Reduction in Root Secondary Growth as a Strategy for Phosphorus Acquisition  

Abstract

We tested the hypothesis that reduced root secondary growth of dicotyledonous species improves phosphorus acquisition. Functional-structural modeling in SimRoot indicates that, in common bean (Phaseolus vulgaris), reduced root secondary growth reduces root metabolic costs, increases root length, improves phosphorus capture, and increases shoot biomass in low-phosphorus soil. Observations from the field and greenhouse confirm that, under phosphorus stress, resource allocation is shifted from secondary to primary root growth, genetic variation exists for this response, and reduced secondary growth improves phosphorus capture from low-phosphorus soil. Under low phosphorus in greenhouse mesocosms, genotypes with reduced secondary growth had 39% smaller root cross-sectional area, 60% less root respiration, 27% greater root length, 78% greater shoot phosphorus content, and 68% greater shoot mass than genotypes with advanced secondary growth. In the field under low phosphorus, these genotypes had 43% smaller root cross-sectional area, 32% greater root length, 58% greater shoot phosphorus content, and 80% greater shoot mass than genotypes with advanced secondary growth. Secondary growth eliminated arbuscular mycorrhizal associations as cortical tissue was destroyed. These results support the hypothesis that reduced root secondary growth is an adaptive response to low phosphorus availability and merits investigation as a potential breeding target.

Most soils on earth have suboptimal phosphorus (P) availability for plant growth (Vance et al., 2003; Lynch and Brown, 2008; Lynch, 2011), as it is only available to plants as inorganic P and is rarely present in concentrations greater than several micromolar in soil solution (Bieleski, 1973). Diffusion of P in soil is greatly outpaced by plant uptake, resulting in the formation of P-depletion zones around roots (Hinsinger et al., 2005). Due to the limited availability and slow movement of P in soil, one of the most effective strategies of increasing P uptake is to increase the volume of soil explored by the root system. This accounts for the increase in the root-shoot ratio under P stress (Fohse et al., 1988). Although increasing resource allocation to root growth improves P acquisition, unbalanced root development reduces overall plant growth due to the increased metabolic cost of added root tissue (Nielsen et al., 1998, 2001; Lambers et al., 2006). Over 50% of daily carbon fixation may be consumed by the root system, with P-stressed plants allocating a larger fraction of their daytime net carbon assimilation than nonstressed plants (Van Der Werf et al., 1988; Lambers et al., 1996; Nielsen et al., 1998, 2001). This cost consists of three main components: the growth of new root tissue, ion uptake and assimilation, and the maintenance of existing root tissue (Nielsen et al., 1998; Fan et al., 2003). Nielsen et al. (1998) found that, in common bean (Phaseolus vulgaris) under P deficit, the proportion of total root respiration allocated to maintenance accounts for approximately 90% of total root respiration. The functional-structural model SimRoot has estimated that the cost of maintenance respiration of the root system constitutes 40% of the total growth reduction under P stress (Postma and Lynch, 2011). Consequently, the greatest opportunity to reduce the metabolic burden of the root system lies in moderating maintenance costs.

To improve the balance between soil exploration and the consumption of growth-limiting resources, a decrease in root secondary growth would reduce the carbon cost of producing and maintaining root length (Lynch, 1995). It has been hypothesized that this may be an adaptive strategy to improve the metabolic efficiency of soil foraging under P stress, where roots will favor primary growth (elongation) over secondary growth (radial thickening) to achieve greater exploration of soil domains that have not been depleted of P (Lynch, 2007, 2011; Lynch and Brown, 2008; De la Riva and Lynch, 2010).

Previous observations of root systems under P stress provide evidence for the importance of root diameter in a diversity of plant species. In the sedge Carex coriacea, specific root length (i.e. root length per mass of root tissue) was negatively correlated with P availability, with a 30% reduction in root diameter from high P to low P (Powell, 1974). In a study of the root morphology of temperate pasture species, a reduction in root diameter, root mass density, and an increase in specific root length were observed under P stress (Hill et al., 2006). Reduced lateral root diameter also has been described under low P in water hyacinth (Eichhornia crassipes) and maize (Zea mays; Xie and Yu, 2003; Zhu and Lynch, 2004). Nevertheless, observations of reductions in root diameter under P stress have not been made within root classes, and published reductions in root diameter of the entire root system may be the product of a greater proportion of higher order lateral roots rather than the effects of altered secondary growth. To determine if the reduction of secondary growth is an adaptive response, an explicit study of secondary growth within root classes under P stress is required (Lynch and Brown, 2008).

During secondary growth, periclinal cellular divisions and differentiation of secondary tissues at the vascular cambium and phellogen cause the splitting and destruction of the epidermis, cortex, and endodermis. While the production of the periderm replaces these primary tissues and helps to protect the vasculature of the root, the bulk of secondary thickening is driven by the production of secondary xylem elements and parenchyma internal to the vascular cambium (Dickison, 2008). This elimination of the primary tissues and proliferation of secondary tissue are observed in transverse sections as the loss of the epidermis, cortex, and endodermis, expansion of the stele, and increases in the abundance and size of metaxylem vessels (Supplemental Fig. S1). Under P deficiency, the observed changes in root anatomy of a variety of species include smaller root diameter and stele diameter, fewer and smaller epidermal cells and metaxylem vessels, reduced percentage of stele area, and fewer cortical cells and xylem vessels (Fohse et al., 1991; Fan et al., 2003; Liu et al., 2004; Sarker et al., 2015).

