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Research Article

Grasses suppress shoot-borne roots to conserve water during drought

Jose Sebastian, Muh-Ching Yee, Willian Goudinho Viana, Rubén Rellán-Álvarez, Max Feldman, Henry D. Priest, Charlotte Trontin, Tak Lee, Hui Jiang, View ORCID ProfileIvan Baxter, Todd C. Mockler, View ORCID ProfileFrank Hochholdinger, View ORCID ProfileThomas P. Brutnell, and View ORCID ProfileJosé R. Dinneny
PNAS first published July 15, 2016; https://doi.org/10.1073/pnas.1604021113
Jose Sebastian
aDepartment of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305;
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Muh-Ching Yee
aDepartment of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305;
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Willian Goudinho Viana
aDepartment of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305;
bCoordination for the Improvement of Higher Education Personnel (CAPES) Foundation, Ministry of Education of Brazil, Brasilia-DF 70.040-020, Brazil;
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Rubén Rellán-Álvarez
aDepartment of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305;
cUnidad de Genómica Avanzada, Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, 36821 Irapuato, Mexico;
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Max Feldman
dDonald Danforth Plant Science Center, St. Louis, MO 63162;
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Henry D. Priest
dDonald Danforth Plant Science Center, St. Louis, MO 63162;
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Charlotte Trontin
aDepartment of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305;
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Tak Lee
eDepartment of Biotechnology, College of Life Sciences and Biotechnology, Yonsei University, Seoul 03722, Korea;
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Hui Jiang
dDonald Danforth Plant Science Center, St. Louis, MO 63162;
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Ivan Baxter
dDonald Danforth Plant Science Center, St. Louis, MO 63162;
fPlant Physiology and Genetics Research, Agricultural Research Unit, US Department of Agriculture, St. Louis, MO 63132;
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Todd C. Mockler
dDonald Danforth Plant Science Center, St. Louis, MO 63162;
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Frank Hochholdinger
gCrop Functional Genomics, Institute of Crop Science and Resource Conservation, University of Bonn, D-53113 Bonn, Germany
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Thomas P. Brutnell
dDonald Danforth Plant Science Center, St. Louis, MO 63162;
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José R. Dinneny
aDepartment of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305;
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  • For correspondence: jdinneny@carnegiescience.edu
  1. Edited by Natasha V. Raikhel, Center for Plant Cell Biology, Riverside, CA, and approved June 2, 2016 (received for review March 10, 2016)

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Significance

Grasses, whose members constitute key food and bioenergy crops worldwide, use unique developmental programs to establish the root system from the shoot. Shoot-borne crown roots originate near the soil surface and provide the main conduits through which the plant takes up water and nutrients. We show that crown root development is the major target of drought stress signaling. Water deficit-triggered crown root arrest provides an important mechanism to conserve water under drought, and this response is widely conserved across grass species. Substantial phenotypic variation exists in maize for this trait, which may be a useful target in breeding efforts to improve drought tolerance.

Abstract

Many important crops are members of the Poaceae family, which develop root systems characterized by a high degree of root initiation from the belowground basal nodes of the shoot, termed the crown. Although this postembryonic shoot-borne root system represents the major conduit for water uptake, little is known about the effect of water availability on its development. Here we demonstrate that in the model C4 grass Setaria viridis, the crown locally senses water availability and suppresses postemergence crown root growth under a water deficit. This response was observed in field and growth room environments and in all grass species tested. Luminescence-based imaging of root systems grown in soil-like media revealed a shift in root growth from crown-derived to primary root-derived branches, suggesting that primary root-dominated architecture can be induced in S. viridis under certain stress conditions. Crown roots of Zea mays and Setaria italica, domesticated relatives of teosinte and S. viridis, respectively, show reduced sensitivity to water deficit, suggesting that this response might have been influenced by human selection. Enhanced water status of maize mutants lacking crown roots suggests that under a water deficit, stronger suppression of crown roots actually may benefit crop productivity.

  • root development
  • drought
  • Poaceae
  • Setaria
  • Zea mays

Drought is the most damaging environmental condition affecting global agricultural productivity, due in large part to the effects of water deficit (WD) (www.fao.org/home/en/; reports.weforum.org/global-risks-2015). Roots provide the main route through which water is absorbed from the soil environment, and thus represent an important target for breeding efforts aimed at improving drought tolerance in crops (1). In the grass lineage, roots develop from multiple sites aboveground and belowground, substantially distinguishing their development from other eudicot models. The initial root system of species such as maize consists of primary and seminal roots, which originate from the embryonic axis (2, 3) (Table S1). This embryonic root system is important for seedling establishment but is largely ephemeral (2, 4, 5). Later during development, roots borne from the crown initiate and emerge from internal tissues. This important developmental transition represents the beginning of the postembryonic shoot-borne root system (4, 6, 7), which will come to dominate the architecture of the plant below ground (2, 8, 9).

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Table S1.

Glossary of commonly used terms describing root system features

The number of crown roots, their growth rate, and their angle of growth with respect to gravity all vary between different inbred lines of maize (10). The physiological impact of variation in crown root growth has been explored through modeling approaches, which indicates that faster growing roots with a steeper gravity setpoint angle promote access to deep-water resources (11, 12). Greater crown root density comes at a cost, however, with recent studies suggesting that higher density is negatively correlated with the efficiency of nitrogen foraging (13).

To understand how WD affects the balance between embryonic and postembryonic parts of the root system and specifically crown root development, we analyzed root growth through excavation from soil and using the GLO-Roots imaging system (14). These studies used the emerging grass model species Setaria viridis, which is a C4-grass model for other agronomically important panicoid grasses, such as maize and sorghum (15, 16). Our results define the primary developmental mechanism through which WD affects root system architecture and reveals the physiological relevance of such a response.

