This article isThis article by protected copyright. All rightsreserved. 10.1111/nph.15516doi: differences to lead between the of thisversion citethisa and Version Record.Please copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which This article acceptedhas been for publication andundergone fullpeer review buthasnot Summary Accepted: Received: 5000 Kingdom United 1BJ Southampton, SO17 Road, University of Southampton, $ 6 5 4 3 2 Southampton Southampton, of University Environment, the and Engineering 1 Tiina Roose J. Laura Cooper Nicolai Koebernick Imaging microstructure of the barley rhizosphere: particle packing and root hair influences : Article type : ID 0000 (Orcid ROOSE DR TIINA School ofAberdeen, University Science, Environmental and Biological Institute of Warwick ofWarwick, University Institute, Mathematics University of Engineering, and Science School of Dundee Institute, Hutton The James Group, Sciences Ecological En Group, Research Sciences Bioengineering C

Accepted University Environment, and Engineering of Faculty , [email protected] author: orresponding Article   .

ORCID of Computing and Engineering and Computing of regimes. The tubes were tubes were The regimes. parent were Roots contradictory. hairs such asroot water an S oil oil 12 June 2018 12 23 September 2018 September 23 1 adjacent to adjacent $ :

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of hairless barley( hairless of 1,4 Regular Manuscript Regular d solute acquisition by plants. Detailed knowledge on how root activity root how on knowledge Detailed plants. by soluteacquisition d , Timothy S.George , Timothy 1 - , Keith R. , Keith Daly 0001 grown in tubes of sieved (<250 (<250 sieved of intubes grown

roots has distinct structural and physical properties from bulk frombulk soil properties physical and distinct structural has roots - 8710

affect affect

- 1063 the the Hordeum vulgare Hordeum 1 - scanned with scanned , Samuel D. Keyes D. , Samuel 0001

3D 3D 2 , Paul D. Hallett D. , Paul , University of West of University , - 8744 pore

- gineering Sciences Academic Unit, Faculty of Faculty Unit, SciencesAcademic gineering structure at structure 2060) s ynchrotron based based X ynchrotron Dundee, Dundee Dundee,

L. cv ‘Optic’) mutant mutant ‘Optic’) cv L.

1 ,  5 Anthony G. Bengough Anthony m) sandy loam soil under two different water water two different under loamsoil m) sandy , Muhammad Naveed , Muhammad

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This article isThis article by protected copyright. All rightsreserved. 2012) al. et 2011;Valentine et al. Acceptedsuch as properties mechanical soil elongation root opposing the impedance that overcomes pressure growth exerta must roots soil, to penetrate In order altered po wh soil, rhizosphere resource acquisition et2014) Aravena al. significantly water uptake rate ofroot simulated the increased expansion root that shown has modelling and combining imaging work instance interface for properties. structural resources all because uptake andsolute water root for importance primary isof ofroots immediate vicinity the in structure pore The activity). ofmicrobial enhancement theassociated and exudates of and hydraulic stresses and mechanical of action the combined well It is transport. andsolute flow to water related directly is soil in porespace oftheavailable The geometry Introduction Article packing particle structure, hairs, Root Keywords:  

and compaction soil th describes that isproposed A model conditions. differences statistically significant distribution observed r at the particles of packing packing dense Less analysed thesoil structure at

entering lea or entering re geometry affects resource capture by roots. byroots. capture affectsresource geometry re

versus versus increased increased

known

ich . Despite the Despite . was narrower narrower was there isstill a there

, The importance of of importance The r was

hizosphere, hizosphere, mechanisms and root traits contribute to contribute root traits and mechanisms distance distance ving the root have to cross this layer this to cross have ving theroot (Jin et al. 2013) et (Jin al.

that plant roots alter the pore geometry of the soil adjacent to roots by to roots adjacent the soil of geometry alterthe pore roots that plant recognized - pore volume volume pore root interface. Pore volume fraction and pore size distribution were sizedistribution pore and fraction volume Pore interface. root the surface surface

