short title: root secondary growth and phosphorus ......2017/11/08 · jonathan p. lynch, email:...
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Short Title: Root Secondary Growth and Phosphorus Acquisition
Corresponding Author Details: Jonathan P. Lynch, Email: [email protected], Tel.: +1 8148632256 Article Title: Reduction in Root Secondary Growth as a Strategy for Phosphorus Acquisition
Authors: Christopher F. Strock, Laurie Morrow de la Riva, Jonathan P. Lynch
Authors’ Affiliation: Department of Plant Science, The Pennsylvania State University, University Park, PA USA
One-sentence summary: Reduced root secondary growth decreases
maintenance and construction costs, allowing greater root elongation and soil exploration, thereby improving P acquisition and plant growth under P stress.
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Authors’ Contributions: C.S. performed all of the experiments, analyzed the data, and wrote the article with contributions of all the authors; L.R. carried out the original screening and foundational research for these published data; J.L. conceived the hypotheses, and supervised the design, experimentation, analysis, and reporting.
Funding Information: This project was supported by the USAID Climate Resilient Beans Feed the Future Legume Innovation Laboratory and the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch project 4582.
Present Address: 221 Tyson Bldg., University Park, PA 16802
Corresponding Author Email: [email protected]
Plant Physiology Preview. Published on November 8, 2017, as DOI:10.1104/pp.17.01583
Copyright 2017 by the American Society of Plant Biologists
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Abstract 1
We tested the hypothesis that reduced root secondary growth of dicotyledonous 2
species improves phosphorus acquisition. Functional-structural modeling in 3
SimRoot indicates that in common bean (Phaseolus vulgaris), reduced root 4
secondary growth reduces root metabolic costs, increases root length, improves 5
phosphorus capture, and increases shoot biomass in low phosphorus soil. 6
Observations from the field and greenhouse confirm that under phosphorus 7
stress, resource allocation is shifted from secondary to primary root growth, 8
genetic variation exists for this response, and reduced secondary growth 9
improves phosphorus capture from low phosphorus soil. Under low phosphorus 10
in greenhouse mesocosms, genotypes with reduced secondary growth had 39% 11
smaller root cross sectional area, 60% less root respiration, 27% greater root 12
length, 78% greater shoot phosphorus content, and 68% greater shoot mass 13
than genotypes with advanced secondary growth. In the field under low 14
phosphorus, these genotypes had 43% smaller root cross sectional area, 32% 15
greater root length, 58% greater shoot phosphorus content, and 80% greater 16
shoot mass than genotypes with advanced secondary growth. Secondary growth 17
eliminated arbuscular mycorrhizal associations as cortical tissue was destroyed. 18
These results support the hypothesis that reduced root secondary growth is an 19
adaptive response to low phosphorus availability and merits investigation as a 20
potential breeding target. 21
Introduction 22
Most soils on earth have suboptimal phosphorus (P) availability for plant growth 23
(Vance et al. 2003; Lynch & Brown 2008; Lynch 2011), as it is only available to 24
plants as inorganic P (Pi), and is rarely present in concentrations greater than 25
several M in soil solution (Bieleski 1973). Diffusion of P in soil is greatly 26
outpaced by plant uptake, resulting in the formation of P depletion zones around 27
roots (Hinsinger et al. 2005). Due to the limited availability and slow movement of 28
P in soil, one of the most effective strategies of increasing P uptake is to increase 29
the volume of soil explored by the root system. This accounts for the increase in 30
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the root:shoot ratio under P stress (Fohse et al. 1988). Although increasing 31
resource allocation to root growth improves P acquisition, unbalanced root 32
development reduces overall plant growth due to the increased metabolic cost of 33
added root tissue (Nielsen et al. 1998, 2001; Lambers et al. 2006). Over 50% of 34
daily carbon fixation may be consumed by the root system, with P stressed 35
plants allocating a larger fraction of their daytime net carbon assimilation than 36
non-stressed plants (Van der Werf et al. 1988; Lambers et al. 1996; Nielsen et al. 37
1998, 2001). This cost consists of three main components: the growth of new 38
root tissue, ion uptake and assimilation, and maintenance of existing root tissue 39
(Nielsen et al. 1998; Fan et al. 2003). Nielsen et al. (1998) found that in common 40
bean under P deficit, the proportion of total root respiration allocated to 41
maintenance accounts for approximately 90% of total root respiration. The 42
functional-structural model Simroot has estimated that the cost of maintenance 43
respiration of the root system constitutes 40% of the total growth reduction under 44
P stress (Postma & Lynch 2011). Consequently, the greatest opportunity to 45
reduce the metabolic burden of the root system lies in moderating maintenance 46
costs. 47
To improve the balance between soil exploration and consumption of growth 48
limiting resources, a decrease in root secondary growth would reduce the carbon 49
cost of producing and maintaining root length (Lynch 1995). It has been 50
hypothesized that this may be an adaptive strategy to improve the metabolic 51
efficiency of soil foraging under P stress, where roots will favor primary growth 52
(elongation) over secondary growth (radial thickening) to achieve greater 53
exploration of soil domains that have not been depleted of P (Lynch & Brown 54
2008; De la Riva & Lynch 2010; Lynch 2007, 2011). 55
Previous observations of root systems under P stress provide evidence for the 56
importance of root diameter in a diversity of plant species. In the sedge Carex 57
coriacea, specific root length (SRL, i.e. root length per mass of root tissue) was 58
negatively correlated with P availability, with a 30% reduction in root diameter 59
from high P to low P (Powell 1974). In a study of the root morphology of 60
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temperate pasture species, a reduction in root diameter, root mass density, and 61
an increase in SRL were observed under P stress (Hill 2006). Reduced lateral 62
root diameter has also been described under low P in water hyacinth (Eichhornia 63
crassipes) and maize (Zea mays L.) (Xie & Yu 2003; Zhu & Lynch 2004). 64
Nevertheless, observations of reductions in root diameter under P stress have 65
not been made within root classes, and published reductions in root diameter of 66
the entire root system may be the product of a greater proportion of higher-order 67
lateral roots, rather than the effects of altered secondary growth. To determine if 68
reduction of secondary growth is an adaptive response, an explicit study of 69
secondary growth within root classes under P stress is required (Lynch & Brown 70
2008). 71
During secondary growth, periclinal cellular divisions and differentiation of 72
secondary tissues at the vascular cambium and phellogen cause the splitting and 73
destruction of the epidermis, cortex, and endodermis. While the production of the 74
periderm replaces these primary tissues and helps to protect the vasculature of 75
the root, the bulk of secondary thickening is driven by the production of 76
secondary xylem elements and parenchyma internal to the vascular cambium 77
(Dikison 2008). This elimination of the primary tissues and proliferation of 78
secondary tissue are observed in transverse sections as the loss of the 79
epidermis, cortex, and endodermis, expansion of the stele, and an increase in 80
the abundance and size of metaxylem vessels (Fig. S1). Under P deficiency, 81
observed changes in root anatomy of a variety of species include smaller root 82
diameter, stele diameter, fewer and smaller epidermal cells and metaxylem 83
vessels, reduced percent stele area, and fewer cortical cells, and xylem vessels 84
(Fohse et al. 1991; Fan et al. 2003; Liu et al. 2004; Sarker et al. 2015). 85
In this study, we utilize functional-structural modeling as well as empirical 86
observations of plants grown in controlled environment mesocosms and in the 87
field to explore the effect of reduced secondary growth of roots on P acquisition. 88
Our goals were to test the hypotheses that 1) secondary growth is suppressed by 89
P stress, 2) genetic variation exists for this response, and 3) reduced secondary 90
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growth of roots improves P acquisition. We address these hypotheses by first 91
utilizing the functional-structural plant model SimRoot to determine the 92
relationship between secondary growth of roots and P acquisition, followed by 93
greenhouse and field studies to validate in silico results. 94
The common bean (Phaseolus vulgaris L.) was used to test these hypotheses 95
