Dolabralexins are secreted outside of maize roots
To examine a possible role of maize diterpenoids in plant-microbiome interactions, we utilized the Zman2 mutant genotype in comparison to its isogenic WT sibling [16, 29]. Previous studies showed that 30-day-old WT maize plants significantly accumulate dolabralexins, and to a lesser extent kauralexins, in roots [16], whereas the Zman2 mutant genotype is almost completely devoid of these diterpenoids [16–18]. Despite the deficiency of both kauralexins and dolabralexins in Zman2 root tissue, mutant plants did not show an apparent phenotype under well-watered conditions (Fig. 2A), consistent with prior reports describing largely unaltered root and shoot weight, developmental features, and GA and zealexin levels in the Zman2 mutant [16]. Zman2 plants were used as a control for analyzing root metabolite exudation due to the deficiency of diterpenoids, and subsequent analysis of the microbiome and metabolome.
To determine a possible ability of maize diterpenoids to affect the rhizosphere microbiome, we first tested if diterpenoids can be exuded from maize roots. For this purpose, 38-day-old maize plants, both Zman2 and its WT sibling, were grown on soil then gently cleaned and subsequently suspended for 48 hours in nutrient water. After removing the plants, metabolites were extracted from the nutrient water using an equal volume of ethyl acetate and analyzed by LC-MS/MS against authentic metabolite standards. As a positive control, the benzoxazinoid 1,3-benzoxazol-2-one (BOA), known to be secreted from maize roots, was measured and used as a standard to detect BOA and other benzoxazinoids metabolites with similar mass spectra. Benzoxazinoids were found to be present in both Zman2 and WT plant exudates, while absent in nutrient water without plants, as expected (Fig. 2B). Of the known dolabralexins for which standards were available – epoxydolabradiene, epoxydolabranol, and trihydroxydolabrene – only trace amounts were detected in nutrient water after incubation of the Zman2 mutant (Fig. 2B), as expected based on the known mutant phenotype. Trihydroxydolabrene and epoxydolabradiene were both significantly enriched in the WT root exudate samples than Zman2 or nutrient water without plants (Fig. 2B). Epoxydolabranol was not detected in mutant nor WT plant exudates.
Maize Root Microbial Communities Are Distinct By Compartment
The Zman2 mutant genotype and its corresponding WT sibling serve as a tool to investigate the effect of diterpenoids, or the lack thereof, on the maize root microbial community [16, 18]. Under abiotic stress conditions, while WT maize plants were shown to have increased levels of kauralexin and dolabralexin metabolites and a greater root/shoot ratio, Zman2 plants were shown to be more susceptible to stress conditions via earlier onset of leaf curling and a reduced root/shoot weight ratio [16]. Based on this knowledge and the growth conditions of previous research on the mutant genotype, we used one-month-old Zman2 and isogenic WT sibling plants to comparatively examine the impact of diterpenoid-deficiency on the maize root microbiome. Half of the plants were treated with drought stress in order to investigate if changes in the microbiome exist under ideal and/or stress conditions, and if these changes may suggest an influence on the Zman2 ability to cope with abiotic stress. Representative plant images are shown in Fig. 2A.
Microbiomes of the rhizosphere (1–2 mm of soil outside the root) and endosphere (inside the root), the latter representing root samples after removal of rhizosphere and rhizoplane microbes through washing and sonication of the roots, were analyzed. Bulk soil without plants was used as a control to examine background soil microbial communities. The 16S rRNA gene (V4 region) was sequenced using Illumina MiSeq and sequences were clustered into operational taxonomic units (OTUs) using the QIIME pipeline and the Greengenes database [32]. After filtering to remove mitochondrial and chloroplast OTUs, 4,259 distinct OTUs remained. OTU counts were then normalized by relative abundance, which was used rather than rarefaction methods so as not to discard low abundance OTUs. In all, four factors were analyzed: compartment, genotype, water status, and the interaction between genotype and water status.
Consistent with previous research in maize and other plant species [7, 33], the microbial communities of the two plant compartments and the bulk soil were all statistically distinct. The alpha diversity, as measured by the Shannon index, showed the greatest diversity of microbes in bulk soil, with reduced diversity in the rhizosphere and further reduction in the endosphere (Fig. 3). A permutational multivariate analysis of variance (PERMANOVA) was used to measure the diversity between samples (beta diversity) and showed that, when accounting for all factors, compartment accounts for 23% of the variation between samples (p < 0.001). This was confirmed by a principle coordinate analysis (PCoA), in which compartment was the greatest source of variation (Fig. 4). A total of 960 OTUs, in 21 phyla (out of 32 total phyla), were enriched in the rhizosphere as compared to the endosphere, whereas 82 OTUs in 9 phyla were enriched in the endosphere as compared to the rhizosphere, as determined using the DESeq2 package in R. Among the 10 most abundant phyla plotted for each sample type, some phyla were found to be enriched in both compartments, whereas the rhizosphere was predominantly enriched for OTUs in the phyla Actinobacteria, Acidobacteria, and Alphaproteobacteria (Fig. 5).