, within 1mmo within potential

as well as morphological properties of the roots. Thicker roots are roots Thicker oftheroots. properties asmorphological well as due to due substantive substantive pore structure, structure, pore Hordeum vulgare Hordeum in the in effec measuring measuring

fraction fraction and radial growth radial and a in pore structure structure in pore

impo de 195 t e creas lack of understanding of the structural properties of properties the structural of ofunderstanding lack at the root surface. at theroot

variation in porosity near roots taking into account account takinginto nearroots inporosity variation f rtance ofthe rtance 0s the root surface. the root immediately next to theepidermis nextto immediately soil strength soil

ed by by small , noninvasive imaging, synchrotron, soil soil imaging,synchrotron, noninvasive , fraction of fraction Richards and Wadleigh (1952) Wadleigh and Richards oot enhanced inter enhanced -

scale - surface surface . Root penetration therefore depends on depends therefore penetration . Root between genotypes or moisture moisture or genotypes between rhizosphere

which has which

, biochemical activity ( activity biochemical

hydraulic hydraulic and and structural structural larger pores. larger pores.

was hypothesised was soil soil - aggregate contact contact aggregate matric potential potential matric distinct physical and and physical distinct gradients at the soil at gradients

pore structure for structurefor pore changes, and how the how and changes, There were no were There (Aravena et2011; al. (Aravena i. . . The

e. the release therelease e. to cause the cause the to More recent (Bengough (Bengough

p caused by caused ore size size ore - root root

This article isThis article by protected copyright. All rightsreserved. Accepted to close roots. porosity they expe macropores with many root that worth mentioning fromthe rhizosp sizesinaggregates pore but greater fungi. root associated than porosity soil on etal.2009) 2006;Hallett etal. (Feeney have in therhizosphere porosities unclear.Increased remained themechanism although rhizosphere, the in porestructure of inthe regulation a crucial role greater air air decreased exhibited parent wildtype with its mutant barley deficient o soilstructure the rhizosphere comparing anexperiment in were made observations Similar root maturation. with andit decreased theroots youngest in observed was gradient porosity poros soil that showed CT imaging by observations Recent growth. of root direction along in the tha and displacement lateral than stronger much was displacement et2010) Vollsnes al. 2017; et (Keyes al. Article surface. simple model 1998) Young 2010; et al. Vollsnes 1990; Hettiaratchi 1996; etal. Bruand as the roots around densification soil of azone predict models physical and studies experimental ofprevious majority into soil. The at soil soil the of pattern the deformation to are related hairs ofroot thepresence by enhanced may be and biopores macropores high localise al. 2010) et Vollsnes 2017; et Keyes al. 2003; al. penetration root to influence shown been caphave root atthe mucilage of and therelease tip at root soil the the by stress loosening axial limiting overcoming of amechanism as been interpreted has soils strong generally S

imilar pattern imilar better at penetrating strong soils penetrating strong better at rience low water potentials potentials water low rience - , filled porosity at the at porosity filled

have shown that densification of rhizosphere soil may not be the general trend. They trend. general be the not may soil ofrhizosphere densification that shown have d mechanical impedance by compensatory extension into loose soil compartments or compartments soil loose into extension compensatory by impedance mechanical d (Bingham and Bengough 2003; Colombi et al. 2017) et al. 2003;Colombi Bengough and (Bingham that that (Hettiaratchi 1990) (Hettiaratchi predict ity decreased with distance from the root surface of tomato. The greatest Thegreatest tomato. of root surface the from ity with distance decreased

- s (Colombi et al. 2017; Kooistra et al. 1992; Schmidt et al. 2012) etal. Schmidt 1992; et Kooistra al. 2017; etal. (Colombi filled porosity at the soil at porosity filled

ha ed ed ve - soil contact can be significantly decreased when roots grow into soil soil growinto roots when decreased contact besignificantly soil can an exponential decrease of soil density with distance from thero from withdistance density soil of decrease exponential an

been observed experimentally using time using experimentally observed been immediate root surface. It was concluded that root might hairs play root that concluded was It root surface. immediate th ey . Furthermore, theshape Furthermore, . (Carminati et al. al. 2013) et (Carminati ingress into soil soil into ingress

. However, However, . and it could be shown that roots had a much stronger impact impact stronger much hada that roots shown be could it and