due to observed genotypic variation in phosphorus acquisition and metabolic 96
efficiency of roots under P stress (Nielsen et al. 2001; Beebe et al. 2006; De la 97
Riva & Lynch 2010; Henry et al. 2010; Miguel et al. 2015). Common bean is also 98
an important food security crop in Africa and Latin America, where its productivity 99
is often limited by low P availability. 100
Results 101
Phenotypic Classification 102
Total cross sectional area (TCSA) (mm2) of the root and the percent stele area 103
from greenhouse grown root segments were used to categorize genotypes into 104
two phenotypic groups that were used as a factor in data analysis; genotypes 105
with a mean TCSA < 0.5 mm2 and < 50% stele in the basal segment at time of 106
flowering (46 DAP) were classified as having reduced secondary growth 107
(“reduced”) (DG6, DG35, L8814, L8863). Genotypes with a mean TCSA > 0.5 108
mm2 and > 50% stele in the basal segment at flowering were categorized as 109
having advanced secondary growth (“advanced”) (DG23, DG51, L8843, L8857). 110
Effects of Reducing Secondary Growth in Silico 111
When root secondary growth was reduced by 50% (intermediate phenotype), by 112
40 DAP under P stress (4 mol/L available P) root respiration per g was reduced 113
by 14%, total root length was increased by 7%, P acquisition increased by 9%, 114
and shoot mass increased by 17% from the “advanced” phenotype (Figs. 1, S2). 115
In the “reduced” phenotype where roots had no secondary growth, root 116
respiration was reduced by 12%, total root length was increased by 14%, net P 117
acquisition increased by 15%, and the shoot mass increased by 31% from the 118
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“advanced” phenotype (Figs. 1, S3). In environments with greater P availability, 119
root respiration rate was suppressed and root length increased to a greater 120
degree, but the impact of these root parameters on improving P capture and 121
shoot biomass was less pronounced (Fig. 1). When P availability was more 122
strongly stratified with depth, the benefits to reducing secondary growth became 123
less pronounced (Fig. S4). 124
Plant Growth in Mesocosms 125
Under high P in mesocosms, no genotypic differences in shoot size and shoot P 126
were observed, while under P stress, reduced genotypes had 68% greater shoot 127
mass and 78% greater shoot P than advanced genotypes by 46 DAP (Figs. 2, 3). 128
Under high P there were no phenotypic differences in root mass, while under P 129
stress, reduced genotypes had significantly greater root mass than advanced 130
genotypes (Fig. S5). Within each treatment, no phenotypic differences in 131
root:shoot ratios were observed (Fig. S5). Allometric analysis revealed that root 132
mass had a hyperallometric relationship to shoot size under both P treatments 133
(Table 1). All genotypes had statistically similar basal root whorl number, basal 134
root number, and adventitious root number. 135
By 46 DAP under P stress, reduced genotypes had 56% greater specific root 136
length and 27% greater basal root length than advanced genotypes, while in the 137
high P treatment, both phenotypic groups had statistically similar specific root 138
length and basal root length (Fig. 3). Although a trend of thinner lateral roots and 139
greater net lateral root length were observed for the reduced genotypes under P 140
stress, statistical analysis did not reveal a significant genotypic effect at p < 0.05 141
(Fig. S6). Under P stress, specific root length and basal root length were both 142
positively correlated with shoot P content by 32 DAP, while in the high P 143
treatment no relationship was observed. 144
Plant Growth in the Field 145
Under high P in the field, no genotypic differences in shoot size and shoot P 146
content were observed, while in the P stress treatment, reduced genotypes had 147
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80% greater shoot mass, 62% greater leaf number, and 58% greater shoot P 148
than advanced genotypes by 49 DAP (Fig. 4). Under P stress, reduced 149
genotypes had 32% greater root length density in the top 40cm of soil than 150
advanced genotypes, while under high P no genotypic differences were 151
detectable (Fig. 4). Root length density in the top 40 cm was positively correlated 152
with total shoot P under P stress while no relationship was observed under high 153
P (Fig. 5). Root length density in the top 40 cm of soil had a hyperallometric 154
relationship with shoot size under P stress, but not under high P (Table 1). All 155
genotypes had statistically similar basal root whorl number, basal root number, 156
adventitious root number, and basal root growth angle. 157
Root Anatomy 158
In mesocosms, total cross sectional area (TCSA), percent stele, metaxylem 159
vessel number, total metaxylem vessel area, and hydraulic conductance 160
increased significantly from 18 to 46 DAP and from the apical to the basal end of 161
the root in both P treatments (Figs. 6, S7, S8, S9, S10). Phosphorus stress 162
significantly reduced these anatomical phenes in both greenhouse and field-163
grown roots (Fig. 7). Differences in TCSA between reduced and advanced 164
genotypes were statistically detectable by 18 DAP under P stress (Fig. 6). 165
Phosphorus treatment, temporal, and phenotypic effects were most detectable in 166
the basal segment, where the greatest secondary growth had occurred. In this 167
segment at 46 DAP, reduced genotypes displayed 29% smaller percent stele 168
area, 21% fewer metaxylem number, 48% less metaxylem area, and 52% less 169
hydraulic conductance than advanced genotypes under P stress (Figs. 7, S7, S8, 170
S9, S10). At 49 DAP in the field, reduced genotypes had 43% smaller TCSA, 171
26% fewer metaxylem number, 41% reduced metaxylem area, and 55% less 172
hydraulic conductance than advanced genotypes under P stress (Figs. 8, S10). 173
Allometric analysis indicates that differences in secondary growth of roots 174
(TCSA, percent stele, metaxylem area, metaxylem number) were not driven by 175
differences in plant size in either the greenhouse or the field (Table 1). Under P 176
stress a negative relationship between basal TCSA and total shoot P was 177
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observed, but under high P, no significant association between basal TCSA and 178
shoot P was detected (Fig. 9). 179
In mesocosms under P stress, TCSA of the basal segment was negatively 180
correlated with basal root length at all time points, while under high P no 181
relationship was statistically observable (Fig. 9). Similarly, at 49 DAP under P 182
deficit in the field basal TCSA was negatively correlated with root length density 183
in the top 40 cm of soil (Fig. 5). This association between the allocation of 184
resources from secondary growth to primary growth can be represented by the 185
ratio of basal TCSA:total basal root length. Mean TCSA:total basal root length at 186
46 DAP in the greenhouse was greater for genotypes classified as advanced 187
than for most genotypes in the reduced group with the exception of L8863 (Fig. 188
S12). Genotypes in the advanced group had mean TCSA:RL > 0.1 while 189
genotypes in the reduced group had a mean TCSA:RL of < 0.1. 190
Root Respiration 191
By 46 DAP in the greenhouse, reduced genotypes had 60% less basal segment 192
respiration, 47% less middle segment respiration, and 69% less apical segment 193
respiration than advanced genotypes under P deficit (Fig. 10). There was a 194
strong positive relationship between respiration rate per unit length and TCSA in 195
all P treatments and time points (Fig. 9). By 32 DAP under P stress, respiration 196
per unit length of all segments was negatively correlated with basal root length, 197
and by 46 DAP (Fig. S13), respiration per unit length of all locations was 198
negatively correlated with shoot mass (Fig. S14). These relationships between 199
root respiration and root length and shoot mass were not present in the high P 200
treatment (Figs. S13, S14). 201
Root Construction Costs 202
Root P concentration decreased significantly with root segment age in the high P 203
treatment but was consistent for all root segments in P-stressed plants (Fig. 204
S15). Nitrogen (N) concentration was significantly greater in the growing root tips 205
than in older root segments and was significantly greater in P stressed roots than 206
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in roots grown under high P (Fig. S15B). Carbon (C) concentration was 207
statistically similar for root segments in all P treatments, time points, and 208
positions along the root. There was no effect of P treatment on the C:H ratio of 209
roots and no detectable change in the C:H ratio among locations, but C:H ratio 210
significantly increased at the basal segment over time. Phosphorus stress 211
significantly reduced C:N and C:N increased with root segment age. These 212
increases in C:N were driven by a reduction in N, not changes in C. No 213
statistically observable differences in the elemental concentration of roots were 214