In this study, we utilize functional-structural modeling as well as empirical observations of plants grown in controlled environment mesocosms and in the field to explore the effect of reduced secondary growth of roots on P acquisition. Our goals were to test the hypotheses that (1) secondary growth is suppressed by P stress, (2) genetic variation exists for this response, and (3) reduced secondary growth of roots improves P acquisition. We address these hypotheses by first utilizing the functional-structural plant model SimRoot to determine the relationship between secondary growth of roots and P acquisition, followed by greenhouse and field studies to validate in silico results.

The common bean was used to test these hypotheses due to observed genotypic variation in P acquisition and metabolic efficiency of roots under P stress (Nielsen et al., 2001; Beebe et al., 2006; De la Riva and Lynch, 2010; Henry et al., 2010; Miguel et al., 2015). Common bean also is an important food security crop in Africa and Latin America, where its productivity often is limited by low P availability.

RESULTS

Phenotypic Classification

Total cross-sectional area (TCSA; mm²) of the root and the percentage of stele area from greenhouse-grown root segments were used to categorize genotypes into two phenotypic groups that were used as a factor in data analysis. Genotypes with mean TCSA < 0.5 mm² and less than 50% stele in the basal segment at the time of flowering (46 d after planting [DAP]) were classified as having reduced secondary growth (reduced; DG6, DG35, L8814, and L8863). Genotypes with mean TCSA > 0.5 mm² and greater than 50% stele in the basal segment at flowering were categorized as having advanced secondary growth (advanced; DG23, DG51, L8843, and L8857).

Effects of Reducing Secondary Growth in Silico

When root secondary growth was reduced by 50% (intermediate phenotype), by 40 DAP under P stress (4 μmol L⁻¹ available P) root respiration per g was reduced by 14%, total root length was increased by 7%, P acquisition was increased by 9%, and shoot mass was increased by 17% from the advanced phenotype (Fig. 1; Supplemental Fig. S2). In the reduced phenotype, where roots had no secondary growth, root respiration was reduced by 12%, total root length was increased by 14%, net P acquisition was increased by 15%, and shoot mass was increased by 31% from the advanced phenotype (Fig. 1; Supplemental Fig. S3). In environments with greater P availability, root respiration rate was suppressed and root length was increased to a greater degree, but the impact of these root parameters on improving P capture and shoot biomass was less pronounced (Fig. 1). When P availability was more strongly stratified with depth, the benefits to reducing secondary growth became less pronounced (Supplemental Fig. S4).

Plant Growth in Mesocosms

Under high P in mesocosms, no genotypic differences in shoot size and shoot P were observed, while under P stress, reduced genotypes had 68% greater shoot mass and 78% greater shoot P than advanced genotypes by 46 DAP (Figs. 2 and 3). Under high P, there were no phenotypic differences in root mass, while under P stress, reduced genotypes had significantly greater root mass than advanced genotypes (Supplemental Fig. S5). Within each treatment, no phenotypic differences in root-shoot ratios were observed (Supplemental Fig. S5). Allometric analysis revealed that root mass had a hyperallometric relationship to shoot size under both P treatments (Table I). All genotypes had statistically similar basal root whorl number, basal root number, and adventitious root number.

Allometric analysis comparing shoot biomass and root phenes in the greenhouse at 46 DAP and in the field at 49 DAP

By 46 DAP under P stress, reduced genotypes had 56% greater specific root length and 27% greater basal root length than advanced genotypes, while in the high-P treatment, both phenotypic groups had statistically similar specific root length and basal root length (Fig. 3). Although a trend of thinner lateral roots and greater net lateral root length was observed for the reduced genotypes under P stress, statistical analysis did not reveal a significant genotypic effect at P < 0.05 (Supplemental Fig. S6). Under P stress, specific root length and basal root length were both positively correlated with shoot P content by 32 DAP, while in the high-P treatment, no relationship was observed.

Plant Growth in the Field

Under high P in the field, no genotypic differences in shoot size and shoot P content were observed, while in the P stress treatment, reduced genotypes had 80% greater shoot mass, 62% greater leaf number, and 58% greater shoot P than advanced genotypes by 49 DAP (Fig. 4). Under P stress, reduced genotypes had 32% greater root length density in the top 40 cm of soil than advanced genotypes, while under high P, no genotypic differences were detectable (Fig. 4). Root length density in the top 40 cm was positively correlated with total shoot P under P stress, while no relationship was observed under high P (Fig. 5). Root length density in the top 40 cm of soil had a hyperallometric relationship with shoot size under P stress but not under high P (Table I). All genotypes had statistically similar basal root whorl number, basal root number, adventitious root number, and basal root growth angle.