Whalley et al.(2005) et Whalley (Chimungu etal. 2015) (Chimungu . In naturally structured soils structured Innaturally . - (Koebernick et al. 2017) et al. (Koebernick root interface, wildtype roots showed a significantly asignificantly showed roots interface, wildtype root Keyes et al. (2017) et Keyes al. here compared to those from the bulk soil. is It bulksoil. the thosefrom to here compared (Aravena et al. 2011; Aravena et al. 2014; et2014; al. 2011;Aravena et al. (Aravena - root interface caused by root penetration by root penetration interface caused root , which would result in a greater greater a result in , which would (Bengough et al. 20 et al. (Bengough Helliwell et al. (2017) et al. Helliwell

of the root tip theroot of been evidenced been evidenced

. The ability to penetrate loose soil soil loose penetrate to Theability . found no differences in porosity in differences no found , indeed thickening of roots in roots of thickening , indeed t particles tended to be dragged todragged be tended t particles

showed that that vertical showed . - . While the hairless mutant mutant the hairless . While lapse imaging techniques imaging lapse Dexter (1987) Dexter ,

roots can overcome can overcome roots (Colombi et al. 2017b) etal. (Colombi in earlier work work in earlier 16) ,

using time using . T f a root hair hair f aroot

or shrink when when shrink or

hese factors hese factors proposed a proposed (Iijima et et (Iijima ot ot - lapse lapse ,

This article isThis article by protected copyright. All rightsreserved. Accepted distance with parameters structural extract soil appliedto was image analysis Digital formation. were treatments moisture root of theimpact visualise (WT) parent its wildtype and (NRH) mutant hairless genotypes, a a Here, wepresent roots. plant suchas biological interfaces near porosity determining considered in not been knowledge con cylindrical extensively a Such the particles. soil the root and of diameters therelative on depending would be behaviour. physical soil local influence subsequently would which in porosity, increases scale particleoraggregate produce largersurfaces. against tightly than together the of the concept illustrates the pac is attention not has that aroot surface to close porosity the aspectdetermining An important exudates of effect / stabilising reversetheweakening may also rhizosphere inthe microaggregates Article of formation roleinthe animportant whichplay andbacteria, fungi including microbiota, soil for source carbon important arean Root exudates soil. whichstabilised frommaize, exudates (2017a) Naveed et al. species the same chemica etal.2018) 2009;Vidal et Hallettal. 2000; andmicrobes plant roots by areexuded which agents binding additionally requiring and aggregation Hallett 2008) particles ofclay shrinkage through ofcrack formation driver asamajor recognised verydryconditions under especially deformation, plastic growing tip Radial uptake. plant or water growth root by the soil of fracturing ismechanical roots to close porosity may impact that mechanism Another

l composition and physical properties depending on thespecies on properties depending physical and composition l expected to take the shape to takethe expected analysed both experimentally and experimentally both analysed (Hettiaratchi 1990) (Hettiaratchi . Wetting and drying cycles in the rhizosphere are also an important driver of driverof an important are also in therhizosphere drying cycles and Wetting . tainers such as chemical reactors reactors chemical as tainers such rhizosheath formation formation rhizosheath

(Mwafulirwa et al. 2016; Naveed et al. 2017a; Naveed et al. 2017b) etal. Naveed al. 2017a; et Naveed et 2016; al. (Mwafulirwa n imaging study with roots of contrasting barley ( barley ofcontrasting roots with imaging study n king of soil particles soil king of

showed that exudates from barley roots weakened s weakened roots barley from exudates that showed D u e to e to comp

activity surface/

such asurface such and root growth may lead to elastic fracturing of the soil instead of of the instead soil fracturing of elastic to may lead root growth and ared ared

on soil pore structure at the soil at pore structure soil on (Vidal et al. et al. (Vidal

of an oscillatory wave with amplitude and frequency frequency and amplitude wavewith an oscillatory of wall effect (Suzuki et al. 2008) where particles pack more more pack particles where 2008) et al. (Suzuki effect wall to assess the impact of soil suction on rhizosphere structure structure rhizosphere suctionon ofsoil the impact assess to