observed between advanced and reduced genotypes. 215
Mycorrhizal Synergism with Secondary Growth 216
Despite observed genotypic differences in secondary growth, no statistically 217
detectable genotypic differences in mycorrhizal symbiosis were observed under 218
P stress. While roots grown under P stress had significantly greater 219
mycorrhization of the basal-most segment, roots grown under high P displayed 220
the opposite pattern of symbiosis with the least abundance of mycorrhizal 221
structures in the basal segment (Fig. 11C). Basal segments from the P stress 222
treatment had 367% greater cortical tissue area than basal segments in the high 223
P treatment and across both P treatments there was a significant positive 224
relationship between cortical tissue area and symbiosis (Fig. 11A, 11B, 11D). 225
Discussion 226
SimRoot predicted that reducing secondary growth of roots reduces metabolic 227
costs, liberates resources for greater primary growth and thereby augments the 228
total quantity of P captured. These predictions were confirmed by in vivo 229
observations under P stress in the greenhouse and field. These results confirm 230
the hypotheses that 1) in low P soil, roots of this dicot species favor primary 231
growth over secondary growth (Figs. 4, 5, 7, 8, 9), 2) genetic variation exists for 232
this response (Figs. 3, 4, 5, 6, 8, S11, S12), and 3) and the reallocation of 233
resources from secondary to primary growth improves P acquisition (Figs. 1, 3, 5, 234
9, S2). 235
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Because SimRoot does not explicitly consider beneficial aspects of secondary 236
root growth such as axial water transport (Valenzuela-Estrada et al. 2008), 237
mechanical support of the shoot, or resistance to herbivores and pathogens 238
(Eissenstat 1992; Valenzuela-Estrada et al. 2008), reduced secondary growth is 239
unconditionally beneficial for improving root length and P acquisition in silico. In 240
vivo, secondary growth is a constitutive characteristic of dicot roots, and the 241
inverse relationship between secondary growth and P acquisition predicted by 242
the model is only present under P stress. Under high P, plants are able to 243
acquire adequate P to support the production of less efficient roots without 244
reducing plant growth. The observation that secondary growth is inhibited only 245
under P stress would suggest that increased root diameter affords increased 246
fitness in fertile environments. 247
Despite possible drawbacks to reduced root diameter, reallocation of resources 248
among different tissues is a hallmark adaptive response to P deficiency (Fohse et 249
al. 1988). This concept is evident in the present study through the greater 250
allometric scaling coefficient for root mass under P stress compared to high P 251
conditions, demonstrating a shift in resources from shoot growth to soil 252
exploration. Although there was an increase in root:shoot ratio under P stress, 253
there was no phenotypic difference for this metric within the P stress treatment. 254
While the relative investment of resources to roots of both phenotypic groups 255
was comparable, the allocation of those resources within the root system of 256
reduced and advanced groups differed. This shift in allocation of resources within 257
the root system is evidenced by the reduction in root diameter and metabolic cost 258
per length of root, and increase in total root length of the reduced genotypes 259
under P stress. Genotypic variation in metabolic efficiency of P. vulgaris roots 260
under P stress has been previously reported in a study by Nielsen et al. (2001), 261
where there was no difference in daily carbon allocated to roots of P-efficient and 262
P-inefficient genotypes of P. vulgaris, but P-efficient genotypes were able to 263
maintain a larger root system per unit of carbon respired than inefficient 264
genotypes. Further evidence for a genetic component to root etiolation is 265
reported in a QTL study by Beebe et al. (2006) for root architectural phenes 266
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using the same DxG RILs as in the present study. This work found that these 267
DxG RILs displayed genetic variation in P accumulation per unit root length and 268
two significant QTLs for P accumulation under P stress were identified in the 269
same regions as QTLs for root length and for specific root length, with joint QTL 270
analysis uncovering a positive relationship between specific root length and P 271
accumulation under P stress (Beebe et al. 2006). 272
The observed effect of P deficit on root length at 18 DAP suggests that the 273
reduction of secondary growth in root segments at later time points may be in 274
part the ancillary effect of shifts in allocation of resources to primary growth. The 275
regulation of root elongation under P deficiency has been previously described 276
and attributed to ethylene signaling pathways in Arabidopsis (Ma et al. 2003). In 277
P. vulgaris, ethylene production is greater in roots grown under P stress and 278
serves to maintain root elongation under P stress while ethylene inhibits 279
elongation under high P conditions (Borch et al. 1999; Liao et al. 2001). 280
Additionally, the reduction in root diameter and respiration rate at the root apex 281
under P stress indicates that the differences in secondary growth are not strictly 282
the result of the gradual accrual of differences in secondary growth in older 283
segments of root over time, but are initiated at the root apex. The anatomy of 284
these thinner apical segments from the P stress treatment did not manifest as an 285
isometric reduction in both cortex and vascular tissue, rather, we observed 286
thinner apical segments that had reduced cortex area, but comparable stele 287
area, metaxylem number, and metaxylem area to apical segments in the high P 288
treatment (Fig. S16). These P stressed root apices in reduced genotypes achieve 289
smaller TCSA and decreased respiration through reduction in cortical tissue 290
while maintaining the same amount of vasculature necessary for axial transport. 291
Unlike roots in the high P treatment, where P concentration of root tissue 292
decreased with secondary growth, under P stress, root P concentration remained 293
stable over time. This may indicate that the P stress was substantial and the P 294
concentration in root tissue was being sustained at the minimal level required to 295
maintain living tissue. While no phenotypic differences in nutrient concentrations 296
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of roots were observed between reduced and advanced groups under P stress, 297
advanced genotypes have greater root diameter and therefore greater total 298
nutrient content per length compared to reduced genotypes. In addition to the 299
smaller respiration rate per length of thinner roots, this savings in construction 300
costs is another avenue for conservation of resources in reduced genotypes 301
under P stress. Furthermore, as secondary growth progresses, the vascular 302
tissue expands and the living cortex is destroyed, thereby shifting the 303
physiological role of roots from resource capture to axial transport (McCully, 304
1999). While reduced genotypes had diminished axial conductance, the delayed 305
transition into the role of axial transport may allow roots to acquire more 306
resources from the surrounding soil for a greater length of time. 307
In addition to the possible benefit of greater direct nutrient uptake by the root, 308
retarded stele development and maintenance of cortical tissue in roots under P 309
stress has a synergistic effect on P uptake through the preservation of arbuscular 310
mycorrhizal associations that colonize the cortex. While it is well known that P 311
availability suppresses mycorrhizal associations in ways unrelated to secondary 312
growth, the significant relationship between cortical area and fungal colonization 313
across P treatments suggests that secondary growth inhibits arbuscular 314
mycorrhizal relationships. These results reinforce Brundrett (2002) who has 315
suggested that plant species with less root cortical volume sacrifice the capacity 316
for arbuscular mycorrhizal associations, and Valenzuela-Estrada et al. (2008), 317
who observed this in Vaccinium, where roots with greater radial growth and 318
reduced specific root length had less mycorrhizal colonization. 319
While suppression of secondary growth appears to facilitate mycorrhizal 320
symbiosis, it may also increase the vulnerability of roots to soil pathogens and 321
herbivores. Although there was no greater incidence of disease observed in roots 322
of reduced genotypes, in soils where pathogens and herbivores are prevalent, 323
roots with advanced secondary growth may have greater longevity than those 324
with reduced secondary growth. This relationship between anatomical 325
development and disease has demonstrated in Malus domestica, where 326