Root Anatomy

In mesocosms, TCSA, percentage stele, metaxylem vessel number, total metaxylem vessel area, and hydraulic conductance increased significantly from 18 to 46 DAP and from the apical to the basal end of the root in both P treatments (Fig. 6; Supplemental Figs. S7–S10). P stress significantly reduced these anatomical phenes in both greenhouse- and field-grown roots (Fig. 7). Differences in TCSA between reduced and advanced genotypes were statistically detectable by 18 DAP under P stress (Fig. 6). P treatment temporal and phenotypic effects were most detectable in the basal segment, where the greatest secondary growth had occurred. In this segment at 46 DAP, reduced genotypes displayed 29% smaller percentage stele area, 21% lower metaxylem number, 48% less metaxylem area, and 52% less hydraulic conductance than advanced genotypes under P stress (Fig. 7; Supplemental Figs. S7–S10). At 49 DAP in the field, reduced genotypes had 43% smaller TCSA, 26% lower metaxylem number, 41% reduced metaxylem area, and 55% less hydraulic conductance than advanced genotypes under P stress (Fig. 8; Supplemental Fig. S11). Allometric analysis indicates that differences in secondary growth of roots (TCSA, percentage stele, metaxylem area, and metaxylem number) were not driven by differences in plant size in either the greenhouse or the field (Table I). Under P stress, a negative relationship between basal TCSA and total shoot P was observed, but under high P, no significant association between basal TCSA and shoot P was detected (Fig. 9).

In mesocosms under P stress, TCSA of the basal segment was negatively correlated with basal root length at all time points, while under high P, no relationship was statistically observable (Fig. 9). Similarly, at 49 DAP under P deficit in the field, basal TCSA was negatively correlated with root length density in the top 40 cm of soil (Fig. 5). This association between the allocation of resources from secondary growth to primary growth can be represented by the ratio of basal TCSA to total basal root length. The mean TCSA-total basal root length ratio at 46 DAP in the greenhouse was greater for genotypes classified as advanced than for most genotypes in the reduced group with the exception of L8863 (Supplemental Fig. S12). Genotypes in the advanced group had a mean TCSA:root length ratio > 0.1, while genotypes in the reduced group had a mean TCSA:root length ratio < 0.1.

Root Respiration

By 46 DAP in the greenhouse, reduced genotypes had 60% less basal segment respiration, 47% less middle segment respiration, and 69% less apical segment respiration than advanced genotypes under P deficit (Fig. 10). There was a strong positive relationship between respiration rate per unit of length and TCSA in all P treatments and time points (Fig. 9). By 32 DAP under P stress, respiration per unit of length of all segments was negatively correlated with basal root length, and by 46 DAP (Supplemental Fig. S13), respiration per unit of length of all locations was negatively correlated with shoot mass (Supplemental Fig. S14). These relationships between root respiration and root length and shoot mass were not present in the high-P treatment (Supplemental Figs. S13 and S14).

Root Construction Costs

Root P concentration decreased significantly with root segment age in the high-P treatment but was consistent for all root segments in P-stressed plants (Supplemental Fig. S15). Nitrogen (N) concentration was significantly greater in the growing root tips than in older root segments and was significantly greater in P-stressed roots than in roots grown under high P (Supplemental Fig. S15B). Carbon (C) concentration was statistically similar for root segments in all P treatments, time points, and positions along the root. There was no effect of P treatment on the C-H ratio of roots and no detectable change in the C-H ratio among locations, but C-H ratio increased significantly at the basal segment over time. P stress significantly reduced C-N ratio, and C-N ratio increased with root segment age. These increases in C-N were driven by a reduction in N, not changes in C. No statistically observable differences in the elemental concentration of roots were observed between advanced and reduced genotypes.

Mycorrhizal Synergism with Secondary Growth

Despite the observed genotypic differences in secondary growth, no statistically detectable genotypic differences in mycorrhizal symbiosis were observed under P stress. While roots grown under P stress had significantly greater mycorrhization of the basal-most segment, roots grown under high P displayed the opposite pattern of symbiosis, with the least abundance of mycorrhizal structures in the basal segment (Fig. 11C). Basal segments from the P stress treatment had 367% greater cortical tissue area than basal segments in the high-P treatment, and across both P treatments, there was a significant positive relationship between cortical tissue area and symbiosis (Fig. 11, A, B, and D).

DISCUSSION

SimRoot predicted that reducing the secondary growth of roots reduces metabolic costs, liberates resources for greater primary growth, and thereby augments the total quantity of P captured. These predictions were confirmed by in vivo observations under P stress in the greenhouse and field. These results confirm the hypotheses that (1) in low-P soil, roots of this dicot species favor primary growth over secondary growth (Figs. 4, 5, and 7–9), (2) genetic variation exists for this response (Figs. 3–6 and 8; Supplemental Figs. S11 and S12), and (3) the reallocation of resources from secondary to primary growth improves P acquisition (Figs. 1, 3, 5, and 9; Supplemental Fig. S2).

