The influence of heavy metal stress on the evolutionary transition of teosinte to maize

Reviewer #1 (Public review):

In this study, Acosta-Bayona et al. aim to better understand how environmental conditions could have influenced specific gene functions that may have been selected for during the domestication of teosinte parviglumis into domesticated maize. The authors are particularly interested in identifying the initial phenotypic changes that led to the original divergence of these two subspecies. They selected heavy metal (HM) stress as the condition to investigate. While the justification for this choice remains speculative, paleoenvironmental data would add value; the authors hypothesize that volcanic activity near the region of origin could have played a role.

The authors exposed both maize and teosinte parviglumis to a fixed dose of copper and cadmium, representing an essential and a non-essential element, respectively. They assessed shoot and root phenotypic traits at a defined developmental stage in plants exposed to HM stress versus controls. They then focused on three genes already known to help plants manage HM stress: ZmHMA1, ZmHMA7, and ZmSKUs5. Two of these genes are located in a genomic region linked to traits selected during domestication. A closer examination of nucleotide variability in the coding and flanking regions of these genes provided evidence of selective pressure among teosinte parviglumis, maize, and the outgroup Tripsacum dactyloides.

They further generated a null mutant for ZmHMA1 and showed, for the first time in maize, a pleiotropic phenotype reminiscent of traits associated with the domestication syndrome. Finally, using qPCR, they reported increased expression of the domestication gene Teosinte branched1 (tb1) in teosinte parviglumis under HM stress. Comparative studies focusing on teosinte parviglumis and the genes ZmHMA1, ZmHMA7, and ZmSKUs5 under HM stress are limited; thus, this phenotypic characterization provides a promising starting point for further understanding the genetic basis of the response.

The dataset is of good quality, but the conclusions are not sufficiently supported by the data. Analyses should be expanded, and additional experiments included to strengthen the findings.

(1) Although the paper presents some interesting findings, it is difficult to distinguish which observations are novel versus already known in the literature regarding maize HM stress responses. The rationale behind focusing on specific loci is often lacking. For example, a statistically significant region identified via LOD score on chromosome 5 contains over 50 genes, yet the authors focus on three known HM-related genes without discussing others in the region. It is unclear why ZmHMA1 was selected for mutagenesis over ZmHMA7 or ZmSKUs5.

(2) The idea that HM stress impacted gene function and influenced human selection during domestication is of interest. However, the data presented do not convincingly link environmental factors with human-driven selection or the paleoenvironmental context of the transition. While lower nucleotide diversity values in maize could suggest selective pressure, it is not sufficient to infer human selection and could be due to other evolutionary processes. It is also unclear whether the statistical analysis was robust enough to rule out bias from a narrow locus selection. Furthermore, the addition of paleoclimate records (Paleoenvironmental Data Sources as a starting point) or conducting ecological niche modeling or crop growth models incorporating climate and soil scenarios would strengthen the arguments.

(3) Despite the interest in examining HM stress in maize and the presence of a pleiotropic phenotype, the assessment of the impact of gene expression is limited. The authors rely on qPCR for two ZmHMA genes and the locus tb1, known to be associated with maize architecture. A transcriptomic analysis would be necessary to 1- strengthen the proposed connection and 2- identify other genes with linked QTLs, such as those in the short arm of chromosome 5.

Reviewer #2 (Public review):

Summary:

This work explores the phenotypic developmental traits associated with Cu and Cd responses in teosinte parviglumis, a species evolutionary related to extant maize crops. Cu and Cd could serve as a proxy for heavy metals present in the soils. The manuscript explores potential genetic loci associated with heavy metal responses and domestication identified in previous studies. This includes heavy metal transporters, which are unregulated during stress. To study that, the authors compare the plant architecture of maize defective in ZmHMA1 and speculate on its association with domestication.

Strengths:

Very few studies covered the responses of teosintes to heavy metal stress. The physiological function of ZmHMA1 in maize also gives some novelty in this study. The idea and speculation section is interesting and well-implemented.

Weaknesses:

The authors explored Cu/Cd stress but not a more comprehensive panel of heavy metals, making the implications of this study quite narrow. Some techniques used, such as end-point RT-PCR and qPCR, are substandard for the field. The phenotypic changes explored are not clearly connected with the potential genetic mechanisms associated with them, with the exception of nodal roots. If teosintes in response to heavy metal have phenotypic similarity with modern landraces of maize, then heavy metal stress might have been a confounding factor in the selection of maize and not a potential driving factor. Similar to the positive selection of ZmHMA1 and its phenotypic traits. In that sense, there is no clear hypothesis of what the authors are looking for in this study, and it is hard to make conclusions based on the provided results to understand its importance. The authors do not provide any clear data on the potential influence of heavy metals in the field during the domestication of maize. The potential role of Tb-1 is not very clear either.