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pathogen colonization is closely linked to the senescence and loss of the root 327
cortex (Emmet et al. 2014). Although P limitation alone does not diminish root 328
survivorship in P. vulgaris grown in sand culture, in the field where soil biota are 329
present, up to 49% of roots are lost by late pod filling (Fisher et al. 2002). 330
These results support growing evidence that root phenes and phene states that 331
reduce the metabolic cost of soil exploration are adaptive in resource-poor soil 332
environments (Chimungu et al. 2014a,b; Lynch 2014; Saengwilai et al. 2014; 333
Chimungu et al. 2015; Miguel et al. 2015; Schneider et al. 2017). In this context, 334
root anatomical phenes merit attention as breeding targets for more stress 335
tolerant crops. 336
Conclusions 337
These results support the hypothesis that reduced root secondary growth 338
increases resources available for primary growth, thereby increasing the total 339
volume of soil explored and acquisition of soil resources. Although all P. vulgaris 340
genotypes tested favor primary growth of roots over secondary growth under P 341
stress, genotypes differ in the intensity of this response. Genotypes with reduced 342
secondary growth had suppressed anatomical development, reduced metabolic 343
and construction costs per length of root, greater soil exploration, and greater P 344
acquisition than genotypes with advanced secondary growth. These results 345
demonstrate the adaptive significance of reduced secondary growth under P 346
stress, but further work to determine the influence of reduced hydraulic 347
conductance in roots with reduced secondary growth on water capture in drying 348
soils would be of merit, as well as a targeted investigation into the relationship 349
between secondary growth and the colonization of arbuscular mycorrhizal. 350
Further research may elucidate if a reduction in root secondary growth improves 351
soil resource capture under drought and other nutrient deficiencies. 352
Materials & Methods 353
Germplasm 354
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Previous work by De la Riva and Lynch (2010) identified two genotypes of P. 355
vulgaris contrasting in P acquisition and root development under P stress. DOR 356
364 is a high yielding genotype developed by breeders at CIAT (Cali, Colombia) 357
(CIAT 1996, p.22-38). Despite exhibiting a strong reduction in secondary growth 358
of roots under low P conditions, it has been identified as being P inefficient due to 359
other components of the root phenotype (Liao et al. 2004). G19833 is a Peruvian 360
landrace from the Andean gene pool (Beebe et al. 1997). This genotype displays 361
less reduction in secondary growth of roots than DOR 364 under P stress (De la 362
Riva & Lynch 2010), but is classified as P efficient due to the contribution of other 363
beneficial root phenes (phene is to phenotype as gene is to genotype (York et al. 364
2013; Serebrovsky 1925) including a shallow basal root angle (Bonser et al. 365
1996), high basal root whorl number (Miguel et al. 2013), and long, dense root 366
hairs (Yan et al. 2004). DOR 364 and G19833 were selected for their contrasting 367
P efficiency and root characteristics and crossed to generate a population of 368
recombinant inbred lines (RILs). The DOR 354 x G19833 (DG) RIL population 369
was then screened for variation in root secondary growth under P stress and four 370
genotypes were selected for their observed differences in secondary growth. 371
These genotypes include DG 6 (reduced secondary growth), DG 35 (reduced 372
secondary growth), DG 23 (advanced secondary growth), DG 51 (advanced 373
secondary growth). Additionally, genotypes from the L88 RIL population 374
(developed by J. Kelly, Michigan State University), generated from a cross 375
between drought resistant B98311 and P-efficient TLP 19 (Frahm et al. 2004), 376
were selected for their differences in secondary growth of roots under low P. 377
These genotypes include L88-14 (reduced secondary growth), L88-63 (reduced 378
secondary growth), L88-43 (advanced secondary growth) and L88-57 (advanced 379
secondary growth). A multiline study by Henry et al. (2010) further supports the 380
purported contrast in secondary growth between these genotypes where under 381
low P in the field, L88-14 had thinner roots and more roots per root core while 382
L88-57 had thicker roots and less roots per core. 383
In Silico Study 384
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The functional-structural plant model SimRoot is able to integrate parameters of 385
root growth, nutrient uptake, and resource allocation from in vivo studies to 386
model the relationship between root growth and performance of P. vulgaris 387
(Postma et al. 2017). To investigate the relationship between secondary growth 388
in roots and P acquisition efficiency, root systems of P. vulgaris were modeled 389
with three different secondary growth rates; root systems with no secondary 390
growth (Reduced), root systems with 50% the secondary growth rates observed 391
under high P conditions (Intermediate), and root systems with the same 392
secondary growth rates observed under high P conditions (Advanced). All other 393
plant properties were held constant in all simulations. For each level of 394
secondary growth, P availability was varied from 0.17 to 5 kg ha-1 across 13 395
levels. Here, phosphorus concentration represents the quantity available to the 396
plant in the soil solution, and the buffer capacity of the soil (ratio between the 397
dissolved and absorbed fraction) is held constant. In total, 39 simulations (3 398
secondary growth rates x 13 P levels) were run on the Pennsylvania State 399
University clusters (http://rcc.its.psu.edu/hpc/systems). Starting from germination, 400
plant growth was simulated for 40 days and root growth was permitted to grow 401
within a 60 x 60cm by 1.5 m deep soil volume. Any roots that intersected the 402
boundary of the soil environment were mirrored back to maintain a total root 403
length similar to that of field conditions. 404
In SimRoot, carbon used for growth comes from either seed reserves or 405
photosynthesis. The model is inclusive of multiple components of metabolic costs 406
stemming from respiration, nitrogen fixation, nutrient uptake, and production of 407
exudates. Root system architecture is represented by a network of root nodes 408
and is modeled in three dimensions. Shoot growth is simulated non-geometrically 409
and is represented by integral parameters such as leaf area and dry shoot mass. 410
Phosphorus uptake at each root node is parameterized using the Barber-411
Cushman model and integrated over the length of the root system (Barber and 412
Cushman 1981). When P availability is inadequate to satisfy optimal growth, leaf 413
area expansion, photosynthesis, and root growth are inhibited. Inter-root 414
competition for P is simulated in one dimension by the Barber-Cushman model 415
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16
and is dependent on the average root density within 1 cm of the root (Postma & 416
Lynch 2011). In SimRoot, sink strength of a given organ is based on the resource 417
requirements for potential growth and maintenance of the tissue. The growth rate 418
of all root classes, leaves, and stem tissue is based on empirical data. Roots of 419
greater thickness have greater longitudinal potential growth rates and 420
consequently have greater sink strength (Pages 2000). Resources required for 421
secondary growth are determined by the volumetric increase associated with 422
class, location, and age of each root segment. Respiration is a function of the 423
root segment biomass and age. Further information on SimRoot, can be found in 424
Postma and Lynch (2011). Overview of SimRoot parameterization is available in 425
the supplementary files (SimRoot Parameterization) and files used to generate 426
this simulation are available at (https://doi.org/10.5281/zenodo.998950). 427
Greenhouse Study 428
This study was conducted in a greenhouse located at The Pennsylvania State 429
University, University Park, Pennsylvania, USA (40.801955° N, -77.862544° W). 430
Plants were grown from April through May 2016 under a 16:8 (light:dark) 431
photoperiod and max/min temperature of 34°C/20°C. Mid-day photosynthetic 432
active radiation (PAR) was approximately 900-1000 mol photons m-2 s-1. Natural 433
light was supplemented from 0600 to 2200 h with 110mol photons m-2 s-1 from 434
LED Illumitex ES2 lights (Illumitex, Inc., Austin, TX, USA). A Complete 435
Randomized Block Design was utilized with two P levels; P stress and high P. 436
The experiment was run for a total of 46 days with destructive measurements 437
taken from all genotypes in all treatments at 18, 32, and 46 days after planting 438
(DAP). Each genotype at each time point and treatment had four replications. 439
Seeds were surface sterilized in a 25% NaOCl solution for 2 minutes, rinsed in 440
deionized water and germinated in 0.5 mM CaSO4 in the dark at 28°C for 24 hrs. 441