Because SimRoot does not explicitly consider beneficial aspects of secondary root growth, such as axial water transport (Valenzuela-Estrada et al., 2008), mechanical support of the shoot, or resistance to herbivores and pathogens (Eissenstat, 1992; Valenzuela-Estrada et al., 2008), reduced secondary growth is unconditionally beneficial for improving root length and P acquisition in silico. In vivo, secondary growth is a constitutive characteristic of dicot roots, and the inverse relationship between secondary growth and P acquisition predicted by the model is only present under P stress. Under high P, plants are able to acquire adequate P to support the production of less efficient roots without reducing plant growth. The observation that secondary growth is inhibited only under P stress would suggest that increased root diameter affords increased fitness in fertile environments.

Despite possible drawbacks to reduced root diameter, reallocation of resources among different tissues is a hallmark adaptive response to P deficiency (Fohse et al., 1988). This concept is evident in this study through the greater allometric scaling coefficient for root mass under P stress compared with high-P conditions, demonstrating a shift in resources from shoot growth to soil exploration. Although there was an increase in root-shoot ratio under P stress, there was no phenotypic difference for this metric within the P stress treatment. While the relative investment of resources to roots of both phenotypic groups was comparable, the allocation of those resources within the root system of reduced and advanced groups differed. This shift in allocation of resources within the root system is evidenced by the reduction in root diameter and metabolic cost per length of root and the increase in total root length of the reduced genotypes under P stress. Genotypic variation in the metabolic efficiency of bean roots under P stress has been reported previously by Nielsen et al. (2001), where there was no difference in daily carbon allocated to roots of P-efficient and P-inefficient genotypes of bean, but P-efficient genotypes were able to maintain a larger root system per unit of carbon respired than inefficient genotypes. Further evidence for a genetic component to root etiolation is reported in a quantitative trait locus (QTL) study by Beebe et al. (2006) for root architectural phenes using the same DOR 354 × G19833 (DG) recombinant inbred lines (RILs) as in this study. This work found that these DG RILs displayed genetic variation in P accumulation per unit of root length, and two significant QTLs for P accumulation under P stress were identified in the same regions as QTLs for root length and for specific root length, with joint QTL analysis uncovering a positive relationship between specific root length and P accumulation under P stress (Beebe et al., 2006).

The observed effect of P deficit on root length at 18 DAP suggests that the reduction of secondary growth in root segments at later time points may be in part the ancillary effect of shifts in the allocation of resources to primary growth. The regulation of root elongation under P deficiency has been described previously and attributed to ethylene signaling pathways in Arabidopsis (Arabidopsis thaliana; Ma et al., 2003). In bean, ethylene production is greater in roots grown under P stress and serves to maintain root elongation under P stress, while ethylene inhibits elongation under high-P conditions (Borch et al., 1999; Liao et al., 2001). Additionally, the reduction in root diameter and respiration rate at the root apex under P stress indicates that the differences in secondary growth are not strictly the result of the gradual accrual of differences in secondary growth in older segments of root over time but are initiated at the root apex. The anatomy of these thinner apical segments from the P stress treatment did not manifest as an isometric reduction in both cortex and vascular tissue; rather, we observed thinner apical segments that had reduced cortex area but comparable stele area, metaxylem number, and metaxylem area to apical segments in the high-P treatment (Supplemental Fig. S16). These P-stressed root apices in reduced genotypes achieve smaller TCSA and decreased respiration through reduction in cortical tissue while maintaining the same amount of vasculature necessary for axial transport.

Unlike roots in the high-P treatment, where P concentration of root tissue decreased with secondary growth, under P stress, root P concentration remained stable over time. This may indicate that the P stress was substantial and the P concentration in root tissue was being sustained at the minimal level required to maintain living tissue. While no phenotypic differences in nutrient concentrations of roots were observed between reduced and advanced groups under P stress, advanced genotypes have greater root diameter and, therefore, greater total nutrient content per length compared with reduced genotypes. In addition to the smaller respiration rate per length of thinner roots, this savings in construction costs is another avenue for the conservation of resources in reduced genotypes under P stress. Furthermore, as secondary growth progresses, the vascular tissue expands and the living cortex is destroyed, thereby shifting the physiological role of roots from resource capture to axial transport (McCully, 1999). While reduced genotypes had diminished axial conductance, the delayed transition into the role of axial transport may allow roots to acquire more resources from the surrounding soil for a greater length of time.

In addition to the possible benefit of greater direct nutrient uptake by the root, retarded stele development and maintenance of cortical tissue in roots under P stress have a synergistic effect on P uptake through the preservation of arbuscular mycorrhizal associations that colonize the cortex. While it is well known that P availability suppresses mycorrhizal associations in ways unrelated to secondary growth, the significant relationship between cortical area and fungal colonization across P treatments suggests that secondary growth inhibits arbuscular mycorrhizal relationships. These results reinforce Brundrett (2002), who suggested that plant species with less root cortical volume sacrifice the capacity for arbuscular mycorrhizal associations, and Valenzuela-Estrada et al. (2008), who observed this in Vaccinium spp., where roots with greater radial growth and reduced specific root length had less mycorrhizal colonization.