The endosphere did not demonstrate significant differences attributed to genotype, water treatment, nor their interaction, as determined by PERMANOVA and PCoA (Fig. 4B). No individual OTUs were significantly enriched or depleted in regards to sample type in the endosphere as analyzed by generalized linear models. Because there was no apparent difference, all further analysis focuses on the rhizosphere compartment only, and the bulk soil and endosphere samples were omitted from further analyses.
Rhizosphere Microbial Communities Are Distinct Under Different Watering Conditions
Drought was defined in this study as a withholding of water for seven days, with a single watering on day four. For both WT and Zman2, drought-treated plants showed severe leaf curling, but were still green and not wilted over completely (Fig. 2A). Volumetric water content (VWC) of the soil surrounding well-watered plants was 10.7 ± 1.9 percent, while drought plants was 6.9 ± 1.9 percent. When accounting for all rhizosphere samples, water status accounted for 12.6% of the observed microbiome variation (p < 0.001), and visibly separated sample types in a principal components analysis (Fig. 4C).
We next identified OTUs enriched or depleted across treatments using the R software package DESeq2. When including all plants, irrespective of genotype, drought-stressed plants were enriched for 51 OTUs, which were predominantly composed of taxa from the Phylum Actinobacteria (Fig. 6A, OTU abundances by sample type in Supplemental Fig. 1). Well-watered plants were enriched for 97 OTUs as compared to drought conditions, mostly represented by Bacteriodetes, Alphaproteobacteria, Gammaproteobacteria, and Betaproteobacteria (Fig. 6A, OTU abundances by sample type in Supplemental Fig. 2). In bulk soil samples, 21 OTUs, including Bacteroidetes and Verrucomicrobia, were enriched, whereas six OTUs, primarily Actinobacteria, were depleted under well-watered conditions (Fig. 6B). Of these, 21 significant OTUs were also enriched or depleted in the rhizosphere samples as compared to the bulk soil control samples, suggesting they are influenced by drought stress regardless of any plant-microbe interactions (Fig. 6C). Together, these results are consistent with the demonstrated contribution of water status to the rhizosphere microbiome composition [33–35], and suggest that environmental as well as host-controlled processes influence the composition of rhizosphere-associated microbiomes under drought stress.
The Zman2 mutant features a distinct microbial community composition
Next, the impact of genotype on the rhizosphere microbiome composition was assessed by investigating each water status separately for genotype effects by water treatment (GxD). Under well-watered conditions, significant differences in the microbiome composition were observed between WT and Zman2 plants, with genotype accounting for 5.8% of the variation for well-watered samples alone (p < 0.05). Zman2 plants harbor a more diverse microbiome as determined by a greater alpha diversity compared to the WT sibling (Fig. 3). Six OTUs were more abundant in WT plants, whereas none were enriched in Zman2 samples (Fig. 7; OTU abundances by sample type plotted in Supplemental Fig. 3). Of the six OTUs, all were assigned to Alphaproteobacteria belonging to the order Sphingomonadales, three of which were assigned to the genus Sphingobium, whereas the remaining OTUs were unclassified at the genus level.
In contrast to well-watered conditions, there is much less variation in the rhizosphere microbiome of WT and Zman2 under drought conditions. Distance-based approaches analyzing beta diversity, analyzing variance between samples, showed them to be indistinguishable. In addition, no differences in the alpha diversity (variance within a sample) were observed between the two genotypes under drought conditions. However, a handful of individual OTUs are significantly differentially abundant between the two genotypes when determining individual OTUs that were significantly different: six were enriched in Zman2 (including Sphingomonadales and Enterobacteriales) and none were significantly enriched in WT (Fig. 7; Supplemental Fig. 3).
Genotype By Environment Interaction Suggested By Microbial Communities
A genotype by environment (GxE) interaction is implied by the different responses of the Zman2 and WT genotypes to drought stress. This is reflected by the interaction of genotype:water status, which accounts for 5.3% of the variation in beta diversity in all rhizosphere samples (p < 0.05) and by the principal component analysis, in which Zman2 and WT are separate under well-watered conditions, but converge to have the same beta diversity under drought conditions (Fig. 4C). Analysis of the GxE interaction using log ratio tests of full and reduced models in DESeq2 determined that one Chitinophagaceae and one Sphingomonadales OTU were significantly impacted by this interaction (OTU abundances plotted by sample type in Supplemental Fig. 4).