Uniform seedlings were transplanted to the greenhouse in opaque, 20 L 442
mesocosms 30 cm in diameter and 44 cm in height, wrapped in silver duct tape 443
to enhance reflectiveness. Mesocosms were filled with a mixture of 40% coarse 444
grade A perlite (Whittemore Co., Inc., Lawrence, MA, USA), 30% medium grade 445
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17
sand (Quikrete Co, Inc., Atlanta, GA, USA), 20% low P soil (Ap2 Hagerstown silt 446
loam (fine, mixed, semi-active, mesic Typic Hapludalf) Available P = 12 ppm) 447
sieved through 6mm mesh, and 10% D3 coarse grade A vermiculite (Whittemore 448
Co., Inc., Lawrence, MA, USA). The soil was incorporated to replicate features 449
found under field conditions such as the presence of organic matter, soil biota, 450
and oxide surfaces that serve to buffer P availability. Mesocosms designated as 451
being part of the high P treatment received 40 g granular triple superphosphate 452
(25% P) incorporated into the media at the time of mixing. Mesocosms assigned 453
to the low P treatment did not receive any supplemental P. Other nutrients were 454
supplied through drip irrigation once daily. At each irrigation event, high and low 455
P mesocosms received 400 ml of nutrient solution. This nutrient solution 456
contained 1.5 mM KNO3, 1.2 mM Ca(NO3)2, 0.4 mM NH4NO3, 0.025 mM MgCl2, 457
0.5 mM MgSO4, 0.3 mM K2SO4, 0.3 mM (NH4)2SO4, 5 M Fe-EDTA, 1.5 M 458
MnSO4, 1.5 M ZnSO4, 0.5 M CuSO4, 0.15 M (NH4)6Mo7O24, and 0.5 M 459
Na2B4O7. The pH of the nutrient solution was adjusted as needed at every other 460
irrigation event to 5.8 with KOH and HCl. 461
At 18, 32, and 46 DAP destructive shoot measurements were taken including leaf 462
number, dry shoot biomass, and leaf tissue P content. Dry mass was determined 463
from tissues dried at 65°C for 7 d. Leaf P content was measured 464
spectrophotometrically after ashing leaf tissue at 500°C for 16 h (Murphy & Riley 465
1962). 466
The root system of each plant was extracted, washed, and basal root whorl 467
number, basal root number, adventitious root number, root respiration rate, root 468
P content, specific root length, basal root length, and anatomical phenes were 469
measured. Root respiration rates were determined immediately after washing for 470
two, 10 cm segments taken from representative basal roots at the 10 cm nearest 471
to the hypocotyl (basal), 10 cm at the middle of the root axis (middle), and 10 cm 472
from the growing tip back (apical) (Fig. S17). To relate differences in respiration 473
rates to secondary growth of the primary root axis, lateral roots were removed 474
from these segments with a razor prior to respiration measurements. Respiration 475
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18
rates were measured using a Li-Cor 6400 gas exchange system with a modified 476
respiration chamber (Li-Cor, Lincoln, NE, USA). Measurements were performed 477
under ambient greenhouse conditions with the sealed chamber being submerged 478
in a water bath kept at 28°C and baseline sample chamber and reference 479
chamber CO2 concentration of 400 mol mol-1. 480
Following respiration measurements, 2.5 cm of each root segment used for 481
respiration measurements was used for characterization of anatomy. These 482
segments were preserved using a Leica EM CPD300 critical point dryer (Leica 483
Microsystems, Inc., Buffalo Grove, IL, USA). Preserved segments were 484
sectioned with laser ablation tomography (LAT) using an Avia 7000, 355 nm 485
pulsed laser and simultaneously imaged with a camera equipped with a 5x zoom 486
lens. Root cross-section images were analyzed using MIPAR software 487
(MIPAR.beta.8, MIPAR, Columbus, OH). Anatomical features measured include 488
total cross sectional area (TCSA), percent stele area, metaxylem number, and 489
metaxylem area. Theoretical axial metaxylem conductance (Kh) (kg m MPa-1 s-1) 490
was calculated for each cross-sectional image using the modified Hagen-491
Poiseuille law (Eq. 1) where d is the diameter of the vessel in meters, is the 492
fluid density (equal to water at 20° C; 1000 kg m-3), and is the viscosity of the 493
fluid (equal to water at 20°C; 1 x 10-9 MPss) (Tyree & Ewers 1991). The 494
remaining 7.5 cm of root segments used in respiration measurements were dried 495
at 65°C for 7 d, and 2.5 mg subsample of this tissue was analyzed for N, C, and 496
H content using an elemental analyzer (Series II CHNS/O Analyzer 2400; 497
PerkinElmer). 498
𝑘ℎ = (𝜋𝜌
128𝜂)∑(𝑑𝑖
4)
𝑛
𝑖=1
Equation 1. Modified Hagen-Poiseuille law
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19
Basal root whorl number, basal root number, and adventitious root number were 499
determined by counting the root whorls and basal roots after washing the root 500
system. One intact, representative basal root that was sectioned into 20 cm 501
segments along its primary axis and each segment was imaged using an EPSON 502
Perfection V700 PHOTO scanner. From the scanned image, total basal root 503
length, including length and diameters of lateral roots, was quantified with 504
WinRhizo software (WinRhizo Pro, Regent Instruments, Quebec City, Quebec, 505
Canada). The scanned basal root was then dried at 65°C and weighed to 506
determine specific root length, calculated by dividing the total root length by the 507
total root dry weight. Root P content was then determined from these dried 508
segments spectrophotometrically after ashing at 500°C for 16 h (Murphy & Riley 509
1962). 510
Field Study 511
This study was conducted at the Russell E. Larson Agricultural Research Farm, 512
at Rock Springs, Pennsylvania, USA (40.709746° N, -77.956965° W) from June 513
through September 2016. A split plot design was utilized with two P levels; two 514
0.05 ha low P fields (10 ppm mean available P by Mehlich-3 (ICP)) split into two, 515
0.025 ha blocks each and two 0.05 ha high P fields (38 ppm mean available P) 516
split into two, 0.025 ha blocks each. Plant genotypes were randomized within 517
each block. Fields were fertilized according to each treatment with soil nutrient 518
levels adjusted to meet P. vulgaris requirements as determined by soil tests at 519
the beginning of each season. Each genotype was planted in a five-row, 3 m long 520
plot with 72 cm row spacing and 10 cm intra-row spacing. During periods of 521
inadequate rainfall, irrigation was supplied through drip tape. The experiment 522
was run until destructive measurements were taken from all genotypes in all 523
treatments at time of flowering (49 DAP). Each genotype had four replications 524
within each P treatment. Average max/min temperature of this site for duration of 525
the experiment were 27°C/17°C, average total rainfall was 18.5 cm, and average 526
light:dark photoperiod was 14.5:9.5. Mid-day photosynthetic active radiation 527
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20
(PAR) was approximately 1500-2000 mol photons m-2 s-1. Soil is a Hagerstown 528
silt loam (fine, mixed, semi-active, mesic Typic Hapludalf). 529
To limit the presence of fungal disease, seed were treated with Captan 50W 530
fungicide at a rate of 0.5 ml/ 100 seeds prior to planting. At time of flowering (49 531
DAP), destructive shoot measurements were taken including leaf number, dry 532
shoot biomass, and leaf tissue P content. Dry mass was determined from tissues 533
dried at 65°C. Leaf P content was measured from 10, 2.5 cm leaf discs taken 534
from throughout the canopy in each plot. 535
At flowering (49 DAP), the crown of the root system for 3 representative plants 536
per plot (i.e. per replicate) were extracted, washed, and basal root whorl number, 537
basal root number, adventitious root number, basal root growth angle were 538
measured. A representative plant is a healthy plant that is comparable in shoot 539
size to the majority of plants throughout the plot. Basal root growth angle was 540
visually scored against a protractor. Three soil cores were taken from each plot 541
to a depth of 40 cm, 10 cm from the base of representative plants toward plants 542
from the neighboring row (Giddings Machine Co., Windsor, CO, USA). Soil cores 543
were 5.1 cm in diameter and were divided into 4, 10 cm increments, washed, and 544
extracted roots from each segment were scanned with an EPSON Perfection 545
V700 PHOTO scanner. From these images root length density (length of root per 546
volume of soil) in the top 40 cm of soil was quantified with WinRhizo software 547
(WinRhizo Pro, Regent Instruments, Quebec City, Quebec, Canada). The 548
anatomy of 5 representative basal roots was analyzed from the segment of root 549
2.5 cm from the hypocotyl. Basal root segments were preserved, sectioned, and 550
anatomical features were measured following the same protocol as described 551
above. 552
Mycorrhizal Study 553
This study was conducted under the same growth conditions as described above 554
for the greenhouse trial. Two genotypes contrasting in secondary growth (DG35 555
and DG51) were used. A complete randomized block design was utilized with 556
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21