Results

Suppression of Crown Root Growth Is a Major Response to Water Deficit in S. viridis.

The S. viridis A10.1 accession was grown in 35.5-cm-deep pots filled with a peat-based soil mixture (Materials and Methods). Seeds were planted in soil at full water-holding capacity (pot capacity). For well-watered (WW) conditions, pot weight was maintained near water-holding capacity throughout the experiment. To induce WD, water was withheld after germination, which caused gradual drying of the soil from the top to the bottom of the pot (Fig. S1 A and B). WD treatment led to a reduction in leaf relative water content (RWC); an acceleration in leaf initiation rate, flowering time (date of panicle emergence), and tiller production; and a dramatic reduction in root mass (Fig. 1 A and D and Fig. S1 C–F).

Fig. 1.
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Fig. 1.

Crown root growth is suppressed in S. viridis as a response to WD. (A) Comparison of whole-root systems of S. viridis grown under WW and WD conditions (30 DAS). (B) Comparison of crown regions of WW- and WD-treated S. viridis plants (25 DAS). (C) Magnified image of WD-treated S. viridis crown region showing the presence of arrested crown roots. (D) Comparison of plant dry weight under WW and WD conditions (34 DAS; n = 15–20 plants per condition). (E) Number of arrested and outgrown crown roots under WW and WD conditions (41 DAS; n = 10–15 plants per condition). (F) Crown root response to WD treatment in 18 S. viridis accessions (40 DAS; n = 5–10 plants per accession). (G) Number of leaves and crown roots quantified in field-grown plants under WW and WD conditions. Data are an average of results from six subplot replicates (n = 120–130 plants per treatment). (H) Time-lapse images of the crown region of a WD-treated plant after rewatering. Labels indicate time after rewatering. Image series shows rapid emergence and elongation of new roots from the crown, whereas previously emerged roots remain arrested. (I) Quantification of outgrown and arrested crown root formation in plants grown under WW (Left), WD (Center), or WD followed by rewatering (Rewatered, Right) (n = 25 plants). Plants in the rewatered condition were WD-treated until the 14th DAS and then rewatered. (Scale bars: 1 cm in A and B, 1.5 mm in C and H.) *P < 0.05, Student’s t test. Error bars represent SE.

Fig. S1.
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Fig. S1.

Crown root growth arrest in S. viridis as a WD response. (A) Pot moisture content measured through a time series showing the gradual drying of the soil with WD treatment (n = 10 pots). Pots with soil weighed on average 586.5 ± 1.3 grams before watering. (B) Soil water content of deep pots at 27 DAS subjected to WW or WD conditions (n = 3). Top, soil from the top half of the pot; bottom, soil from the bottom half of the pot. (C) Relative water content of leaves from WW and WD conditions at 41 DAS (n = 10). (D–F) Comparison of leaf initiation rate (D), flowering time (E), and tiller production (F) in S. viridis grown under WW and WD conditions (n = 10–15 plants). (G) A time-series analysis of crown root emergence in S. viridis. Plants are grown in deep pots under WW conditions. (H) S. viridis crown region showing primary (arrowhead) and crown roots at 36 DAS. (I and J) Image of an S. viridis plant grown under WD conditions at 22 °C (I) and a magnified image of the crown region showing arrested crown roots and the primary root (arrowhead) (J) (40 DAS). (K) Comparison of arrested vs. outgrown crown roots in S. viridis plants grown under WW or WD conditions at 22 °C (n = 12–14 plants). *P < 0.05, Student's t test. (Scale bars: 1 cm.) Error bars represent SE. WW, watered; WD, water deficit.

The first crown roots emerged 7 to 9 days after sowing (DAS) under WW conditions, and new roots emerged continuously thereafter (Fig. S1G). Excavation of the root system at 30 DAS revealed that most branches were crown root-derived under WW conditions, while under WD, crown roots were completely absent (Fig. 1 B and E and Fig. S1H). Close examination of the crown showed an accumulation of arrested roots, which were absent under WW conditions (Fig. 1 B, C, and E). Interestingly, the number of arrested crown roots was greater than outgrown roots under WW conditions, suggesting that WD may promote crown root initiation (Fig. 1E). Similar effects were observed in a survey of WD responses in 18 other S. viridis accessions and in plants grown at a lower temperature (Fig. 1F and Figs. S1 I–K and S2A). Importantly, the observed suppression of crown root growth also occurred under field conditions simulating WW and WD conditions (Fig. 1G and Fig. S2 B–F). Together, these results show that the postemergence suppression of crown root growth represents a major response to WD in the S. viridis root system and is responsible for the dramatic decrease in root system size.

Fig. S2.
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Fig. S2.

S. viridis accessions and analysis of crown root growth under field conditions. (A) A global map showing the approximate location (yellow markers) of the different S. viridis accessions used in this study (Ames 21519, China; Ames 21520, Russia; Ames 28193, Kazakhstan; PI202407, Chile; PI204624, Turkey; PI204625, Turkey; PI204727, Turkey; PI204730, Turkey; PI212625, Afghanistan; PI221960, Afghanistan; PI223677, Azerbaijan; PI230134, Iran; PI230135, Iran; PI408810, China; PI649320, Mongolia; ME042-1, Canada; VB80-1, United States; and RO10106, United States). Fig. S2A © 2016 Google – Map data © 2016 Google. (B and C) Relative moisture content in the topsoil measured using a Diviner 2000 probe for WW (B) and WD (C) field plots. (D) RWC (leaf) of field-grown plants (n = 36 plants). (E and F) Images of S. viridis plants from the field experiment under WW (E) and WD (F) conditions. (Scale bars: 1 cm.) Error bars represent SE.

