Glutaredoxin regulation of primary root growth confers early drought stress tolerance in pearl millet

Reviewer #1 (Public Review):

The authors use a combination of crop modeling and field experiments to argue that drought during seedling establishment likely severely impacts the yield of pearl millet, an important but understudied cereal crop, and that rapid seedling root elongation could play a major role in mitigating this. They further argue that this trait has a strong genetic basis and that major polymorphisms in candidate genes can be identified using standard methods from modern genetics and genomics. Finally, they use homology with the model plant Arabidopsis thaliana to argue that the function of one putatively causal gene is to regulate root cell elongation.

The major strength of this paper is that it convincingly demonstrates how modern methods from plant breeding and model organisms can be combined to address questions of great practical importance in important but poorly understood crops. The notion that it is possible to connect single-locus polymorphism and cellular biology to drought tolerance and crop yield in pearl millet is not a trivial one.

The weakness is obvious: while the argument made is convincing, it must be recognized that the strength of the evidence is by no means of the level expected in a model organism. Conclusions could easily be wrong, and there is no direct evidence that regulatory variation in PgGRXC9 leads to higher crop yield via cell elongation and seedling drought tolerance. However, generating such evidence in a poorly studied crop would be a monumental undertaking, and should probably not be the priority of people working on pearl millet!

The utility of this work is that it suggests that it is practicable to gain valuable insight into crop adaptation by clever use of modern methods from a variety of sources.

Reviewer #2 (Public Review):

Carla de la Fuente et al., utilize a diversity of approaches to understand which plant traits contribute to the stress resilience of pearl millet in the Sahelian desert environment. By comparing data resulting from crop modeling of pearl millet growth and meteorological data from a span of 20 years, the authors clearly determined that early season drought resilience is contributed by accelerated growth of the seedling primary root, which confirms a hypothesis generated in a previous study, Passot et al., 2016. To determine the genetic basis for this trait, they performed a combination of GWAS, QTL analysis, and RNA sequencing and identified a previously unannotated coding sequence of a glutaredoxin C9-like protein, PgGRXC9, as the strongest candidate. Phenotypic analysis using a mutant of the closest Arabidopsis homolog AtROXY19 suggests the broad conservation of this pathway. Comparisons between the transcript of PgGRXC9 by in situ hybridization (this work) and AtROXY19 pattern expression (Belin et al., 2014) support the hypothesis that this pathway acts in the elongation zone of the root. Additional analysis of cell production and elongation rates in root apex in both pearl millet and A. thaliana suggests that PgGRXC9 specifically regulates primary root through the promotion of cell elongation. While several studies have established the connection between redox status of cells and root growth, the current study represents an important contribution to the field because of the agricultural importance of the plant studied, and the connection made between this developmental trait and stress resilience in a specific and stressful environmental context of the Sahelian desert.

While the study presents a compelling narrative that is based on a diverse range of approaches, some aspects require further refinement to be fully convincing.

First, while it is appreciated that working with pearl millet presents certain technical challenges regarding genetic characterization, and the authors have done outstanding work by combing the power of GWAS and QTL mapping to reproducibly identify genetic loci associated with root growth, the related work in Arabidopsis is not fully substantiated. In particular, only one mutant allele was utilized to test the function of this gene in root growth. The lack of a second characterized allele or evidence of genetic complementation makes it difficult to definitively contribute the root developmental defects to the characterized mutation in ROXY19.

The role of redox status in contributing to root growth differences between accessions was not directly tested here. The manuscript is not able to mechanistically link the molecular function of ROXY19 to the change in root growth rate, however, this limitation of the study was not clearly described in the text.

The authors state the use of cell elongation rate (Morris and Silk, 1992) as a parameter to estimate the difference in root growth between contrasted pearl millet lines and A. thaliana roxy19 mutant versus wild type; however, there are inconsistencies in what data are presented. First, in Figure 2E, regarding the comparison between different genotypes of pearl millet lines, they use the parameter of maximum cell length but when authors compare cell elongation between A. thaliana genotypes, in Figure 4D, they use the elongation rate parameter. Second, while the cell elongation rate is based exclusively on the cell length data of the "elongation only" zone (Morris and Silk, 1992), the authors profile the cell length in the whole root apex, from the quiescent center to the beginning of the differentiation zone and it is not clear how they discriminate between each developmental zone and what data was used to estimate elongation rate.