two P levels; P stress and high P, and two vesicular-arbuscular mycorrhizae 557
(VAM) levels; inoculated and mock-inoculated. Each genotype had three 558
replications in each of the VAM levels within each P treatment. 559
Glomus intraradices (now known as Rhizophagus irregularis, Tisserant et al., 560
2013) promotes P acquisition in P. vulgaris (Nielsen et al, 1998) and was used 561
for this study. To facilitate even distribution of spores, the inoculant (Premier 562
Tech Biotechnologies, Rivière-de-Loup, Québec, Canada), consisting of R. 563
irregularis spores, was mixed thoroughly with 1.5 kg of sterilized sand before 564
being mixed into the bulk growth media prior to planting. The final inoculation 565
intensity for mesocosms assigned to the inoculated treatment was 200 spores 566
per liter of growth media. For the mock-inoculated treatment, the same amount of 567
the liquid inoculant was filtered through Whatman filter papers #1 and #42 (Li et 568
al., 2012), mixed with 1.5 kg sterilized sand, and added to the growth media in 569
mock inoculated mesocosms to introduce inoculum factors other than VAM fungi. 570
At 49 DAP, root and shoot measurements were taken as described in the above 571
greenhouse study. VAM colonization was quantified using the magnified 572
intersections method (McGonigle et al. 1990). Two 10 cm segments of root were 573
harvested from the basal, middle, and apical ends of 2 basal roots from each 574
plant. Segments were cleared in 10% KOH, stained in a 5% ink-vinegar solution 575
(Vierheilig et al. 1998). A minimum of 50 intersections per sample were observed 576
and the incidence of hyphae, arbuscules, and vesicles was scored. The 577
percentage incidence of each structure over total intersections was calculated. 578
Statistical Analysis 579
All statistical analyses were performed using RStudio Version 0.99.903 (RStudio, 580
Inc.). Normality and homoscedasticity of the data were determined using the 581
Shapiro-Wilk test and the non-constant error variance test respectively. Where 582
data did not meet these assumptions, a box-cox or log transformation was used 583
to normalize the data. Analysis of variance (ANOVA), Tukey HSD, and 584
regression analysis were performed with significant effects considered at P 585
0.05. Because plants grown under high P were larger than those under P stress, 586
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22
allometric relationships between root phenotypes and shoot mass were explored 587
as in Burridge et al. (2017). The relationship between the decadic logarithm of 588
the root phene and shoot biomass were fitted by linear regression. Log 589
transformation of data prior to the regression analysis is necessary to normalize 590
any multiplicative relationships that may exist between the shoot biomass and 591
value of a given metric. The scaling coefficient of = 0.33 is considered to be the 592
threshold where root phenes with ≥ 0.33 have scaled faster than shoot size 593
and are considered hyperallometric, while root phenes where < 0.33 scale at a 594
slower rate than shoot size and are considered hypoallometric. Statistical 595
analysis of SimRoot output was not performed, as modeling output is most suited 596
for qualitative comparisons rather than statistical tests designed for empirical 597
data. Performing statistical tests on modeling output results in artificially high p 598
values, regardless of effect size, as differences in replicates are simply the result 599
of random number generators within the model. Additionally, because the 600
contrasting parameters are programmed into the model, it is known before the 601
model is run that the null hypothesis is false (White et al. 2014). 602
Acknowledgements: We thank James Burridge for his assistance with field 603
research, Bob Snyder for oversight of lab and field activities, Johannes Postma 604
and Harini Rangarajan for support with SimRoot, Airong Li for guidance with 605
mycorrhizal research, and Michael Williams for assistance with elemental 606
analysis. 607
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23
Table 1. Allometric analysis comparing shoot biomass to root phenes in the greenhouse at 46 DAP, and in the field at 49 DAP. Root phenes include total cross sectional area (TCSA; mm2), metaxylem vessel area (MXA; mm2), metaxylem vessel number (MXN), axial hydraulic conductance (cond.; kg m MPa-
1 s-1), basal root length measured in the greenhouse (BRL; cm)/ root length density (RLD; cm/cm3), basal root whorl number (BRWN), basal root number (BRN), adventitious root number (ARN), stele cross sectional area (SCSA; mm2),
percent stele area (% Stele), basal respiration rate (Resp.; mol CO2 cm-1 s-1), total root length (Tot. RL; cm), specific root length (SRL; cm/g), and total root dry mass (R Mass; g). Anatomical data were means from the basal segment for each mesocosm/plot. Adjusted coefficient of determination (R2), y intercept (Int.),
scaling coefficient (), and P-value (p) for the regression line are shown.
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24
Fie
ld
Hig
h P
p
0.4
4
0.3
5
0.7
9
0.2
5
0.9
5
0.0
6
0.0
9
0.3
6
()
0.0
8
0.0
7
0.0
3
0.0
6
-0.0
1
0.6
6
0.3
0
0.1
9
Int.
1.2
2
1.2
8
1.1
6
1.6
7
1.2
2
1.0
2
1.0
1
1.0
3
R2
-0.0
1
-0.0
04
-0.0
3
0.0
1
-0
.03
0.1
0
0.0
8
-0.0
05
P S
tre
ss
p
0.0
6
0.1
3
0.3
9
0.0
4
0.0
1
0.6
1
0.2
5
0.5
7
()
-0.2
6
-0.1
9
-0.1
4
-0.1
8
0.7
1
0.4
5
0.4
8
-0.2
1
Int.
0.9
9
0.8
1
1.2
4
-0.4
0
1.2
3
0.8
6
0.6
3
1.2
4
R2
0.0
9
0.0
4
-0.0
08
0.1
0
0.2
0
-0.0
2
0.0
1
-0.0
2
G
ree
nh
ou
se
Hig
h P
p
0.9
8
0.8
1
0.5
1
0.9
9
0.3
0
0.2
0
0.0
2
0.4
8
0.9
7
0.6
9
0.2
3
0.0
4
0.9
8
<0.0
1
()
0.0
03
0.0
2
0.0
8
0.0
01
-0.1
8
0.2
6
0.5
7
-0.0
5
0.0
04
0.2
8
0.1
0
0.1
8
0.0
04
0.5
0
Int.
1.5
9
1.6
0
1.4
3
1.5
9
1.9
2
1.4
9
1.0
6
1.6
3
1.5
8
1.0
2
1.9
2
0.9
6
1.5
7
1.1
0
R2
-0.0
3
-0.0
3
-0.0
2
-0.0
3
0.0
03
0.0
2
0.1
4
-0.0
2
-0.0
3
-0.0
3
0.0
2
0.1
0
-0.0
3
0.6
0
P S
tre
ss
p
0.2
0
0.7
9
0.7
3
0.6
4
<0.0
1
0.1
6
0.0
5
0.0
8
0.4
1
0.8
3
<0.0
1
0.0
1
<0.0
1
<0.0
1
()
-0.2
7
-0.4
-0.0
6
0.0
5
1.9
0
0.6
2
0.9
0
-0.4
8
-0.1
0
-0.0
5
-0.3
2
0.3
5
0.6
8
0.6
7
Int.
0.5
0
0.5
1
0.6
6
0.9
6
-2.8
7
0.3
6
-0.2
8
1.0
7
0.5
2
0.6
6
-0.8
7
-0.4
7
-2.3
2
0.5
2
R2
0.0
2
-0.0
3
-0.0
3
-0.0
3
0.5
2
0.0
3
0.0
9
0.0
7
-0.0
1
-0.0
3
0.2
3
0.1
6
0.1
9
0.6
9
TCSA
MX
A
MX
N
Co
nd
.
BR
L/R
LD
BR
WN
BR
N
AR
N
SCSA
% S
tele
Re
sp.
Tot.
RL
SRL
R M
ass
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25
Figure Legends
Figure 1. SimRoot results showing meanSE root respiration rate (g C/g root-1 day-1) (A), total root length (m) (B), total P uptake (mmol) (C), and shoot biomass (g) (D) in roots systems with three levels of secondary growth (advanced,
intermediate, reduced) under P stress (4 m P) and high P (30 m P) at 40 days of growth.
Figure 2. MeanSE shoot mass (g) (A), leaf number (B), and leaf area (cm2) (C) at 18, 32, and 46 DAP under P stress in the greenhouse. Comparisons are made across all timepoints.
Figure 3. MeanSE specific root length (m/g) (A), axial basal root length (cm) (B), and total shoot P (mg) (C) at 18, 32, and 46 DAP under P stress in the greenhouse. Comparisons are made across all timepoints.
Figure 4. Field data comparing basal TCSA (mm2) (A), root length density (RLD) (cm/cm3) (B), and total shoot P (mg) (C) between advanced and reduced phenotypes under P stress. Comparisons for each variable were made across phenotypic groups and P treatments.
Figure 5. Correlation between mean basal TCSA (mm2) and mean root length density (RLD) (cm/cm3) (A, B), as well as mean root length density (RLD) (cm/cm3) and total shoot P (mg) (C, D) for each plot under P stress and high P treatments in the field. Red lines indicate a significant correlation at a confidence
levels of p 0.05 using Pearson’s product-moment correlation analysis. n = 32
Figure 6. MeanSE TCSA (mm2) of basal root at three locations along the root axis taken at 18 DAP (A), 32 DAP (B), and 46 DAP (C) under P stress in the greenhouse. Comparisons are made within each timepoint.
Figure 7. Comparison of basal root anatomy between reduced and advanced groups under high P and P stress in greenhouse conditions at 46 DAP. All cross
sections are at the same scale (bar is 100 m).
Figure 8. MeanSE basal root TCSA (mm2) (A), metaxylem number (B), total metaxylem area (mm2) (C), and axial hydraulic conductance (kg m MPa-1 s-1) (D) at 49 DAP in the field. Comparisons for each variable were made across phenotypic groups and P treatments.
Figure 9. Correlation between basal TCSA (mm2) and basal respiration rate (μmol CO2 cm-1 s-1) (A), total basal root length (cm) (B), and total shoot P (mg) (C) under P stress and high P treatments at 46 DAP in the greenhouse. Red
lines indicate a significant correlation at a confidence levels of p 0.05 using Pearson’s product-moment correlation analysis. n = 32
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26
Figure 10. MeanSE respiration rate (mol CO2 cm-1 s-1) of the basal (A), middle (B), and apical (C) positions at 18, 32, and 46 DAP under P stress in the greenhouse. Comparisons are made within each timepoint.