Author response:

Reviewer 1:

The selection of heavy metal stress as the condition to investigate is not speculative. The elucidation of the genome from the Palomero toluqueño maize landrace revealed heavy metal effects during domestication (Vielle-Calzada et al., 2009). Differences concordant with its ancient origin identified chromosomal regions of low nucleotide variability that contained the three domestication loci included in this study; all three are involved in heavy-metal detoxification. Results presented in Vielle-Calzada et al 2009 indicated that environmental changes related to heavy metal stress were important selective forces acting on maize domestication. Our study expands those results by starting to elucidate the function of these heavy metal response genes and their role in the evolutionary transition from teosinte parviglumis to maize.

Although the paper presents some interesting findings, it is difficult to distinguish which observations are novel versus already known in the literature regarding maize HM stress responses. The rationale behind focusing on specific loci is often lacking. For example, a statistically significant region identified via LOD score on chromosome 5 contains over 50 genes, yet the authors focus on three known HM-related genes without discussing others in the region. It is unclear why ZmHMA1 was selected for mutagenesis over ZmHMA7 or ZmSKUs5.

We appreciate the value of this comment. We will modify the manuscript to clearly show which phenotypic observations are novel and which were previously reported for maize grown under HM stress. The rationale for focusing on three specific loci is related to results from Vielle-Calzada et al. 2009 (see comment above). Although we demonstrated that these three loci show unusual reduction in genetic variability when compared to the rest of chromosome 5 – including a separate class of genes previously identified as being affected by domestication (Hufford et al., 2012) -, we will expand the genetic and expression analysis to all genes included in a region precisely defined via LOD scores of five QTL 1.5-LOD support intervals that overlap with ZmHMA1.Within this region of 1.5 to 2 Mb, we will compare nucleotide variability and gene expression in response to HMs. Contrary to major domestication loci showing a single highly pleiotropic gene responsible for important domestication traits, in this chr.5 genomic region phenotypic effects are due to multiple linked QTLs (Lemmon and Doebley, 2014). The mutagenic analysis of ZmHMA7 and ZmSKUs5 will be included in a different publication; we can anticipate that the results reinforce the conclusions of this study.

The idea that HM stress impacted gene function and influenced human selection during domestication is of interest. However, the data presented do not convincingly link environmental factors with human-driven selection or the paleoenvironmental context of the transition. While lower nucleotide diversity values in maize could suggest selective pressure, it is not sufficient to infer human selection and could be due to other evolutionary processes. It is also unclear whether the statistical analysis was robust enough to rule out bias from a narrow locus selection. Furthermore, the addition of paleoclimate records (Paleoenvironmental Data Sources as a starting point) or conducting ecological niche modeling or crop growth models incorporating climate and soil scenarios would strengthen the arguments.

We agree that lower nucleotide diversity values in maize are not sufficient to infer human selection and could be due to other evolutionary processes. As a matter of fact, since these same HM response loci also show unusually low nucleotide variability in teosinte parviglumis (Fig 2), we cannot discard the possibility that natural selection forces related to environmental changes could have affected native teosinte parviglumis populations in the early Holocene, before maize emergence. This possibility supports a speculative model suggesting that phenotypic changes caused by HM stress could have preceded human selection and its consequences, contributing to initial subspeciation; the model is proposed in the “Ideas and Speculation” section of the manuscript. Fortunately, as suggested by the reviewer, a large body of paleoclimatic records and paleoenvironmental data is available for the Trans-Mexican Volcanic Belt in the Holocene, including geographic regions where the emergence of maize presumably occurred. We will include an extensive analysis of available paleoenvironmental data and discuss it at the light of our current results regarding the effects of HM stress. We are also expanding the physical range of our statistical analysis to cover at least 60 Kb per locus - including neighboring genes for all three loci - to determine if our results could be due to narrow locus selection.

Despite the interest in examining HM stress in maize and the presence of a pleiotropic phenotype, the assessment of the impact of gene expression is limited. The authors rely on qPCR for two ZmHMA genes and the locus tb1, known to be associated with maize architecture. A transcriptomic analysis would be necessary to 1- strengthen the proposed connection and 2- identify other genes with linked QTLs, such as those in the short arm of chromosome 5.

Although real-time qPCR is an accurate and reliable approach to assess the expression of specific genes such as ZMHMA1 and Tb1, we will explore the possibility of complementing our analysis with available RNA-seq results that are pertinent for this study (see for example Li et al., 2022 and Zhang et al., 2024) and further explore causative effects between HM stress, Tb1 and ZmHMA1 expression. As also pointed by Reviewer#1, TEs are known to influence gene expression under abiotic stress and RNA-Seq analysis would allow to determine if TE activity could lead to similar outcomes.