Since the microbiome compositions between Zman2 and WT genotypes differed much more under well-watered but not drought conditions, we analyzed both water statuses separately for each genotype to look at drought effects on each genotype (DxG). This analysis revealed that drought had a larger effect on WT, in which 70 OTU were enriched and seven were depleted under well-watered conditions as compared to drought stress, and drought-exposed plants had a greater alpha diversity. In comparison, for Zman2, only 12 OTU were enriched and six depleted in well-watered conditions, and drought treatment did not impact the alpha diversity.
Dolabralexins, but not other specialized metabolites, are more abundant in wild type plants
To verify that differences in microbiome composition can be attributed to a deficiency in diterpenoids in the roots of Zman2 plants, metabolite profiling using both targeted and untargeted LC-MS/MS analysis was performed on the same root samples used for microbial composition analysis. Targeted metabolite analysis of the major dolabralexin metabolite, trihydroxydolabrene (THD) confirmed via a standard that THD was near absent in the Zman2 mutant while present in WT under well-watered conditions (Fig. 8). Epoxydolabranol was not found in either mutant nor WT plants (Supplemental Fig. 5), while epoxydolabradiene was found to be lowly abundant in both mutant and WT plants (Fig. 8), presumably because of their conversion to THD. Dolabralexins were not significantly enriched in WT versus Zman2 under drought conditions, due to notable variation between individual WT plants, but were still markedly trace in Zman2 drought plants (Fig. 8). Mirror plots demonstrate these identifications, as well as their absence in the mutant plants (Supplemental Fig. 5). This observation is consistent with previous research, showing low levels of dolabralexin and kauralexin metabolites in Zman2, predictably due to ent-CDP derived from ZmAn1 activity [16]. As a control, benzoxazinoid abundance was calculated using BOA as a standard, and found to be present in both Zman2 and WT roots without significant differences in abundance between well-watered and drought-stressed plants. Using BOA for LC-MS/MS generates multiple peaks with similar mass spectra due to various benzoxazinoids compounds, and their total area was analyzed here (Supplemental Fig. 5).
Parallel untargeted metabolomics analysis also did not indicate any significant variance in the metabolite profiles of the WT and Zman2 plants beyond the focal diterpenoids (Fig. 9A and B). Two observed data points of significant difference represented the same plants in both positive and negative mode (Fig. 9A and B). PERMANOVA analysis based on all dominant mass ions and corresponding specific retention times demonstrated neither genotype nor water status significantly impacted the metabolome using either positive or negative ionization modes. This result was further corroborated by a PCoA (Fig. 9A and B). Although the PERMANOVA and PCoA demonstrated no significant difference overall between sample types, a generalized linear model was used to identify individual metabolites that may be significantly enriched or depleted. A total of 111 metabolites were enriched in WT as compared to 85 enriched in Zman2 under well-watered conditions using positive ionization mode (Fig. 9C, Supplemental Table 1). Consistent with the targeted metabolite profiling, THD was among the 111 enriched metabolites in WT roots. Annotation of the remaining metabolites with distinct profiles in WT and Zman2 roots by comparison to mass spectral databases identified significant matches for 53 compounds, representing 27% of the differentially abundant metabolites (Supplemental Table 1). Further metabolite annotation using the PACTOLUS method generated significant matches for an additional 113 compounds (Supplemental Table 1). Although a few metabolites were enriched or depleted in abundance between WT and Zman2 plants (Fig. 9C, Supplemental Table 1), no other alterations in the metabolite profile were identified as significant, and the distinct abundance of dolabralexins was the dominant change in the overall metabolome composition of Zman2 and WT roots. The analysis was repeated for differences between the genotypes under drought conditions (in which the microbiomes are more similar), and less metabolites were significantly different (Fig. 9C, Supplemental Table 2).
Similar to genotype, drought stress did not significantly affect the metabolite profiles of the WT or Zman2 roots (Fig. 9A and B), as determined by PERMANOVA and PCoA. While the metabolite compositions variance between genotypes was not greater than the variance within the genotypes, there was a handful of metabolites that were determined to be significantly enriched or depleted, as determined by linear models. There was a significant enrichment of 99 metabolites in WT plants and 283 metabolites in Zman2 plants under drought stress as compared to well-watered plants (Fig. 9C, Supplemental Tables 3 and 4). By comparison, 36 and 88 metabolites were enriched under well-watered conditions in WT and Zman2 roots, respectively, as compared to drought (Fig. 9C, Supplemental Tables 3 and 4). While there are limited numbers of metabolites that change their abundance between drought and well-watered conditions, the overall metabolomes and variance between watering statuses remains statistically indistinguishable by PERMANOVA and PCoA.