While suppression of secondary growth appears to facilitate mycorrhizal symbiosis, it also may increase the vulnerability of roots to soil pathogens and herbivores. Although there was no greater incidence of disease observed in roots of reduced genotypes, in soils where pathogens and herbivores are prevalent, roots with advanced secondary growth may have greater longevity than those with reduced secondary growth. This relationship between anatomical development and disease was demonstrated in Malus domestica, where pathogen colonization is closely linked to the senescence and loss of the root cortex (Emmett et al., 2014). Although P limitation alone does not diminish root survivorship in bean grown in sand culture, in the field where soil biota are present, up to 49% of roots are lost by late pod filling (Fisher et al., 2002).

These results support growing evidence that root phenes and phene states that reduce the metabolic cost of soil exploration are adaptive in resource-poor soil environments (Chimungu et al., 2014a, 2014b, 2015; Saengwilai et al., 2014; Lynch, 2015; Miguel et al., 2015; Schneider et al., 2017). In this context, root anatomical phenes merit attention as breeding targets for more stress-tolerant crops.

CONCLUSION

These results support the hypothesis that reduced root secondary growth increases the resources available for primary growth, thereby increasing the total volume of soil explored and the acquisition of soil resources. Although all bean genotypes tested favor primary growth of roots over secondary growth under P stress, genotypes differ in the intensity of this response. Genotypes with reduced secondary growth had suppressed anatomical development, reduced metabolic and construction costs per length of root, greater soil exploration, and greater P acquisition than genotypes with advanced secondary growth. These results demonstrate the adaptive significance of reduced secondary growth under P stress, but further work to determine the influence of reduced hydraulic conductance in roots with reduced secondary growth on water capture in drying soils would be of merit, as well as a targeted investigation into the relationship between secondary growth and the colonization of arbuscular mycorrhiza. Further research may elucidate if a reduction in root secondary growth improves soil resource capture under drought and other nutrient deficiencies.

MATERIALS AND METHODS

Germplasm

Previous work by De la Riva and Lynch (2010) identified two genotypes of common bean (Phaseolus vulgaris) contrasting in P acquisition and root development under P stress. DOR 364 is a high-yielding genotype developed by breeders at CIAT in Cali, Colombia (CIAT, 1996). Despite exhibiting a strong reduction in secondary growth of roots under low-P conditions, it has been identified as being P inefficient due to other components of the root phenotype (Liao et al., 2004). G19833 is a Peruvian landrace from the Andean gene pool (Beebe et al., 1997). This genotype displays less reduction in secondary growth of roots than DOR 364 under P stress (De la Riva and Lynch 2010) but is classified as P efficient due to the contribution of other beneficial root phenes (phene is to phenotype as gene is to genotype; Serebrovsky, 1925; York et al., 2013), including a shallow basal root angle (Bonser et al., 1996), high basal root whorl number (Miguel et al., 2013), and long, dense root hairs (Yan et al., 2004). DOR 364 and G19833 were selected for their contrasting P efficiency and root characteristics and crossed to generate a population of RILs. The DG RIL population was then screened for variation in root secondary growth under P stress, and four genotypes were selected for their observed differences in secondary growth. These genotypes include DG 6 (reduced secondary growth), DG 35 (reduced secondary growth), DG 23 (advanced secondary growth), and DG 51 (advanced secondary growth). Additionally, genotypes from the L88 RIL population (developed by J. Kelly, Michigan State University), generated from a cross between drought-resistant B98311 and P-efficient TLP 19 (Frahm et al., 2004), were selected for their differences in secondary growth of roots under low P. These genotypes include L88-14 (reduced secondary growth), L88-63 (reduced secondary growth), L88-43 (advanced secondary growth), and L88-57 (advanced secondary growth). A multiline study by Henry et al. (2010) further supports the purported contrast in secondary growth between these genotypes, where under low P in the field, L88-14 had thinner roots and more roots per root core while L88-57 had thicker roots and fewer roots per core.

In Silico Study

The functional-structural plant model SimRoot is able to integrate parameters of root growth, nutrient uptake, and resource allocation from in vivo studies to model the relationship between root growth and performance of bean (Postma et al., 2017). To investigate the relationship between secondary growth in roots and P acquisition efficiency, root systems of bean were modeled with three different secondary growth rates; root systems with no secondary growth (reduced), root systems with 50% of the secondary growth rates observed under high-P conditions (intermediate), and root systems with the same secondary growth rates observed under high-P conditions (advanced). All other plant properties were held constant in all simulations. For each level of secondary growth, P availability was varied from 0.17 to 5 kg ha⁻¹ across 13 levels. Here, P concentration represents the quantity available to the plant in the soil solution, and the buffer capacity of the soil (the ratio between the dissolved and absorbed fraction) is held constant. In total, 39 simulations (three secondary growth rates × 13 P levels) were run on the Pennsylvania State University clusters (https://ics.psu.edu/wp-content/uploads/2015/09/Lion-X-Manual.pdf). Starting from germination, plant growth was simulated for 40 d and root growth was permitted to grow within a 60- × 60-cm by 1.5-m deep soil volume. Any roots that intersected the boundary of the soil environment were mirrored back to maintain a total root length similar to that of field conditions.