(A and agglomerates lbalasmeh and Ghezzehei 2014; Watt et et al.1994) 2014;Watt Ghezzehei lbalasmeh and . These root exudates may differ significantly in their intheir differsignificantly may exudates root . These expans Immediately

effect, r effect, mathema 2018) (Mueller 2010; Suzuki et al. 2008) et al. 2010;Suzuki (Mueller ion ion of the root may may theof root . Microbial decomposition of root exudates root of decomposition . Microbial adial void distribution about a root surface surface a root about distribution adial void tically in packed beds of spheres in spheresin of beds inpacked tically (Ruiz et al. al. 2015) et (Ruiz

at theroot at arranged about arranged

(Naveed etal. (Naveed 2017a) grown grown - Hordeum vulgare Hordeum root interface. Two contrasting contrasting Two interface. root - loosen soil in soil

in soil microcosms to microcosms in soil and among genotypes of of genotypes among and

. Water uptake by roots is roots by Water uptake . the root surface. root the oil as opposed to opposed as oil terface, this could could this terface,

the soil ahead of the aheadof the soil phenomenon phenomenon , yet yet

but has to our to but has (Yoshida and (Yoshida (Czarnes et al. et (Czarnes al. . .

received much received For instance, instance, For

L. cv. Optic) Optic) cv. L. , has been has Fig. Fig. 1 This article isThis article by protected copyright. All rightsreserved. ( (WT) barley of three of day everysecond reservoir the byremoving dry”), generated (“wet and drying wetting of cycle andii) rack, a holder underthe sample continuously were kept used, were treatments moisture contrasting Two head of ina resulting 20cm of atadistance water reservoirs open into the wicks byhanging provided was barrels the syringe of racks.Watering sample holder of content gravimetric water a to andwatered fine sand filled with assembly was seedling the of compartment The seed in described as connect ofseven and ingroups clustered were syringe barrels range size to theparticle in thefield Mgm ± 0.03 1.31 of dry bulk density a packing at a homogeneous resultedin procedure This again. tapped gently and refilled soil with space was thre barrel thesyringe tapping bygently wasconsolidated soil barrel w fine sand mL a through passed and one end on to aknot tied were =1mm) wick (ø carbonfibre of long strings the packing in bed aggregate an restructure deform and ratherthan soil, aggregate to roots the capacity of explore to 2017) et al. (Koebernick earlierwork thanour range particle size UK Dundee, Institute, Hutton a The was soil S Materials and Methods ma which the across rhizosphere, soluteflow water and surface. the root particles about spherical soil of geometry packing theparticle and extension by root compression soil account into which takes the roots. from Accepted Articleample syringe barrel barrel syringe by passing it through the funnel until the syringe barrel was completely filled with soil. The filled soil. with completely was barrel syringe until the thefunnel itthrough passing by plant treatments was randomly assigned to each assembly: i) a root hair bearing wild bearing hair i)aroot each assembly: to assigned randomly was treatments plant

preparation

immediately after tillage after immediately as poured in poured as sandy loam texture loam sandy root microcosms root Koebernick et al. (2017) et al. Koebernick Hordeum vulgare Hordeum We propose a novel simplified model of porosity distribution in the inthe rhizosp distribution porosity of model anovel simplified propose We (ø =4.2

to

studied and intentional removal of larger aggregated structures. aggregated oflarger removal intentional and studied mm

the syringe barrel viaa barrel the syringe . ) 0.2 g

The s The such that that such . To ensure even wate even To ensure

L. d

oil was air dried and airdried was oil Dystric Cambisol collected at Cambisol Dystric cv. Optic), ii) a Optic), ii) cv. , but this includes macropores that do not exist in our study due study due our not exist in do that macropores thisincludes , but - 1 . A total of 30 assemblies were prepared and plac and prepared were 30assemblies total of . A .

they they did not slip through the slipthrough did not Our findings have implications on the dynamics of the dynamics on implications have Our findings

- 3 i) a constant treatment (“wet”), where reservoirs reservoirs where (“wet”), treatment constant i) a f . This soil typically has a bulk density of 1.2 Mg m 1.2 density of hasa bulk typically soil . This hairless hairless unnel. Next, the soil was packed into the syringe the syringe waspacked into soil the Next, unnel. ring y affect y affect plant sieved to < to sieved

with a controlled water potential, water acontrolled with mutant (NRH), and iii) unplanted controls. controls. iii)unplanted and mutant (NRH),