Local Perception of Water Rapidly Induces New Crown Root Development.

Irrespective of the degree or duration of the WD regime, if plants were still alive, they could produce new crown roots on rewatering (Fig. S3A). Newly emerged crown roots appeared between 4 and 8 h after treatment (Fig. 1H). All of the newly formed crown roots emerged de novo after rewatering and not as a result of recovery of growth of arrested crown roots (Fig. 1 H and I).

Fig. S3.
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Fig. S3.

Local perception of moisture by roots in the crown. (A) Number of crown roots formed at 24 h after rewatering. Plants were subjected to varying durations of WD before rewatering (n = 6–10 plants). (B–E) Images of the crown region. Soil has been removed to visualize the roots. Plants were grown under WW conditions (B), WD conditions (C), WD conditions for 3 wk then rewatered from the bottom of the pot (base) (D), or fully rewatered (brought back to pot capacity) (E). The arrowhead indicates the crown region. (F and G) Number of outgrown crown roots (F) and leaf RWC (G) in plants grown under WW or WD conditions for 3 wk before being rewatered from the bottom of the pot (base), rewatered at the crown region (10 mL added around the crown region using a pipette), fully rewatered (RW), or continuous WD (n = 10 plants). (H) Division of regions of the pot for analysis of soil moisture content in I. (I) Pot soil moisture content under various watering regimes (n = 3 samples per section). Error bars represent SE. *P < 0.05, Student’s t test. ns, not significant.

Plants in which water was applied at the bottom of the pot recovered their water status but did not induce crown root growth, likely owing to the lower water content at the crown region under this watering regime (Fig. S3 B–I). In contrast, plants for which water was applied directly to the crown region itself rapidly reinitiated crown root growth. These data suggest that water availability is locally sensed by the crown to regulate crown root growth and may be partly independent of the overall water status of the shoot. Whether such sensing occurs in the entire crown or in initiated crown roots is not clear at present.

The Crown Region Is Highly Transcriptionally Responsive to WD.

We performed a transcriptomic analysis of the S. viridis crown tissue to elucidate the molecular pathways associated with WD responses. Crown tissue samples were collected from plants grown under either WW or WD conditions for 6 and 9 DAS. At 6 DAS, neither sample type had produced any emerged crown roots, whereas at 9 DAS, only WW plants formed outgrowing crown roots (Fig. 2A and Fig. S4 A–C). We also sampled a 2-mm region of the stem apical to the crown region for comparison. We found that the crown region at 9 DAS had the most differentially expressed genes between WW and WD conditions (Fig. 2B and Dataset S1), indicating a large relative change in the cellular state of these tissues.

Fig. 2.
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Fig. 2.

The crown region is highly transcriptionally responsive to WD. (A) Images of WW- and WD-treated S. viridis plants at 9 DAS. Dashed lines indicate the sampled regions (C, crown; S, stem) for RNAseq analysis. (B) Numbers of differentially expressed genes from pairwise comparisons of WW and WD in the two regions and time points. (C) Heatmap showing the GO category enrichment in the S. viridis RNAseq analysis. Blue indicates significant enrichment of GO category in down-regulated genes; orange indicates significant enrichment in up-regulated genes (P < 0.05, corrected P value). (D) qRT-PCR showing the relative expression levels of peroxidase (Sevir.5G028500) and NO APICAL MERISTEM (Sevir.7G133100) genes under WW, WD, and RW conditions. (E) qRT-PCR showing the relative expression levels of mi5205b and miR43 under WW, WD, and RW conditions. (Scale bars: 1 cm.) *P < 0.05, Student’s t test. Error bars represent SE. ns, not significant.

Fig. S4.
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Fig. S4.

Developmental stages used for transcriptional analysis of WD responses in crown. Picture of S. viridis plants subjected to WW (A), WD (9 DAS) (B), and RW (10 DAS) (C) conditions. (Scale bars: 1 cm.)

Genes that were highly down-regulated in the crown region at 9 DAS are significantly associated with the oxidative-stress response (P = 4.7e-20) and oxidation-reduction reactions (P = 1.2e-18) and include several putative peroxidases (Fig. 2C and Datasets S1 and S2). Peroxidase activity promotes root growth, and their repression under WD may prevent continued growth of crown roots (17, 18). Sexual reproduction is the most enriched Gene Ontology (GO) enrichment term for up-regulated genes, which is consistent with the accelerated flowering exhibited by S. viridis under WD conditions (Fig. S1E). MapMan pathway analysis showed significant enrichment for genes associated with degradation of branched-chain amino acids in WD-repressed genes of the crown (Dataset S3). This alternative respiration pathway has recently been shown to affect drought tolerance in Arabidopsis (19).

We found that 5% of the differentially expressed genes in the crown region are predicted targets of miRNAs, including Sevir.5G028500, a class III peroxidase that is the most down-regulated gene under WD, and Sevir.7G133100, a NO APICAL MERISTEM (NAM) gene (refs. 20 and 21 and Dataset S4). NAM family genes are plant-specific transcriptional regulators that control growth and development (22, 23). We analyzed levels of the miRNAs, mi5205b, targeting the peroxidase gene. and miR43, targeting the NAM gene, and found that both were significantly induced under WD (Fig. 2 D and E). At 4 h after rewatering, the miRNA levels were down-regulated and the peroxidase gene expression was up-regulated; however, NAM gene expression showed no significant response (Fig. 2 D and E). These data show coordinated regulation of miRNAs and their predicted targets, identifying a potential regulatory cascade affecting growth in the crown region; however, further functional studies are warranted to directly test this hypothesis.