Reviewer #2:

The authors explored Cu/Cd stress but not a more comprehensive panel of heavy metals, making the implications of this study quite narrow. Some techniques used, such as end-point RT-PCR and qPCR, are substandard for the field. The phenotypic changes explored are not clearly connected with the potential genetic mechanisms associated with them, with the exception of nodal roots. If teosintes in response to heavy metal have phenotypic similarity with modern landraces of maize, then heavy metal stress might have been a confounding factor in the selection of maize and not a potential driving factor. Similar to the positive selection of ZmHMA1 and its phenotypic traits. In that sense, there is no clear hypothesis of what the authors are looking for in this study, and it is hard to make conclusions based on the provided results to understand its importance. The authors do not provide any clear data on the potential influence of heavy metals in the field during the domestication of maize. The potential role of Tb-1 is not very clear either.

Thank you for these comments. We will clearly emphasize our hypothesis that HM stress was an important factor driving the emergence of maize from teosinte parvglumis through action of HM response genes. A comprehensive panel of heavy metals would not be more accurate in terms of simulating the composition of volcanic soils evolving across 9,000 years in the region where maize presumably emerged. Copper (Cu) and cadmium (Cu) correspond each to a different affinity group for proteins of the ZmHMA family. ZmHMA1 has preferential affinity for Cu and Ag (silver), whereas ZmHMA7 has preferential affinity to Cd, Zn (zinc), Co (cobalt), and Pb (lead). Since these P1b-ATPase transporters mediate the movement of divalent cations, their function remains consistent regardless of the specific metal tested, provided it belongs to the respective affinity group. By applying sublethal concentrations of Cd (16 mg/kg) and Cu (400 mg/kg), we caused a measurable physiological response while allowing plants to complete their life cycle, including the reproductive phase, facilitating a comprehensive analysis of metal stress adaptation.

Although real-time qPCR is an accurate and reliable approach to assess gene expression, we agree that RNA-Seq results would improve the scope of the analysis and better assess the role of Tb1 in relation to HM response (see comments for Reviewer#1). There are two phenotypic changes clearly connected with the genetic mechanisms involved in the parviglumis to maize transition: plant height and the number of seminal roots (not nodal roots). We will emphasize these phenotypic changes in a modified version of the manuscript. There is a possibility for HM stress to represent a confounding factor in the selection of maize and not a driving factor; however, if such is the case, we think it is rather unlikely that the real driving factor could have acted through mechanisms not related to abiotic stress or HM response. To address the possibility that HM stress was a cofounding factor, we will extensively analyze genetic diversity and gene expression in all loci containing genes mapping in close proximity to peak LOD scores of all 1.5-LOD support intervals located in chromosome 5 and showing pleiotropic effects on domestication traits (Lemmon and Doebley, 2014). These will also include those mapping in close proximity to ZmHMA1. The potential influence of heavy metals in the field is being investigated through the analysis of paleoenvironmental data (see response to Reviewer#1); we will include our results in a modified version of the manuscript.

We thank both reviewers for their detailed revision the manuscript and their pertinent recommendations to improve its presentation and reading.

References:

Hufford, Matthew B., Xun Xu, Joost Van Heerwaarden, Tanja Pyhäjärvi, Jer-Ming Chia, Reed A. Cartwright, Robert J. Elshire, et al. 2012. Comparative population genomics of maize domestication and improvement. Nature Genetics 44(7): 808-11.

Lemmon Zachary H., Doebley John F. 2014. Genetic dissection of a genomic region with pleiotropic effects on domestication traits in maize reveals multiple linked QTL. Genetics 198(1): 345-353.

Lin Kaina, Zeng Meng, Williams Darron V., Hu Weimin, Shabala Sergey, Zhou Meixue, Cao Fangbin, et al. 2022. Integration of transcriptome and metabolome analyses reveals the mechanictic basis for cadmium accumulation in maize. iScience 25(12): 105484.

Vielle-Calzada JP, De La Vega OM, Hernández-Guzmán G, Ibarra-LacLette E, Alvarez-Mejía C, Vega-Arreguín JC, Jiménez-Moraila B, Fernández-Cortés A, Corona-Armenta G, Herrera-Estrella L, Herrera-Estrella A. 2009. The Palomero genome suggests metal effects on domestication. Science 326: 1078.

Zhang Mengyan, Zhao Lin, Yun Zhenyu, Wu Xi, Wu Qi, et al. 2024. Comparative transcriptome analysis of maize (Zea mays L.) seedlings in response to copper stress. Open Life Sciences 19(1): 20220953.