- and then replacing after 24 hours after24 then replacing and

2 kPa at the bottom of the syringes. thesyringes. of bottom atthe kPa 2 ed to a 3D printed seedling assembly seedlingassembly a3Dprinted ed to e times with a spatula. The freed up up The freed timeswithaspatula. e

South Bullio South 250 , and to achieve consistent consistent to achieve , and syringe syringe productivity. µ m. m. We selected a selected We n nozzle. nozzle. field at the James theJames at field

A ed into two into ed 1mm layerof 1mm

I finer finer ndividual ndividual here, here,

25 cm - type type . One - 1 - 3

This article isThis article by protected copyright. All rightsreserved. at 4×magnification. particles. soil adhered barrels fromthe syringe out were Roots washed microscope. sel ofthree roots out onwashed wasmeasured hair morphology h. 48 Cfor 80˚ dryingat oven after and before syringe in each soil the weighing by measured was content Water measurements. After range. dynamic slices stacksof2160 to converted algorithm and (FOV) o view fieldof 1.6µmwitha was size pixel Resulting detector. 5.5CMOS edge withaPCO recorded and magnification) optical (4× system microscope a using magnified was light The scintillated of totalduration to a sleading of0.15 time exposure withan were recorded 180˚ through projections roo finding of the chance to maximise barrel theof syringe end upper the near mm apart) (3.5 heights different two at scanned were barrels Syringe parafilm. sealed with and the growth assembly from excised UK. Source, Oxfordshire, based X Synchrotron Synchrotron based X d an of75%, humidity relative and night,respectively, with operating Canada) Ltd., Winnipeg, chamber climate in 12d for a weregrown Plants compartment. each seed into pipetted per 2mlofwater film, another ofthecling Afterremoval oftheseedlings. emergence planted conditions. the of compartment a control) NRH, (wet,wet treatment moisture for levels design 2 with factorial al. 2004) Seeds were ensity

Accepted~ Article 4 min per scan. X perscan. 4 min

f 4 × 3.5 mm. Reconstruct mm. × f 4 3.5 during the day. day. during the

described previously by previously described ts.

obtained from the barley mutant population at The at population mutant the barley from obtained Scans nd 5 replicates per condition. One pre One condition. per replicates nd 5 w

- - ray CT scanning (SRCT) was performed at the I13 beamline at Diamond Light Light at Diamond beamline theI13 at performed (SRCT)was scanning CT ray scanning, rays were scintillated using a 500 µmcadmium a500 using were scintillated rays ere conducted ere conducted R

oots were placed on microscope slides and imaged with a light microscope microscope alight imaged with slides and microscope on wereplaced oots - P ray CTscanning lants were carried live to live carried were lants Shoot length and fresh weight was recorded on recorded weight was fresh and length Shoot

syringes were snap frozen and stored at at stored and frozen weresnap syringes ion of 3D images was carried out with a filteredback with a out carried imageswas of3D ion (Brown etal.2012) (Brown using ‘pink light’ at energies ofca.15 at energies light’ ‘pink using

a photoperiod of 14 h, temperature of 23˚/18˚ C during day Cduring day 23˚/18˚ of temperature of14h, a photoperiod Seed

compartments were sealed with cling film until cling until film sealedwith were compartments ,

each comprising 2560 × 2560 pixels with 32 pixels with × 2560 comprising each

the beamline the d a d . - The resulting experimental design was a 2 a was design experimental The resulting germinated germinated t 500 µmol m t µmol 500 and and

- dry) James Hutton Institute Institute Hutton James ected samples using a light using alight samples ected

and i sonicated for 5 mins for sonicated s , eed was placed in each seed seed in each eed placed was 3 levels for plant treatment (WT, (WT, plant treatment for 3 levels -

2 tungstate (CdWO tungstate

s ndividual syringe barrels were syringe barrels ndividual - 1 -

80˚ C for waterconten Cfor 80˚ photosynthetic photon f photon photosynthetic - 20 keV. keV. 20 excised shoots. shoots. excised A total of total of A