WD Inhibits the Transition to a Crown-Root Dominated Root System in S. viridis.

To explore acclimatization to water-limited growing conditions at a whole root system level, we used the newly developed luminescence-based imaging system GLO-Roots (Growth and Luminescence Observatory for Roots), which enables imaging of root systems in sheets of peat-based soil (14). Growth of plants in soil-filled rhizotrons enabled implementation of a similar WD regime as used in our pot-based experiments (Fig. S5). We generated transgenic S. viridis plants that constitutively expressed the LUCIFERASE2 codon-optimized transgene (ZmUbi1::LUC2o) and identified lines that had no measurable growth differences compared with the wild type (WT) A10.1 progenitor background (Fig. S5 F–H). Root systems were imaged at two developmental phases: an early stage, at 11 DAS, when the primary and associated lateral roots form a major portion of the root system and a later stage, at 17 DAS, when crown root-derived branches predominate (Fig. 3 A and B). At 11 DAS, WD-treated root systems grew to encompass a similar area of the soil as WW plants; however, the proliferation of roots occurred deeper in the soil profile (Fig. 3 C–F and Fig. S5 I and K).

Fig. 3.
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Fig. 3.

Transition to a crown-root dominated root system is suppressed in response to WD in S. viridis. (A and B) Luminescence-based images of S. viridis root systems at 11 DAS (A) and 17 DAS (B) subjected to WW and WD conditions. The blue arrowhead indicates the crown region. (C) Quantification of total root system area using ImageJ at 11 and 17 DAS (n = 9–10 plants). (D) Root system width in WW- and WD-treated S. viridis plants measured at varying depths of the rhizotron where lateral roots are present (11 DAS; n = 10). (E) Number of crown roots observed in plants grown in rhizotrons at 11 and 17 DAS (n = 8–11 plants). (F) Analysis of root system directionality in S. viridis root systems at 11 and 17 DAS (n = 8–11 plants). Gray regions indicate the 95% confidence interval. (G) Time-lapse series showing the rapid induction of crown root growth on rewatering. Plants were rewatered at 17 DAS. (Scale bars: 1 cm.) *P < 0.05, Student’s t test. Error bars represent SE.

Fig. S5.
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Fig. S5.

Growth of S. viridis in GLO-Roots rhizotrons. (A) Rhizotron with a S. viridis plant. The black sheet blocking light exposure of soil was removed to visualize the soil. (B) S. viridis plants subjected to WW conditions growing in the rhizotron box (11 DAS). (C) Shoot of WW plant at 15 DAS. (D) Plants subjected to WD conditions growing in the rhizotron box (11 DAS). (E) Plant grown under WD at 15 DAS. (F–H) Comparison of growth characteristics of leaf initiation rate (F), flowering time (G), and crown root number (H) between the WT and S. viridis transgenic line expressing LUC2o (ZmUbi1:: LUC2o) (n = 10 per genotype). (I and J) Analysis of leaf RWC of plants grown in rhizotrons at 11 DAS (I) and 17 DAS (J) under WW or WD conditions (n = 5). (K) Primary root lengths in plants grown in rhizotrons under WW or WD conditions at 11 DAS (n = 10). *P < 0.05, Student’s t test. ns, not significant. Error bars represent SE.

At 17 DAS, a significant difference in the size of the root systems was apparent (Fig. 3 C, E, and F and Fig. S5J). The origin of roots was also markedly different, where WD-treated roots showed proliferation of primary root-derived branches, whereas WW root systems were dominated by crown root-derived branches (Fig. 3 B and E). Rewatering of WD-treated plants revealed a remarkable transformation of these root systems, with crown root growth filling the soil volume within 6 d after watering (Fig. 3G). These data indicate that crown root development is heavily dependent on the availability of water, and that the primary root-derived root system expands to complement the suppression of shoot-borne roots. Thus, the transition from a primary root-dominated system to a crown root-dominated system typical of grasses actually may be an environment-dependent transition and an adaptation that allows grasses to rapidly increase root growth in response to recent precipitation events.

Variation in Crown Root Growth Under WD Conditions Explained by a Small Number of Quantitative Trait Loci.

Unlike S. viridis, cultivated foxtail millet, Setaria italica, maintained an ability to produce a small number of crown roots under WD conditions (Fig. 4 A and B). This difference in crown root number under WD between S. italica and S. viridis is not likely to have an allometric basis, because S. italica has a lower shoot dry weight at this stage (Fig. S6A). S. italica is domesticated from its wild ancestor S. viridis, and the two species are intercrossable (24). We performed a quantitative trait loci (QTL) analysis using a recombinant inbred line (RIL) population from a cross between S. viridis and S. italica. We phenotyped a panel of 153 RIL lines under WW and WD conditions for crown root number and total root system dry weight. Overall, 86.5% of the variation in crown root number is explained by treatment. Two QTLs explaining 7.8% and 11.2% of crown root number variation under WW conditions were identified on chromosomes 5 and 6 (CR-WW5 and CR-WW6) (Fig. 4C and Dataset S5). We found no significant QTLs under WD using total crown root number as a continuous trait; however, when treating the data as a binary trait (presence or absence of crown root), we identified a QTL on chromosome 5 (CR-WD5) (Fig. 4D). Because of the 10% overlap between CR-WW5 and CR-WD5 confidence intervals, whether CR-WD5 is WD-specific is unclear (Dataset S5). Of the 380 genes in the confidence interval of CR-WD5, 28 are differentially expressed in the crown region under WD in our S. viridis RNAseq dataset, thus identifying potential candidates for further study (Dataset S6).

Fig. 4.
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Fig. 4.