In SimRoot, carbon used for growth comes from either seed reserves or photosynthesis. The model is inclusive of multiple components of metabolic costs stemming from respiration, nitrogen fixation, nutrient uptake, and production of exudates. Root system architecture is represented by a network of root nodes and is modeled in three dimensions. Shoot growth is simulated nongeometrically and is represented by integral parameters such as leaf area and dry shoot mass. P uptake at each root node is parameterized using the Barber-Cushman model and integrated over the length of the root system (Barber and Cushman, 1981). When P availability is inadequate to satisfy optimal growth, leaf area expansion, photosynthesis, and root growth are inhibited. Interroot competition for P is simulated in one dimension by the Barber-Cushman model and is dependent on the average root density within 1 cm of the root (Postma and Lynch, 2011). In SimRoot, the sink strength of a given organ is based on the resource requirements for potential growth and maintenance of the tissue. The growth rate of all root classes, leaves, and stem tissue is based on empirical data. Roots of greater thickness have greater longitudinal potential growth rates and, consequently, have greater sink strength (Pages, 2000). Resources required for secondary growth are determined by the volumetric increase associated with the class, location, and age of each root segment. Respiration is a function of the root segment biomass and age. Further information on SimRoot is provided by Postma and Lynch (2011). An overview of SimRoot parameterization is available in Supplemental Data S1, and files used to generate this simulation are available at https://doi.org/10.5281/zenodo.998950.

Greenhouse Study

This study was conducted in a greenhouse located at Pennsylvania State University in University Park (40.801955°N, 77.862544°W). Plants were grown from April through May 2016 under a 16/8-h (light/dark) photoperiod and maximum/minimum temperatures of 34°C/20°C. Midday photosynthetic active radiation was approximately 900 to 1,000 μmol photons m⁻² s⁻¹. Natural light was supplemented from 6 am to 10 pm with 110 μmol photons m⁻² s⁻¹ from LED Illumitex ES2 lights (Illumitex). A complete randomized block design was utilized with two P levels: P stress and high P. The experiment was run for a total of 46 d, with destructive measurements taken from all genotypes in all treatments at 18, 32, and 46 DAP. Each genotype at each time point and treatment had four replications.

Seeds were surface sterilized in a 25% NaOCl solution for 2 min, rinsed in deionized water, and germinated in 0.5 mm CaSO₄ in the dark at 28°C for 24 h. Uniform seedlings were transplanted to the greenhouse in opaque, 20-L mesocosms 30 cm in diameter and 44 cm in height, wrapped in silver duct tape to enhance reflectiveness. Mesocosms were filled with a mixture of 40% coarse grade A perlite (Whittemore), 30% medium-grade sand (Quikrete), 20% low-P soil (Ap2 Hagerstown silt loam [fine, mixed, semiactive, mesic Typic Hapludalf]; available P = 12 ppm) sieved through 6-mm mesh, and 10% D3 coarse grade A vermiculite (Whittemore). The soil was incorporated to replicate features found under field conditions, such as the presence of organic matter, soil biota, and oxide surfaces that serve to buffer P availability. Mesocosms designated as being part of the high-P treatment received 40 g of granular triple superphosphate (25% P) incorporated into the medium at the time of mixing. Mesocosms assigned to the low-P treatment did not receive any supplemental P. Other nutrients were supplied through drip irrigation once daily. At each irrigation event, high- and low-P mesocosms received 400 mL of nutrient solution. This nutrient solution contained 1.5 mm KNO₃, 1.2 mm Ca(NO₃)₂, 0.4 mm NH₄NO₃, 0.025 mm MgCl₂, 0.5 mm MgSO₄, 0.3 mm K₂SO₄, 0.3 mm (NH₄)₂SO₄, 5 μm Fe-EDTA, 1.5 μm MnSO₄, 1.5 μm ZnSO₄, 0.5 μm CuSO₄, 0.15 μm (NH₄)₆Mo₇O₂₄, and 0.5 μm Na₂B₄O₇. The pH of the nutrient solution was adjusted as needed at every other irrigation event to 5.8 with KOH and HCl.

At 18, 32, and 46 DAP, destructive shoot measurements were taken, including leaf number, dry shoot biomass, and leaf tissue P content. Dry mass was determined from tissues dried at 65°C for 7 d. Leaf P content was measured spectrophotometrically after ashing leaf tissue at 500°C for 16 h (Murphy and Riley, 1962).

The root system of each plant was extracted and washed, and basal root whorl number, basal root number, adventitious root number, root respiration rate, root P content, specific root length, basal root length, and anatomical phenes were measured. Root respiration rates were determined immediately after washing for two 10-cm segments taken from representative basal roots at the 10 cm nearest to the hypocotyl (basal), 10 cm at the middle of the root axis (middle), and 10 cm from the growing tip back (apical; Supplemental Fig. S17). To relate differences in respiration rates to secondary growth of the primary root axis, lateral roots were removed from these segments with a razor prior to respiration measurements. Respiration rates were measured using a Li-Cor 6400 gas-exchange system with a modified respiration chamber (Li-Cor). Measurements were performed under ambient greenhouse conditions, with the sealed chamber being submerged in a water bath kept at 28°C and baseline sample chamber and reference chamber CO₂ concentration of 400 μmol mol⁻¹.