-

day were day were 4 projection projection to remove to remove ) scintillator. scintillator. ) (Caldwell et (Caldwell (Conviron (Conviron 1601 1601 - Root Root bit t × lux lux

3 This article isThis article by protected copyright. All rightsreserved. Accepted(i.e. thickness non within recorded from the were root distance with changes the root surface. voxelto soil ofany distance w transform distance Euclidean 3D The voxels. than50000 less volume of a radius anopen Subsequently, method Otsu’s computed using was global threshold a and applied voxels)was filter (σ=2 the were images ofroots, segmentation the For Fig.5). (Fig.3d, images binary onthe applied (Fig. 3c). ofpores segmentation each from 1979) (Otsu segmented Soilwas σ=1voxel radius. filter a with 1280 afactor of2 by respectively, directions, usin enhanced contrast was Article Image generator. number using arandom randomly 5werechosen requirements, fulfilled the were there others combination onetreatment For 2017) et al. studies previous done in aswas hairs root the segment to impossible centre fromthe with distance ( images CT the axis of centre the of mm from further an grew roots syringes, many ofthe of drop Inc., (TheMathWorks 2016a Matlab All image Image analysis × 1280 f

o of of For the quantification of pore size ofpore thequantification For f processing and and processing a of the 29 the 29 of σ= × . (Fig. barrels thesyringe theedge of valuesat grey lysis . The threshold was determined wasdetermined The threshold .

1079

4 voxels. The particle analyser in particle analyser The 4 voxels.

10 voxels) about the root. the root. about 10 voxels)

by selecting only by selecting

voxels at isotropic voxel size of 3.2 µm. Downscaling effectively applied a mean mean a applied effectively µm. Downscaling of3.2 size voxel atisotropic voxels

at least 5 replicates. For treatment combinations where more more where combinations treatment For replicates. least5 at replicate scan replicate see supporting information, information, supporting see . -

the syringe. This region was defined by calculating a projection along the the z along aprojection bycalculating defined region was This the syringe. close operation was performed using an octagonal performed an using was operation close A

analysis analysis 3D median filter (σ=2 voxels) was applied on the resulting binary images images binary the resulting on wasapplied voxels) (σ=2 filter median 3D g histogram equalization g histogram .

Due to the fine textured nature of the soil used i used the soil of nature thefinetextured to Due ( WT wet those those close to close to steps were steps s as applied to the segmented root image root appliedtothe segmented as .

The identical threshold was applied to all images for for toallimages wasapplied threshold identical The Cambridge, Cambridge, resulting resulting

images, where roots were growing within a cylinder of 3.8 of cylinder a within weregrowing roots where images, to reduce computational costs computational to reduce To minimise any edge effects imposed by the syringe walls, syringe bythe effects imposed any edge To minimise - dry the syringe walls. These These walls. the syringe

distribution on

)

performed in ImageJ ImageJ in performed Pore volume fraction and pore size distribution distribution and poresize fraction volume Pore only 4 replicates fulfilled the the replicates fulfilled 4 only ImageJ

a reference volume generated from 20 random slices slices 20 random from generated volume a reference image was then upscaled by a factor of 2 (Fig. 3b). 2(Fig. 3b). of by afactor upscaled wasthen image further UK)

into pore and solid phases using Otsu’s method Otsu’s using solid phases poreand into Fig. Fig. . .

In the reconstructed CT volumes, a volumes, CT reconstructed In the

Images were downscaled in thex, in downscaled Images were was used to remove any particles with a a with particles remove any used to was S , the “local thickness” thickness” , the“local

2) downscaled by a factor of 2. A 3D median 3Dmedian 2.A of afactor by downscaled , and measuring the average grey value greyvalue average the measuring , and

1) 1)

- overlapping annuli annuli of overlapping could be samples samples (Schindelin et al. 2012) etal. (Schindelin (Keyes et al. 2013; Koebernick Koebernick al. 2013; et (Keyes . This resulted in a FOV of FOV ina resulted . This observed. Additionally, in Additionally, observed. were excluded were structuring element element with structuring

filtering criteria filtering to determine the the determine to tool tool n this study, it it was n thisstudy, than 5 than in ImageJ 32 - replicates -