Inhibition of postemergence crown root growth is a conserved WD response in the Poaceae that conserves water. (A) Image of WD-treated S. italica crown region showing crown root growth (21 DAS). (B) Comparison of arrested vs. outgrown crown roots in S. viridis and S. italica plants grown under WW and WD conditions (30 DAS; n = 15 plants). (C and D) Logarithm of odds (LOD) score plots showing the QTL regions affecting crown root growth under WW (C) and WD (D) conditions. The scanone 95% threshold is shown (red line) for reference. The number above the peak represents the chromosomal location and cM position. (E) Crown region of maize (B73) plant grown under WW (Left) and WD (Right) conditions (16 DAS). (F) Crown region of Z. mays ssp. parviglumis plants grown under WW (Left) and WD (Right) conditions (16 DAS). (G) Comparison of crown root development between Z. mays ssp. parviglumis and Z. mays ssp. mexicana (16 DAS; n = 10 plants per condition). The asterisk indicates significant difference in the percentage of arrested crown roots by Student’s t test. (H) Crown region of the rtcs mutant lacking crown roots. The arrowhead indicates the crown region. (I) Relative leaf water content of WT or Rtcs/rtcs, and rtcs/rtcs mutant plants (17 DAS; n = 15 for rtcs/rtcs and n = 45 WT/Rtcs/rtcs plants per condition). **Significant genotype × treatment interaction (P < 0.05), two-way ANOVA. (J) Soil moisture content of deep pots with WT or Rtcs/rtcs and rtcs/rtcs maize plants subjected to WW or WD conditions (n = 15). *P < 0.05, Student’s t test. (Scale bars: 1.5 mm in A, 1 cm in E, F, and H.) Error bars represent SE. rtcs/+, Rtcs/rtcs.

Fig. S6.
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Fig. S6.

Variation in shoot and root biomass under WD conditions between S. viridis and S. italica. (A and B) Comparison of shoot (A) and root (B) system dry weight between S. viridis and S. italica grown under WW and WD conditions (30 DAS; n = 5 plants for WW, 15 plants for WD). (C) LOD score plot showing the QTL region involved in regulation of the root biomass under WD. The scanone 95% threshold is indicated for reference. The number above the peak represents the chromosomal location and cM position. Error bars represent SE. *P < 0.05, Student’s t test. ns, not significant.

Total root system biomass in S. italica is significantly higher than that of S. viridis (Fig. S6B). Some 79% of the variation in root weight observed in the RILs is explained by the interaction between the genotype and the treatment, suggesting that the genotypes respond differently to the WD condition. Our analysis revealed one QTL on chromosome 4 (RW-WD4) explaining 12.4% of the total root system biomass variation under the WD condition, whereas no significant QTL was identified under the WW condition (Fig. S6C and Dataset S5). Of the 1,665 genes in this interval, 63 are differentially expressed in our RNAseq dataset, including four putative peroxidases that are strongly down-regulated under the WD condition (Dataset S7). These data indicate that domestication of S. italica may have involved changes at specific loci that contribute to total root system mass and crown root responses to WD.

Inhibition of Postemergence Crown Root Growth Is a Conserved WD Response in the Poaceae.

We analyzed crown root growth in four additional species under WD conditions to test the conservation of the responses observed in S. viridis (Materials and Methods). Sorghum (Sorghum bicolor), switchgrass (Panicum virgatum), and Brachypodium distachyon each showed strong suppression of crown root growth in response to WD, similar to S. viridis (Fig. S7). Five different accessions of sorghum and switchgrass species were analyzed, and all showed similar responses (Fig. S7 A–D). In contrast, the Zea mays (maize) inbred B73 maintained an ability to form some outgrown crown roots under WD. This response differed from the wild relatives of maize, teosinte (Zea mays ssp. mexicana and Z. mays ssp. parviglumis), which showed near-complete suppression of crown root growth (Fig. 4 E–G).

Fig. S7.
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Fig. S7.

Inhibition of postemergence crown root growth is a conserved WD response in the Poaceae. (A) Crown region of sorghum (S. bicolor) plants subjected to WW or WD treatments. (B) Number of arrested vs. outgrown crown roots in WW- and WD-treated plants (21 DAS; n = 5 plants per accession per condition). (C) Crown region of switchgrass (P. virgatum) plants subjected to WW or WD treatment. (D) Number of arrested vs. outgrown crown roots in WW and WD-treated plants (21 DAS; n = 5–8 plants per accession per condition). (E) Crown region of Brachypodium (B. distachyon) plants subjected to WW or WD treatment. (F) Number of arrested vs. outgrown crown roots in WW- or WD-treated plants (38 DAS; n = 10 plants per condition). (Scale bars: 1 cm.) Error bars represent SE.

To explore whether the response of the B73 inbred was representative of maize, we surveyed the response of the nested association mapping (NAM) founder inbred lines, which represent a large portion of the genetic diversity of maize (25). Plants were phenotyped at 12 DAS for WW conditions and 16 DAS for WD, to ensure similar developmental stages were compared. Under WW conditions, the timing of crown root emergence showed limited variation among inbreds [coefficient of variation (CV) = 0.10], whereas the number of outgrown crown roots showed greater variability (CV = 0.28) (Fig. S8 A and B). WD resulted in an increase in phenotypic variation in crown root traits among inbreds. The number of arrested (CV = 0.69) and outgrown crown roots (CV = 0.49) varied substantially across inbred lines. NAM founders, such as M37 and CML69, showed a near-complete resistance to WD-triggered crown root growth arrest, whereas HP301 and NC358 exhibited near-complete arrest of crown root growth comparable to that of teosinte subspecies (Fig. S8B). In contrast to crown root traits, developmental stage of the shoot (V-stage: WW, CV = 0.18; WD, CV = 0.15) and the number of leaves produced showed lower variation across inbreds (WW, CV = 0.11; WD, CV = 0.13) (Fig. S8 C and D). These data show that crown root development remains a highly variable trait in maize inbred lines relative to other developmental traits, particularly under WD stress. Taken together, our results show that inhibition of postemergence crown root growth is a conserved WD response in the Poaceae, and that significant genetic variation exists for this response in maize.