Following respiration measurements, 2.5 cm of each root segment used for respiration measurements was used for the characterization of anatomy. These segments were preserved using a Leica EM CPD300 critical point dryer (Leica Microsystems). Preserved segments were sectioned with laser ablation tomography using an Avia 7000, 355-nm pulsed laser and simultaneously imaged with a camera equipped with a 5× zoom lens. Root cross-section images were analyzed using MIPAR software (MIPAR.beta.8). Anatomical features measured include TCSA, percentage stele area, metaxylem number, and metaxylem area. Theoretical axial metaxylem conductance (k_h; kg m MPa⁻¹ s⁻¹) was calculated for each cross-sectional image using the modified Hagen-Poiseuille law (Eq. 1), where d is the diameter of the vessel in meters, ρ is the fluid density (equal to water at 20°C; 1,000 kg m⁻³), and η is the viscosity of the fluid (equal to water at 20°C; 1 × 10⁻⁹ MPs s⁻¹; Tyree and Ewers, 1991). The remaining 7.5 cm of root segments used in respiration measurements was dried at 65°C for 7 d, and a 2.5-mg subsample of this tissue was analyzed for N, C, and H content using an elemental analyzer (Series II CHNS/O Analyzer 2400; PerkinElmer).

Basal root whorl number, basal root number, and adventitious root number were determined by counting the root whorls and basal roots after washing the root system. One intact, representative basal root was sectioned into 20-cm segments along its primary axis, and each segment was imaged using an EPSON Perfection V700 PHOTO scanner. From the scanned image, total basal root length, including length and diameters of lateral roots, was quantified with WinRhizo software (WinRhizo Pro; Regent Instruments). The scanned basal root was then dried at 65°C and weighed to determine specific root length, calculated by dividing the total root length by the total root dry weight. Root P content was then determined from these dried segments spectrophotometrically after ashing at 500°C for 16 h (Murphy and Riley, 1962).

Field Study

This study was conducted at the Russell E. Larson Agricultural Research Farm at Rock Springs, Pennsylvania (40.709746°N, 77.956965°W), from June through September 2016. A split-plot design was utilized with two P levels: two 0.05-ha low-P fields (10 ppm mean available P by Mehlich-3 [ICP]) split into two, 0.025-ha blocks each and two 0.05-ha high-P fields (38 ppm mean available P) split into two, 0.025-ha blocks each. Plant genotypes were randomized within each block. Fields were fertilized according to each treatment, with soil nutrient levels adjusted to meet bean requirements as determined by soil tests at the beginning of each season. Each genotype was planted in a five-row, 3-m-long plot with 72-cm row spacing and 10-cm intrarow spacing. During periods of inadequate rainfall, irrigation was supplied through drip tape. The experiment was run until destructive measurements were taken from all genotypes in all treatments at the time of flowering (49 DAP). Each genotype had four replications within each P treatment. Average maximum/minimum temperatures of this site for the duration of the experiment were 27°C/17°C, average total rainfall was 18.5 cm, and average light/dark photoperiod was 14.5/9.5 h. Midday photosynthetic active radiation was approximately 1,500 to 2,000 μmol photons m⁻² s⁻¹. Soil was a Hagerstown silt loam (fine, mixed, semiactive, mesic Typic Hapludalf).

To limit the presence of fungal disease, seeds were treated with Captan 50W fungicide at a rate of 0.5 mL per 100 seeds prior to planting. At the time of flowering (49 DAP), destructive shoot measurements were taken, including leaf number, dry shoot biomass, and leaf tissue P content. Dry mass was determined from tissues dried at 65°C. Leaf P content was measured from 10 2.5-cm leaf discs taken from throughout the canopy in each plot.

At flowering (49 DAP), the crown of the root system for three representative plants per plot (i.e. per replicate) was extracted and washed, and basal root whorl number, basal root number, adventitious root number, and basal root growth angle were measured. A representative plant is a healthy plant that is comparable in shoot size to the majority of plants throughout the plot. Basal root growth angle was visually scored against a protractor. Three soil cores were taken from each plot to a depth of 40 cm, 10 cm from the base of representative plants toward plants from the neighboring row (Giddings Machine). Soil cores were 5.1 cm in diameter and were divided into four 10-cm increments, washed, and extracted roots from each segment were scanned with an EPSON Perfection V700 PHOTO scanner. From these images, root length density (length of root per volume of soil) in the top 40 cm of soil was quantified with WinRhizo software (WinRhizo Pro; Regent Instruments). The anatomy of five representative basal roots was analyzed from the segment of root 2.5 cm from the hypocotyl. Basal root segments were preserved and sectioned, and anatomical features were measured following the same protocol as described above.

Mycorrhizal Study

This study was conducted under the same growth conditions as described above for the greenhouse trial. Two genotypes contrasting in secondary growth (DG 35 and DG 51) were used. A complete randomized block design was utilized with two P levels: P stress and high P; and two vesicular-arbuscular mycorrhizae (VAM) levels: inoculated and mock inoculated. Each genotype had three replications in each of the VAM levels within each P treatment.

