conclusions. Fig. 1 has been moved to supplemental information.
5. Figure  6. For all figures, the shorthand names given in the text (eg Os1400) should be explained in the figure captions where appropriate. I would prefer if proper gene identifier numbers were also given in the caption or used instead of the shorthand names.
According to the reviewer 2 comment, we have added the SL biosynthetic pathway with full number of gene ID Os01g0701400 in Fig. 1. However, we would remain the shortened name such as Os1400 in other figures because the name was used in other some papers that have been published.
7. In general, there are a lot of figure panels that convey very little information. I recommend combining as many figures as possible and moving redundant panels or panels with negative results to the supplement if possible.
According to the reviewer comment, figures have been re-organized. Fig. 1 has been moved to supplemental information. Fig. 3 has removed. Finally, fig. 7C and 8C have combined and the other data set have been moved to supplemental information Reviewer 2 1. As you have lots of information about strigolactones biosynthesis, it will be much better to draw a model of SL biosynthetic pathway in planta. This would help readers understand present work easily. The SL biosynthetic pathway has been shown in Fig. 1 3. P5, L155 and L156, it seemed D10, Os900, Os1500, Os1900 expressed significantly different between +S and -S conditions at day 16? (Fig.S2C). You can check these data again.
We have checked the data of Fig. S2C. However, there are no significant difference between +S and -S on D10, Os900, Os1500, and Os1900 expression in Tukey's HSD analysis.
4. P6, L157. Present work focused on the sulfur deficiency and strigolactones levels. I can not understand what the authors want to say for the results of D3 and D14 expression.
We have analyzed the SL-signaling genes D3 and D14 expression as well as expression of the SL biosynthetic genes. In S sufficient condition, expression levels of the SL biosynthetic genes are low, whereas D3 and D14 expression are rather up-regulated.
These results indicate that rice seedlings may increase the SL sensitivity because SL levels are low in S sufficient condition. We have added these sentence in Discussion (See l.260-263).
5. P7 L194, the d27 mutant was not pre-cultured for these experiments? No, the d27 mutant was also pre-cultured in the experiment. We have added d27 in the sentence (See l.230 and 239).
6. P8-P9, L255-L279. Although it is great to propose a hypothetical model of plant adaptation through SLs under -S, you have not any other data such as NSP1, NSP2, Aux/IAA gene to support the model in your planta. I do not think lots of inference is good here Our hypothetical model is just speculation in this time. Thus, most part of the model has been removed. However, NSP1 and NSP2 regulate D27 expression and AM fungi can supply S to the host plant, provided by some papers. We would remain the description (See l.291-306).
We are very sorry. A comma has been inserted between d10 and d14 (See l.116-117).
8. Check the format of literature cited. The journal "Plant Cell" should be replaced with "The Plant Cell". 'Plant Cell' has been changed to 'The Plant Cell'. In addition, 'Plant Journal' has been changed to 'The Plant Journal' (See REFERENCES).
9. It is interesting to investigate the effects of combinations of N, P and S deficiencies on 4DO levels. P-deficiency increased higher level of 4DO than S-deficiency. Is there any different mechanism to induce 4DO biosynthesis between P-deficiency and S-deficiency? However, combination of P-deficiency with S-deficiency induced less 4DO content than P-deficiency alone, but a bit more 4DO content than S-deficiency. Do you have any ideals to explain it? We think there are different mechanism between P and S deficiencies because all SL biosynthetic genes are up-regulated under P deficiency (Umehara et al. 2010), whereas we found that only D27 is up-regulated under S deficiency in this study . Up-regulating system is depending on the type of deficient macronutrient. Expression levels of D27 decreased that of the other SL biosynthetic genes increased under PS deficiencies in comparison with S deficiency, increased the flow of SL biosynthesis. Thus, 4DO content of PS deficiencies might be higher than that of S deficiency (See l.276-279).
10. The Figs can be re-organized. For example, Fig. 1 and Fig. 2 can be combined together as one Figure.