Fig. S8.
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Fig. S8.

Variation in crown root development under WD in maize inbred lines. (A) Survey of variation in crown root emergence among maize NAM founders. Data indicate DAS at which 50% or more plants exhibit emerged crown roots (n = 10 plants). (B) Extent of crown root growth in different maize NAM founders (n = 10 plants). Data collected at 12 DAS for WW and 16 DAS for WD conditions. (C and D) Comparison of V-stage (C) and leaf number (D) of plants grown under WW and WD conditions at 12 and 16 DAS. Error bars represent SE.

Suppression of Crown Root Development Under WD Conditions Preserves Shoot Water Status.

To understand the physiological significance of changes in crown root development during WD, we used the maize rootless concerning crown and seminal roots (rtcs) mutant, which completely abolishes crown root growth while having no significant effect on primary and lateral root growth (26, 27) (Fig. 4H and Fig. S9A). A segregating population of rtcs mutants was germinated and grown under either WW or WD conditions for 17 DAS. RWC measurements demonstrated that homozygous rtcs mutant plants maintained shoot water status better than WT and Rtcs/rtcs plants (Fig. 4I and Fig. S9 C and D). Soil moisture content was greater for rtcs mutant plants compared with WT and Rtcs/rtcs plants (Fig. 4J). Importantly, shoot biomass was not significantly different between genotypes (Fig. S9B), consistent with the root-specific expression of the Rtcs gene (Fig. S9E). Taken together, these data suggest that rtcs mutants conserve more water under WD conditions, likely owing to reduced water uptake by a smaller root system. We hypothesize that for wild species such as S. viridis, suppression of crown root growth under WD may have a similar effect as the rtcs mutant and prevent overdrawing of soil water resources (28), an adaptive strategy known as water banking. The suppression of crown root growth itself also may conserve water, because root tissues require water for cell expansion (29).

Fig. S9.
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Fig. S9.

Analysis of maize rtcs plants subjected to WW and WD conditions. (A) Crown root number between WT/het and rtcs/rtcs maize plants (17 DAS; n = 15 plants). (B) Shoot dry weight of WT/het and rtcs/rtcs maize plants after 17 DAS (n = 15 plants). (C) WT/het and rtcs/rtcs mutant plants at 17 DAS grown under WW conditions. (D) Unlike the rtcs/rtcs mutant, WD-treated WT/het plants showed wilting by 17 DAS. (E) Graph showing the expression pattern of the rtcs gene in different tissues of the maize plant (15). *P < 0.05, Student’s t test. het, heterozygous for the rtcs mutation; ns, not significant. Error bars represent SE. rtcs/+, Rtcs/rtcs.

Discussion

Here we demonstrate the importance of shoot-borne roots in enabling members of the grass family to acclimate to changing water availability in their environment. We find that a key site for sensing water availability is the crown region, and demonstrate that withholding water can dramatically inhibit the postemergence development of crown roots and maintain the root system in its initial primary root-dominated state. We hypothesize that such severe reductions in shoot-borne root growth are crucial to prevent overdrawing of water from the soil and water loss through crown root growth.

Crown roots are necessary to provide mechanical support to the shoot and prevent lodging (7), as well as a level of redundancy required to minimize the damaging effects of biotic stress (30). Based on the results presented here, we can also infer that they provide a strong benefit to the plant in terms of water uptake, given that their development is rapidly activated after the crown locally senses an increase in moisture. The multiaxial nature of the grass root system may enable rapid capture of water from recent precipitation events and provide redundant routes by which water and nutrients can be transported to the shoot. Unlike in eudicots, secondary growth is absent in grasses, which otherwise would increase the vascular capacity of the shoot-root nexus (31).

Perhaps most intriguing from an agricultural standpoint is the difference in WD sensitivity of crown roots in wild and domesticated species of Setaria and maize. The large variation in crown root sensitivity to WD in inbreds of maize suggests that breeders might have inadvertently selected for different response strategies based on the water availability dynamics of the field conditions where selection occurred. In this regard, it is interesting to note that sorghum, which completely abolishes crown root growth under WD, is known to exhibit significant drought tolerance for a crop species (32). Perhaps more targeted breeding for varieties with enhanced WD responses of crown roots may benefit maize productivity when resources are scarce or highly variable.

Materials and Methods

Plant materials and methods for physiological and genetic analysis, plant growth conditions, transgene construction, plant transformation, and the GLO-Roots methodology are described in SI Materials and Methods. Table S1 provides a list of terms used in this work to describe root types, brief definitions of the terms, and associated references. Datasets S1–S8 provide processed RNAseq gene expression values, lists of differentially expressed genes and significantly enriched GO category and MapMan ontology terms, predicted miRNA targeted genes, summarized QTL analysis results, genes within QTL intervals, and a list of primer sequences used in this study.

SI Materials and Methods

Plant Materials, Experimental Conditions, and Measurements.

The S. viridis reference accession A10.1 was used in this study unless indicated otherwise. S. viridis accessions and sorghum, switchgrass, and teosinte lines were obtained from the the US Department of Agriculture’s North Central Regional Plant Introduction Station, Iowa State University. Maize NAM founders were obtained from Torbert Rocheford (Purdue University). Brachypodium seeds were obtained from the laboratory of John Vogel (Department of Energy, Joint Genome Institute).