Glomus intraradices (now known as Rhizophagus irregularis; Tisserant et al., 2013) promotes P acquisition in bean (Nielsen et al., 1998) and was used for this study. To facilitate the even distribution of spores, the inoculant (Premier Tech Biotechnologies), consisting of R. irregularis spores, was mixed thoroughly with 1.5 kg of sterilized sand before being mixed into the bulk growth medium prior to planting. The final inoculation intensity for mesocosms assigned to the inoculated treatment was 200 spores per 1 L of growth medium. For the mock-inoculated treatment, the same amount of the liquid inoculant was filtered through Whatman filter papers #1 and #42 (Li et al., 2012), mixed with 1.5 kg of sterilized sand, and added to the growth medium in mock-inoculated mesocosms to introduce inoculum factors other than VAM fungi.

At 49 DAP, root and shoot measurements were taken as described in the above greenhouse study. VAM colonization was quantified using the magnified intersections method (McGonigle et al., 1990). Two 10-cm segments of root were harvested from the basal, middle, and apical ends of two basal roots from each plant. Segments were cleared in 10% KOH and stained in a 5% ink-vinegar solution (Vierheilig et al., 1998). A minimum of 50 intersections per sample were observed, and the incidence of hyphae, arbuscules, and vesicles was scored. The percentage incidence of each structure over total intersections was calculated.

Statistical Analysis

All statistical analyses were performed using RStudio version 0.99.903 (RStudio). The normality and homoscedasticity of the data were determined using the Shapiro-Wilk test and the nonconstant error variance test, respectively. Where data did not meet these assumptions, a box-cox or log transformation was used to normalize the data. ANOVA, Tukey’s honestly significant difference, and regression analysis were performed, with significant effects considered at P ≤ 0.05. Because plants grown under high P were larger than those grown under P stress, allometric relationships between root phenotypes and shoot mass were explored as described by Burridge et al. (2017). The relationship between the decadic logarithm of the root phene and shoot biomass was fitted by linear regression. Log transformation of data prior to the regression analysis is necessary to normalize any multiplicative relationships that may exist between the shoot biomass and the value of a given metric. The scaling coefficient of α = 0.33 is considered to be the threshold, where root phenes with α ≥ 0.33 scale faster than shoot size and are considered hyperallometric, while root phenes with α < 0.33 scale at a slower rate than shoot size and are considered hypoallometric. Statistical analysis of SimRoot output was not performed, as modeling output is most suited for qualitative comparisons rather than statistical tests designed for empirical data. Performing statistical tests on modeling output results in artificially high P values, regardless of effect size, as differences in replicates are simply the result of random number generators within the model. Additionally, because the contrasting parameters are programmed into the model, it is known before the model is run that the null hypothesis is false (White et al., 2014).

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Transverse section of a basal root at different developmental stages to highlight changes in tissue as secondary growth progresses.

• Supplemental Figure S2. SimRoot results for three bean root systems with three levels of secondary growth.

• Supplemental Figure S3. Model of bean root systems with two levels of secondary growth.

• Supplemental Figure S4. SimRoot results showing mean ± se total P uptake and shoot biomass under three levels of P stratification in soil profile.

• Supplemental Figure S5. Mean ± se root mass and root mass : shoot mass ratio of genotypes with advanced and reduced secondary growth in the greenhouse at 46 DAP.

• Supplemental Figure S6. Mean ± se net length of lateral roots per basal root.

• Supplemental Figure S7. Mean ± se percentage stele area of a cross section of basal, middle, and apical positions.

• Supplemental Figure S8. Mean ± se metaxylem vessel number of basal, middle, and apical positions.

• Supplemental Figure S9. Mean ± se net metaxylem vessel area of basal, middle, and apical positions.

• Supplemental Figure S10. Mean ± se theoretical axial hydraulic conductance of basal, middle, and apical positions.

• Supplemental Figure S11. Root crowns of genotypes with advanced and reduced secondary growth excavated at 49 DAP from the field under P stress.

• Supplemental Figure S12. Mean ± se basal TCSA-root length ratio of eight genotypes in the greenhouse at 32 and 46 DAP under P stress.

• Supplemental Figure S13. Correlation between total basal root length and respiration rate of the apical, middle, and basal segments under P stress and high-P treatments.

• Supplemental Figure S14. Correlation between dry shoot weight and respiration rate of the apical, middle, and basal segments under P stress and high-P treatments at 46 DAP in the greenhouse.

• Supplemental Figure S15. Mean ± se TCSA, N concentration, and P concentration of the basal segment in each phenotypic group.

• Supplemental Figure S16. Mean ± se TCSA, percentage stele area, cortex area, and theoretical hydraulic conductance of the apical segment.

• Supplemental Figure S17. Diagram of basal root segment locations for anatomy, respiration, and elemental analysis.

• Supplemental Data S1. Summarized hierarchical input file showing the context of SimRoot parameters.

ACKNOWLEDGMENTS

We thank James Burridge for assistance with field research, Bob Snyder for oversight of laboratory and field activities, Johannes Postma and Harini Rangarajan for support with SimRoot, Airong Li for guidance with mycorrhizal research, and Michael Williams for assistance with elemental analysis.

LITERATURE CITED

Author notes