µm y systematic systematic .

and and from from - , for , for , and z , and

was was

all -

- A This article isThis article by protected copyright. All rightsreserved. Acceptedwhere neglected the root to adjacent immediately inporosity onlytheincrease simplify theapproximation to Inorder the root. from of theregion considering by beachieved can fromthe root distance of a function as porosity the Calculating root. by the porosity point Atthis packing. the where the root, to adjacent dashed seen When observing It is the with porosity Article of composed particles a of geometry surface root of thevariation for accounts that model p of The variation Analysis of porosity variation about the root hairs micrographs mm alonga1segment root root hairs bycounting estimated was density Root hair morphology. hair root for wereanalysed samples WT selected of images Three light microscope region. ofthe analysed volume bythe divided pores detected volume of the quadrant only

surface/ assume

that in ImageJ. in ImageJ.

line in Figure 1. This oscillatory behaviour will occur everywhere except immediately immediately except occureverywhere will behaviour oscillatory This Figure1. line in

. The result is a modified expression for theporosity for expression modified is a result . The

d . wall effect, but it butit wall effect, oscillates due to the varying volume fraction taken up by the large soil particles, red particles, soil large by the taken up volume fraction the varying to due oscillates . Root hair length was measured with the segmented line tool linetool the segmented with measured was Root hair length .

It that the soil particles form a hexagonal sphere packing, illustrated in 2D in Figure 1. Figure 1. 2Din in illustrated packing, sphere a hexagonal particles form soil that the large, mono

i

s assumed that the variation in porosity at the root surface is related to the to related surface is root atthe porosity in variation that the s assumed

the porosity the porosity . ore volume fraction about the root surface was analysed using a new a new using analysed surface was the root about fraction volume ore In soils a wider range of soil particle sizes and shapes may diminish the impact of theimpact maydiminish shapes and particle sizes soil of range awider soils In furthest furthest

is considered is

gainst

- space occupied by the large spherical soil particles as a function of distance distance function of a as soilparticles spherical the large by occupied space sized from the wall was analysed. wasanalysed. wall the from

a surface. a surface.

is

increases

expected to havea expected to at a distance distance at a spherical rigid particles of radius ofradius rigidparticles spherical surface/wall effect of the the effect of surface/wall

a

nd the oscillations in porosity further from further inporosity the oscillations nd

For simplicity, the simplicity, For

void distribution caused by the by caused distribution void

due to the absence of soil particles in the region occupied occupied theregion particlesin ofsoil theabsence due to

major from the root line root (green the from

Pore volume fraction was recorded as the asthe recorded was fraction volume Pore

influence right at the root atthe influence right root surface root

model model

soil soil

and a sub resolution mixed phase phase mixed resolution and asub

used to illustrate this point this point illustrate used to

forces an incomplete sphere incomplete an forces , s

urface/ f or n=18 selected root selectedroot n=18 or in in

the the root Figure 1) 1) Figure wall effect at at the wall effect

in three three in - soil interface. soil

modified modified are it be it can packing packing is (

1

)

This article isThis article by protected copyright. All rightsreserved. a using models tothe fitted datawere Experimental where is givenby of for proxy a is usedas size the pore study parameters air the related to w fromthe root distance region. analysed size Pore d Analysis of pore size distributi inMatlab. nonlinear solver Dexter’s using fitted datawere Experimental where at the gradient suchthat is constrained where

here

Accepted Article

is inversely related to pore size topore related is inversely . Alternatively

is below is below

is the matric potential, potential, matric is the

is the bulk porosity, porosity, bulk is the

istribution was derived from the histogram of the ‘local thickness’ map within map thickness’ the‘local of the histogram derived from was istribution is the matric matric is the