Deep pots (D25L; Stuewe & Sons) were used for growing plants unless indicated otherwise. Experiments were replicated at least three times. Seeds were germinated in a peat-based soil mixture containing 75% Pro-Mix PGX (Premier Tech) and 25% river sand, imbibed in water to pot capacity. Pot capacity, defined as the amount of water that soil in a pot can hold against the pull of gravity, was estimated to be 230 mL. Plants were grown in a growth chamber (12 h light at 31 °C and 12 h dark at 23 °C with constant relative humidity at 54–55%) and were watered once every third day unless noted otherwise. For WD experiments, seeds were sown in soil imbibed to pot capacity, and no further water was added. To prevent water loss, the bottom of each pot was covered with a plastic bag. To monitor the amount of water loss over the duration of the experiment, pot weight was measured every second day. For WW conditions, the amount of water lost was replenished every third day. Plants were grown in 14-cm-deep pots for the time series analysis of outgrown and arrested crown root formation under WW, WD, and RW conditions. Experimental conditions for teosinte and maize were the same as for S. viridis, except that a soil containing 100% Pro-Mix PGX was used, and seeds were germinated at 50% of pot capacity for WD treatments. Soil moisture content measurements (gravimetric) were performed as follows: Soil samples collected from pots were weighed both before (fresh weight) and after drying (dry weight). Soil samples were dried in an oven at 60 °C for 3 days. Soil moisture (%) was calculated using the following formula: soil moisture (%) = 100 × [(fresh weight – dry weight)/fresh weight].

Field experiments were carried out at the Department of Plant Biology, Carnegie Institution for Science between June 19, 2015, and July 29, 2015. Plants were grown in two equal-sized (1 m × 3 m) raised-bed plots filled with field soil. Each plot was further divided into six subplots, each containing ∼25 plants. Both plots were watered up to 14 DAS, and water was withheld from the WD plot for the next 21 d. Soil moisture was measured regularly using a Diviner 2000 probe (Sentek Technologies). Arrested crown roots were observed and quantified using an Olympus SZ61 stereo microscope.

RWC measurements were carried out as described previously (33). Four to five plants were sampled per treatment and genotype. For each plant, the apical-most fully expanded leaf was sampled for the analysis. Leaf tissue was harvested with surgical scissors from the area between the midvein and the edge. Samples were collected in preweighed Eppendorf tubes and immediately processed. All weight measurements were performed using a microbalance. RWC was calculated using the following formula: RWC (%) = [(W − DW)/(TW − DW)] × 100, where W is sample fresh weight, TW is sample turgid weight, and DW is sample dry weight.

Maize rtcs Genotyping.

Maize rtcs plants were genotyped using the Phire Plant Direct PCR kit (F-130, Thermo Fisher Scientific) with the primers listed in Dataset S8. WT samples yielded a 323-bp PCR product with the rtcs-ATG-79-fw/rtcs-ATG+242-rv primer pair. rtcs/rtcs samples yielded a 325-bp PCR product with the rtcs-ATG-79-fw/rtcs-5bpinsert-rv primer pair.

Transcriptomic Analysis.

Crown tissue samples were collected from plants grown under either WW or WD conditions to 6 and 9 DAS in deep pots. Three biological replicates consisting of 20 plants per sample were collected at 6 and 9 DAS. The 2-mm region containing the crown section and any attached crown roots was cut and placed into a Covaris TT2 tissue bag. The 2-mm region above the crown was cut and placed into a separate bag. Samples were kept at −70 °C until being prepped. S. viridis crowns in tissue bags were frozen in liquid nitrogen and smashed to powder in a Covaris CryoPREP impactor (power setting 4). RNA was extracted using Zymo ZR Plant RNA MiniPrep (R2024). Ribosomal RNA was depleted using Ribozero Plant/Root/Seed (MRZSR116) and strand-specific, barcoded RNAseq libraries were created using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420S; New England BioLabs). Libraries were quantified by qPCR using the Kapa Library Quantification Kit (KK4824; Kapa Biosystems) and pooled in equimolar groups of 12. Pooled libraries were sequenced with HiSeq 2 × 101-bp paired end reads. Reads were mapped to the S. viridis v1.1 genome and analyzed via the Tuxedo pipeline (34).

All RNA-seq data were quality controlled via fastqc. Adapters were filtered using cutadapt, low-quality 3′ sequences were filtered using the fastx toolkit (discarding nucleotides <Q35, reads shorter than 50 nt), and read singletons were discarded. Read pairs that survived the foregoing process were aligned against the S. viridis v1.1 genome (https://phytozome.jgi.doe.gov/) (35) using BWA-MEM (36).

Alignments of RNA-seq data were grouped by tissue/treatment/duration combinations (e.g., the three biological replicates derived from WW upper root samples at 6 DAS were treated as a group). Replicate groups were compared using the Cuffdiff algorithm (i.e., estimating differential expression via dispersion modeling on a Poisson distribution) (34). All possible pairwise comparisons between replicate groups were conducted, and gene loci exhibiting a change in expression with a false-discovery–corrected P value < 0.05 were identified as differentially expressed. Differentially expressed genes were grouped by their directionality of differential expression and analyzed for overrepresentation of GO terms (37) using the topGO R package (38) from Bioconductor (39). GO terms with a false-discovery–corrected P value <0.05 were deemed overrepresented.

The MapMan ontology enrichment analysis was conducted using Oryza sativa annotations (Osa_MSU_v7). Mapping O. sativa to S. viridis (v1.1) genes was done by finding reciprocal blast hits between the two genomes. Pathway enrichment of genes were measured by calculating P values using Fisher’s exact test for each sub-bin.