Figure 11. Cortical tissue containing fungal hyphae being shed during secondary growth (A). Cross section of basal root axis at 46 DAP in the greenhouse showing difference in cortical tissue abundance under high P and P stress (B).
MeanSE percent colonization of basal root axis at three positions of the root under high P and P stress (C). Relationship between mean cortical tissue abundance in cross section and mean percentage colonization for roots grown under high P and P stress (n = 36) (D).
Supplemental Data
Supplemental Figure S1. Transverse section of basal root at different developmental stages to highlight changes in tissue as secondary growth progresses.
Supplemental Figure S2. SimRoot results for three P. vulgaris root systems with three levels of secondary growth.
Supplemental Figure S3. Model of P. vulgaris root systems with two levels of secondary growth.
Supplemental Figure S4. SimRoot results showing meanSE total P uptake, and shoot biomass in roots systems with no secondary growth.
Supplemental Figure S5. MeanSE root mass of genotypes with advanced and reduced secondary growth in the greenhouse at 46 DAP.
Supplemental Figure S6. MeanSE net length of lateral roots per basal root.
Supplemental Figure S7. MeanSE percent stele area of cross section of basal, middle, and apical positions.
Supplemental Figure S8. MeanSE metaxylem vessel number of basal, middle, and apical positions.
Supplemental Figure S9. MeanSE net metaxylem vessel area (mm2) of basal, middle, and apical positions.
Supplemental Figure S10. MeanSE theoretical axial hydraulic conductance (kg m MPa-1 s-1) 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.
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27
Supplemental Figure S12. MeanSE basal TCSA:root length (mm2/m) of eight genotypes in the greenhouse at 32 and 46 DAP under P stress.
Supplemental Figure S13. Correlation between total basal root length (cm) and respiration rate (μmol CO2 cm-1 s-1) of the apical, middle, and basal segments under P stress and high P treatments.
Supplemental Figure S14. Correlation between dry shoot weight (g) and respiration rate (μmol CO2 cm-1 s-1) of the apical, middle, and basal segments under P stress and high P treatments at 46 DAP in the greenhouse.
Supplemental Figure S15. MeanSE TCSA (mm2) (A), N concentration (%), and P concentration (mg P/g tissue) of the basal segment in each phenotypic group.
Supplemental Figure S16. MeanSE TCSA (mm2), percent stele area (%), cortex area (mm2), and theoretical hydraulic conductance
Supplemental Figure S17. Diagram of basal root segment locations for anatomy, respiration, and elemental analysis.
Supplemental Data. Summarized hierarchical input file showing context of SimRoot parameters.
Supplemental Figure S1. Transverse section of basal root at different developmental stages to highlight changes in tissue as secondary growth progresses. Bars are 0.5 mm; images are all to the same scale.
Supplemental Figure S2. SimRoot results for three P. vulgaris root systems with three levels of secondary growth (advanced, intermediate, and reduced) grown
over 40 days under 4m available P where reducing secondary growth of roots results in reduced metabolic cost (A), greater total root length (B), greater P uptake (C), and greater shoot biomass (D).
Supplemental Figure S3. Model of P. vulgaris root systems with two levels of
secondary growth (reduced and advanced) at 40 DAP under 4 m available P where reducing secondary growth of roots results in greater allocation of resources to increase root length. The reduced phenotype has 14% greater root length than advanced phenotype at this P availability.
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28
Supplemental Figure S4. SimRoot results showing meanSE total P uptake (A), and shoot biomass (B) in roots systems with no secondary growth under three levels of P stratification at 40 days of growth. Simulation with no stratification had
4 m P homogenously distributed throughout soil profile. Simulation with weak
stratification had 4 m P in top 15 cm, 1.33 m P from 16 to 29 cm, and 0.27 m
P from 30 cm and below. Simulation with strong stratification had 4 m P in top 5
cm, 1.33 m P from 6 to 10 cm, and 0.27 m P from 11 to 29 cm and 0.24 m P from 30 cm and below.
Supplemental Figure S5. MeanSE root mass (g) of genotypes with advanced
and reduced secondary growth in the greenhouse at 46 DAP (A). MeanSE root mass : shoot mass ratio (g/g) of genotypes with advanced and reduced secondary growth at 46 DAP in the greenhouse (B).
Supplemental Figure S6. MeanSE net length of lateral roots per basal root (cm) (A), percentage of the total basal root length comprised of lateral roots (%) (B), and percentage of lateral roots <0.5mmin diameter (%) (C) in genotypes with advanced and reduced secondary growth under high P and P stress in the greenhouse at 46 DAP.
Supplemental Figure S7. MeanSE percent stele area of cross section of basal, middle, and apical positions at 18 (A), 32 (B), and 46 DAP (C) under P stress in the greenhouse. Comparisons are made within each timepoint.
Supplemental Figure S8. MeanSE metaxylem vessel number of basal, middle, and apical positions at 18 (A), 32 (B), and 46 DAP (C) under P stress in the greenhouse. Comparisons are made within each timepoint.
Supplemental Figure S9. MeanSE net metaxylem vessel area (mm2) of basal, middle, and apical positions at 18 (A), 32 (B), and 46 DAP (C) under P stress in the greenhouse. Comparisons are made within each timepoint.
Supplemental Figure S10. MeanSE theoretical axial hydraulic conductance (kg m MPa-1 s-1) of basal, middle, and apical positions at 18 (A), 32 (B), and 46 DAP (C) under P stress in the greenhouse. Comparisons are made within each timepoint.
Supplemental Figure S11. Root crowns of genotypes with advanced and reduced secondary growth excavated at 49 DAP from the field under P stress. Cross sections are representative of the mean TCSA of the basal segment of each genotype under P stress.
Supplemental Figure S12. MeanSE basal TCSA:root length (mm2/m) of eight genotypes in the greenhouse at 32 and 46 DAP under P stress (A). Mean basal TCSA (mm2) plotted against mean total basal root length (m) in greenhouse at 32 and 46 DAP under P stress (B). Genotypes in the advanced group had mean TCSA:RL > 0.1 while genotypes in the reduced group had a mean TCSA:RL of <
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29
0.1. The dotted line denotes the threshold of 0.1 that separates the phenotypic groups.
Supplemental Figure S13. Correlation between total basal root length (cm) and respiration rate (μmol CO2 cm-1 s-1) of the apical, middle, and basal segments under P stress and high P treatments at 18 (A, B), 32 (C, D), and 46 DAP (E, F) in the greenhouse. Red lines indicate a significant correlation at a confidence levels of p ≤ 0.05 using Pearson’s product-moment correlation analysis. n = 96
Supplemental Figure S14. Correlation between dry shoot weight (g) and respiration rate (μmol CO2 cm-1 s-1) of the apical (A, B), middle (C, D), and basal segments (E, F) under P stress and high P treatments at 46 DAP in the greenhouse. Red lines indicate a significant correlation at a confidence levels of p ≤ 0.05 using Pearson’s product-moment correlation analysis. n = 32
Supplemental Figure S15. MeanSE TCSA (mm2) (A), N concentration (%) (B), and P concentration (mg P/g tissue) (C) of the basal segment in each phenotypic group under P stress (L) and high P (H) treatments at 18, 32 and 46 DAP.
Supplemental Figure S16. MeanSE TCSA (mm2) (A), percent stele area (%) (B), cortex area (mm2) (C), and theoretical hydraulic conductance (kg m MPa-1 s-
1) of the apical segment under P stress and high P at 46 DAP.
Supplemental Figure S17. Diagram of basal root segment locations for anatomy, respiration, and elemental analysis.
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30
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A
B
C
D
Figure 1. SimRoot results showing mean±SE root respiration rate (g C/g root-1 day-1) (A), total root length (m) (B), total P uptake (mmol) (C), and shoot biomass (g) (D) in roots systems with three levels of secondarygrowth (advanced, intermediate, reduced) under P stress (4μm P) and high P(30μm P) at 40 days of growth.
