A Tomato Putative Metalloprotease SlEGY2 Plays a Positive Role in Thermotolerance
Round 1
Reviewer 1 Report
Review of the manuscript entitled: A tomato putative metalloprotease SlEGY2 plays positive role 2 in thermotolerance.
The authors investigate the intramembrane protease EGY2 in Solanum Lycopersicum. The article contains a wide characteristic of the protease and its cDNA sequence including:
- Sequencing of the cDNA encoding the protease
- Phylogenetic analysis of the cDNA sequence
- Investigation of subcellular localization of the protein
- Analysis of the expression of the SIEGY2 gene in different tissues ( roots, hypocotyls, stems, leaves, flowers, and fruits) and response to different treatments like abiotic stresses (heat and drought) as well as in response to hormone treatments (ABA and MeJa).
The authors obtain also three siegy2 antisense transgenic lines and investigate their sensitivity to heat treatment. The MDA, as an indicator of membrane peroxidation, hydrogen peroxide, and superoxide anion were measured as well as relative electrical conductivity, net photosynthesis rate, and the maximum efficiency of PSII.
The experiments seem to be carried out correctly, but in many cases, the methodological description is insufficient, as it is limited to references only. This applies mainly to subsections 2.6 to 2.9 in the “Materials and methods” section. Authors should supplement these sections with short descriptions.
The obtained results are very interesting and in my opinion, they constitute a significant scientific novelty. The authors indicate that their results are in some points, different from those obtained for AtEGY2. In the authors' opinion, it may be because Arabidopsis is a chilling-resistant plant, while tomato is a chilling sensitive plant. That is a very interesting conclusion. Authors refer however only to the aspect of photoinhibition. It would be advisable to extend the discussion. since also the results concerning the expression level of SIEgy2 gene are in some points different than results obtained for Arabidopsis available in public databases like BAR or eFP Browser.
The work is written coherently and clearly but requires minor linguistic corrections. For example, in the 90 it should be Solanum lycopersicum, and the word “ obviously” in line 194 seems to be used incorrectly. Also, the word "aggravated" seems to be out of context. For example in subtitle 3.4 the words “increased’ or “enhanced” seems more suitable.
The above remarks do not lower the high quality of the article.
Author Response
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Reviewer 2 Report
The paper is relevant, presenting valuable genetic information involving the metalloprotease family M50 in tomato plants, specially this thermotolerance. The manuscript has been written, the data was good presented and discussed. So, I recommend more accurate English language review.
Author Response
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Reviewer 3 Report
Zhang and coworkers characterized and revealed a role of a chloroplast metalloprotease, EGY2, in thermotolerance in tomato. Tomato EGY2 was found to share similarities with M50 family of metalloproteases, containing conserved HEXXH and NPDG motifs and is localized in chloroplast. EGY2 expresses largely in leaves and shows presence in flowers as well. Interestingly, EGY2 shows transcriptional upregulation with abiotic stresses, including heat and drought, influenced by phytohormones ABA and JA. For further characterization authors’ developed antisense lines. Like Arabidopsis egy2 mutant, EGY2 antisense transgenic tomato plants show shorter hypocotyls. As anticipated, reduced expression of EGY2 in antisense lines led to their susceptibility toward heat stress, showing reduced biomass, chlorophyll content, PSII efficiency, photosynthetic rate and antioxidant activity, and an increased ROS accumulation, oxidative damage, and consequent cell death. Collectively, the data nicely demonstrate that plants require EGY2 for chloroplast function and tolerance toward heat stress.
The manuscript is well written. Experiments are well designed and nicely executed. There are few queries/concerns, as follows:
1. Why were AS lines tested with heat stress only? ABA and MeJA rather resulted in much higher expression. Drought implicated with increased ABA, could also be tested.
2. EGY2 is linked with chloroplast functioning, especially with light reaction machinery (PMID: 30268696); why wouldn’t authors’ tested high light and photo inhibitory stresses.
3. Why AS lines exhibited no obvious phenotype under normal conditions? Any explanation for that?
In some experiments (Figs. 4, 5), it seems AS lines, especially A3 and A4 have obvious phenotypes under normal conditions as well.
Author Response
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