Real-Time qPCR Analysis.

Crown tissue samples were collected into Covaris TT2 tissue bags from 30 plants per sample in three biological replicates from WW or WD plants grown in 35.5-cm-deep pots for 9 d after sowing. RW plants were grown in WD conditions for 9 d, then watered and harvested 4 h later. Crown tissue was smashed in a cryoPREP impactor, then extracted with TRIzol (Thermo Fisher Scientific) and purified using the Direct-zol RNA MiniPrep Kit (Zymo Research) to maximize the yield of small RNAs. cDNAs of large transcripts were made using iScript Reverse Transcription Supermix (1708841; Bio-Rad) with 1 µg total RNA as input. qPCR was performed using the Bioline SensiFAST SYBR No-ROX Kit in a Roche Light Cycler using NV9 (Sevir.6G210400) as a control. cDNA of miRNA was prepared as described previously (40), except using 2 µg of total RNA as input. qPCR was performed as above using 5.8S rRNA as a control. The qPCR primer sequences are listed in Dataset S8.

GLO-Roots Imaging System for S. viridis.

To visualize Setaria root systems in soil using the GLO-Root imaging system, transgenic plants expressing the luciferase gene were generated. A plant codon-optimized luciferase gene, LUC2o (14), was cloned into the monocot binary vector pANIC10 (41). Transgenic S. viridis plants harboring ZmUbi1::LUC2o were regenerated from callus as described previously (42). Transgenic T2 plants were surface-sterilized and germinated in tissue culture plates as described previously (43). For comparison of growth characteristics of WT and transgenic lines, plants were grown in deep pots filled with Pro-Mix soil. For growth of plants in rhizotrons, seeds were first germinated on agar media and then, at 3 DAS, transferred to rhizotrons and maintained in the growth chamber (12 h light at 31 °C and 12 h dark at 23 °C with constant relative humidity at 54–55%). The WW-treated plants were watered daily using a transfer pipet, but the WD-treated plants received no further watering from 5 DAS onward. Plant imaging and image processing were done as described previously (14). Directionality analysis, which computes the mean direction of roots in the root system, was done as described previously using the GLO-RIA ImageJ plug-in (14). Total root system area was calculated using ImageJ (44).

QTL Analysis.

RILs were grown as described above. One plant per RIL per condition was phenotyped. QTL mapping was performed using the R/qtl package (45) on raw experimental values. The analysis was performed on datasets collected within each treatment block and on the numerical difference of values between treatments using 153 RILs and a genetic map containing 1,595 SNP markers. Haley–Knott regression was used to perform a single QTL scan at each marker individually. One thousand permutations were performed to determine significance thresholds for inclusion additive QTL at α = 0.05. Automated stepwise model selection was used to determine a final additive QTL model. After refinement of QTL position estimates, phenotypic values were fit to the QTL model using ANOVA to assess the proportion of variance explained and effect size of each locus. All putative protein coding genes (S. viridis genome v1.1) found within a 1.5-LOD confidence interval were reported for each QTL.

Acknowledgments

We thank Neil Robbins II and Josep Vilarrasa-Blasi for comments on the manuscript; Therese LaRue for help with figure preparation; Neil Robbins II for bulking of rtcs/+ seeds; the US Department of Agriculture (North Central Regional Plant Introduction Station, Iowa State University), Torbert Rocheford (Purdue University), and John Vogel for seeds; and Todd Tobeck (Department of Global Ecology, Carnegie Institution for Science) for help with field soil moisture measurements. Funding was provided by the US Department of Energy’s Biological and Environmental Research program (Grant DE-SC0008769, to T.P.B., T.C.M., I.B., and J.R.D.) and the National Science Foundation’s Plant Genome Research Program (Grant IOS-PGRP 420-40-45A, to J.R.D.).

Footnotes

  • ↵1J.S. and M.-C.Y. contributed equally to this work.

  • ↵2To whom correspondence should be addressed. Email: jdinneny{at}carnegiescience.edu.
  • Author contributions: J.S., M.-C.Y., and J.R.D. designed research; J.S., M.-C.Y., W.G.V., and M.F. performed research; R.R.-A., M.F., H.D.P., T.L., H.J., T.C.M., F.H., and T.P.B. contributed new reagents/analytic tools; J.S., M.-C.Y., R.R.-A., M.F., H.D.P., C.T., T.L., I.B., and J.R.D. analyzed data; and J.S., M.-C.Y., R.R.-A., M.F., H.D.P., C.T., T.L., F.H., and J.R.D. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE78054).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1604021113/-/DCSupplemental.

Freely available online through the PNAS open access option.

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Grasses suppress shoot-borne roots during drought
Jose Sebastian, Muh-Ching Yee, Willian Goudinho Viana, Rubén Rellán-Álvarez, Max Feldman, Henry D. Priest, Charlotte Trontin, Tak Lee, Hui Jiang, Ivan Baxter, Todd C. Mockler, Frank Hochholdinger, Thomas P. Brutnell, José R. Dinneny
Proceedings of the National Academy of Sciences Jul 2016, 201604021; DOI: 10.1073/pnas.1604021113

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Grasses suppress shoot-borne roots during drought
Jose Sebastian, Muh-Ching Yee, Willian Goudinho Viana, Rubén Rellán-Álvarez, Max Feldman, Henry D. Priest, Charlotte Trontin, Tak Lee, Hui Jiang, Ivan Baxter, Todd C. Mockler, Frank Hochholdinger, Thomas P. Brutnell, José R. Dinneny
Proceedings of the National Academy of Sciences Jul 2016, 201604021; DOI: 10.1073/pnas.1604021113
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