4 μmol P 30 μmol P
Sh
oot
Mass
(g)
Tota
l P U
pta
ke(m
mol)
Tota
l R
oot
Length
(m
pla
nt-1
)R
oot
Resp
irati
on
(g C
/g r
oot-1
day
-1)
Advanced Intermediate Reduced
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**
*
*
*
**
*A
B
C
Figure 2. Mean±SE shoot mass (g) (A), leaf number (B), and leaf area (cm2) (C) at 18, 32, and 46 DAP under P stress in the greenhouse. Comparisons are made across all timepoints.
18 32 46DAP
1
3
5
7
14
10
6
2
1550
950
350
Leaf
Are
a (
cm2)
Leaf
Num
ber
Sh
oot
Mass
(g)
e
e
a
bc
dfa
bc
de
a
bc
df
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*
*
* *
**
*
Shoot
PB
asa
l R
oot
Leng
thSpeci
fic
Root
Length
(mg)
(cm
)A
B
C
100
150
200
250
300(m
/g)
70
55
40
12
9
6
3
0
Figure 3. Mean±SE specific root length (m/g) (A), axial basal root length (cm) (B), and total shoot P (mg) (C) at 18, 32, and 46 DAP under P stress in the greenhouse. Comparisons are made across all timepoints.
18 32 46
a
ab
a a
bcbc
c
b
cd
a
a
ab
bc
cdd
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A
B
C
Tota
l S
hoot
P (
mg
)R
LD
(cm
/cm
3)
Basal TC
SA
(m
m2)
Advanced Reduced
bab
a
c
High P
P Stress
aba
ab b
a
b
a
c
Figure 4. Field data comparing basal TCSA (mm2) (A), RLD (cm/cm3) (B), and total shoot P (mg) (C) between ASG and RSG phenotypes under P stress. Comparisons for each variable were made across phenotypic groups and P treatments.
c
a a
b
aab
bab
aab b
c
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P Stress High PP Stress High PA B
C D
Figure 5. Correlation between mean basal TCSA (mm2) and mean RLD (cm/cm3) (A, B), as well as mean RLD (cm/cm3) and total shoot P (mg) (C, D) for each plot under P stress and high P treatments in the field. Red lines indicate a significant correlation at a confidence levels of P ≤ 0.05 using Pearson’s product-moment correlation analysis. n= 32
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**
*
* **
**
*
A
B
C
Roo
t TC
SA
(m
m2)
Figure 6. Mean±SE TCSA (mm2) of basal root at three locations along the root axis taken at 18 (A), 32 (B), and 46 DAP (C) under P stress in the greenhouse. Comparisons are made within each timepoint.
Basal Middle Apical
0.6
0.5
0.4
0.3
0.6
0.4
0.2
0.8
0.6
0.4
0.2
0
TCS
A (
mm
2)
b
a
c
b
d
a
b
a
b
a
c
a
b
a
b
a
b
a
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Adv
ance
d R
educ
ed
P stress
Apical Middle Basal
P stress P stress High P High P High P
Figure 7. Comparison of basal root anatomy between reduced and advanced groups under high P and P stress in greenhouse conditions at 46 DAP. Roots under P stress have smaller TCSA, smaller percent stele area, fewer xylem vessels, less vessel area, and reduced hydraulic conductance compared to roots grown under high P. All cross sections are at the same scale (bar is 100μm).
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TCS
AM
eta
xyle
m N
um
ber
Meta
xyle
m A
rea
Conduct
ance
kg m
MPa
-1 s
-1m
m2
mm
2a
b
ca
a
b
a
c
aa
b
c
a
c
a
b
A
B
C
D
Figure 8. Mean±SE basal root TCSA (mm2) (A), metaxylem number (B), totalmetaxylem area (mm2) (C), and axial hydraulic conductance (kg m MPa-1 s-1)(D) at 49 DAP in the field. Comparisons for each variable were made acrossphenotypic groups and P treatments.
Advanced Reduced
TCS
A(m
m2)
Meta
xyle
m N
um
ber
Meta
xyle
m A
rea
(mm
2)
Cond
uct
ance
(kg
m M
Pa-1 s
-1)
High PP Stress
cccccccccccccccccccccccccccccccc
cccccccccccccccccccccccccc
a
bb
c
c
ab
a
a
bb
c
c
bb
a
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Figure 9. Correlation between basal TCSA (mm2) and basal respiration rate (μmol CO2 cm-1 s-1) (A), total basal root length (cm) (B), and total shoot P (mg) (C) under P stress and high P treatments at 46 DAP in the greenhouse. Red lines indicate a significant correlation at a confidence levels of P ≤ 0.05 using Pearson’s product-moment correlation analysis. n=32
Basal Respiration (μmol CO2 cm-1 s-1)
A B
C D
E F
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Resp
irati
on (μ
mol C
O2
cm
-1 s
-1)
Figure 10. Mean±SE respiration rate (μmol CO2 cm-1 s-1) of the basal, middle, and apical positions at 18 (A), 32 (B), and 46 DAP (C) under P stress in the greenhouse. Comparisons are made within each timepoint.
A
B
C
a a
a
a
bc
ab
c
d d
a
a
b
bcc c
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High P P Stress
A C
DB
Colonization (%)
Co
rtic
al T
issu
e (m
m2 )Cortical Tissue
Cortical Tissue Hyphae Secondary Tissue
Colo
niz
ati
on
(%
)
Basal Middle Apical
High PP Stress
Colo
niz
ati
on
(%
)
bc bc
aba
c cc
Colonization (%)
Cort
ical Tis
su
e (
mm
2)
Figure 11. Cortical tissue containing fungal hyphae being shed during secondary growth (A). Cross section of basal root axis at 46 DAP in the greenhouse showing difference in cortical tissue abundance under high P and P stress (B). Mean±SE percent colonization of basal root axis at three positions of the root under high P and P stress (C). Relationship between mean cortical tissue abundance in cross section and mean percentage colonization for roots grown under high P and P stress (n = 36) (D).
DHigh PP Stress
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Parsed CitationsBarber S, Cushman J (1981) Nitrogen uptake model for agronomic crops. In: Iskandar I. ed. Modeling waste water renovation: landtreatment. New York: Wiley Interscience, 382–409.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Beebe S, Toro O, Gonzalez AV, Chacon MI, Debouck DG (1997) Wild-weed-crop complexes of common bean (Phaseolus vulgaris L,Fabaceae) in the Andes of Peru and Colombia, and their implications for conservation and breeding. Genetic Resources and CropEvolution 44: 73-91
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Beebe SE, Rojas-Pierce M, Yan XL, Blair MW, Pedraza F, Munoz F, Tohme J, Lynch JP (2006) Quantitative trait loci for rootarchitecture traits correlated with phosphorus acquisition in common bean. Crop Science 46: 413-423
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bieleski RL (1973) Phosphate pools, phosphate transport, and phosphate availability. Annual Review of Plant Physiology and PlantMolecular Biology 24: 225-252
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bonser AM, Lynch J, Snapp S (1996) Effect of phosphorus deficiency on growth angle of basal roots in Phaseolus vulgaris. NewPhytologist 132: 281-288
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Borch K, Bouma TJ, Lynch JP, Brown KM (1999) Ethylene: a regulator of root architectural responses to soil phosphorus availability.Plant Cell and Environment 22: 425-431
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Brundrett MC (2002) Coevolution of roots and mycorrhizas of land plants. New Phytologist 154: 275-304Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Burridge JD, Schneider HM, Huynh BL, Roberts PA, Bucksch A, Lynch JP (2017) Genome-wide association mapping and agronomicimpact of cowpea root architecture. Theoretical and Applied Genetics 130: 419-431
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chimungu JG, Brown KM, Lynch JP (2014a) Reduced root cortical cell file number improves drought tolerance in maize. PlantPhysiology 166: 1943-1955
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chimungu JG, Brown KM, Lynch JP (2014b) Large root cortical cell size improves drought tolerance in maize (Zea mays L.). PlantPhysiology 166: 2166-2178
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chimungu JG, Maliro MFA, Nalivata PC, Kanyama-Phiri G, Brown KM, Lynch JP (2015) Utility of root cortical aerenchyma under waterlimited conditions in tropical maize (Zea mays L.). Field Crops Research, 171: 86-98
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
CIAT (1996) Bean program annual report. CIAT, Cali, Columbia.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
De la Riva LM, Lynch JP (2010) Root etiolation as a strategy for phosphorus acquisition in common bean (Masters dissertation). Thehttps://plantphysiol.orgDownloaded on November 13, 2020. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
Pennsylvania State University Library (Call Number: Thesis 2010mDeLaR,LM)Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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