,

is a fitting parameter. The result is a set of 5 fitting parameters parameters of5fitting isaset The result parameter. is afitting

can be interpreted as the as can beinterpreted

- To entry pressure, and pressure, entry imaging imaging

for comparison for

analyse

the v the suction

resolution and hence and resolution

any changes changes any

an

, Genuchten model Genuchten

is the Dexter decay constant and and constant decay the is Dexter

, th

on

is residual water content, is residual

through theYoung through

is the

e Brooks

and has aunitof has and

is related to pore size distribution. size to pore distribution. related is

in

relative relative

air

pore sizedistribution pore

-

(1987)

entry

-

Corey model was model was Corey

is negative, which amounts to the condition to whichamounts is negative,

assumed to be to assumed

pore volume pore

was used was

pressure, pressure,

model and the new, modified approach using a approach modified new, the model and

least

,

-

Laplace Laplace equation

-

square fit square

, in which case the resulting case theresulting , in which , i.e.,

and

below a threshold pore size. pore a threshold below

0.

is the saturated water content, content, water is the saturated ,

applied t

between between he water release curve release he water

is the pore size distribution. distribution. poresize is the

in Matlab. Matlab. in is the total volume of p volumeof is thetotal is the root radius. The equation equation The root radius. is the

, . Therefore, the inverse of theinverse . Therefore,

where water water where

treatments and with andwith treatments

Translated to to Translated

,

,

, release curve release

imaging imaging is given given by is , and

the has the unit the unit has

ores and ores In our In our

(2)

. ( ( ( 5 4 3

is ) ) )

This article isThis article by protected copyright. All rightsreserved. difference between nosignificant was significantly smaller (Figure 3) controls unplanted a greater had and WT) (NRH treatments Planted Detectable p mm 3.7 hairs 50.7± was density hair µm.Root 991 was length recorded maximum the root hairs), µm 230 (n=18 t (paired significant thewet in ± and24.5 2.5% treatment, the wet (F=0.39, length and shoot p=0.77) (F=0.08, mass fresh onboth effect significant no had treatment watering more the Results of Plant performance and soilwater content measurements Results at repository Southampton of University the from onrequest areavailable thisstudy All data supporting availab Data statistical ofgroup analysis ( Wilk test. Matlab performed in was analysis Statistical Statistical analysis Accepted ArticleANOVA

shoot fresh mass (F=15.18, (F=15.18, mass fresh shoot ) with ) P<

. Multiple comparison of plant treatments showed that the unplanted controls had had controls unplanted the that showed treatments of plant comparison . Multiple S tests, a significance level level of asignificance tests,

0.39). 0.39). tatistical differences between group between differences tatistical ility - https://doi.org/xxxx/soton/yyyy 1

p

Fisher’s Least Significant Differences ( Differences Significant Least Fisher’s root length. root ore volume fraction lant measurements are summarized i aresummarized measurements lant

- The average gravimetric water content at the time of the scans was 25.6 was the scans time atthe of content water gravimetric average The means between means

- (F=8.74, (F=8.74, test, p=0.18). p=0.18). test,

(0.39 ± 0.01) than both than ± 0.01) (0.39

P< 0.05) P<

Average root hair length of WT plants on plants on WT of length Average hair root two , while , while 0.05) and shoot length (F=23.93, (F=23.93, shoot length 0.05) and

P

NRH (0.42 ± 0.01) and WT barley (0.41 ± 0.01) ± barley (0.41 WT and ± (0.42 NRH 0.01) groups, independent two independent groups, =0.05 was applied. applied. =0.05 was moisture treatments treatments moisture

2016a .

planted treatments (Fisher’s LSD, (Fisher’s treatments planted

- dry treatment, differences were not statistically notstatistically were differences treatment, dry - means

mean total pore volume fraction volume fraction pore total mean . Data were checked for normality checked for were Data LSD n Table Table n

were analysed using analysis of variance variance of analysis using were analysed )

for post for

1

. The The - had no impact noimpact had sample t sample - hoc multiple comparisons hoc multiple

NRH genotype had had genotype NRH P< 0.05) than the WT, while the the while theWT, than 0.05) - test was was test micrographs was 521± was micrographs (F=1.42, p=0.24) (F=1.42, P< ( 0.05)

applied ) .

using Shapiro using

than the than about 40% 40% about ,

but there but . Inall . ± 1.4% in 1.4%in ± . For

- This article isThis article by protected copyright. All rightsreserved. (LSD conditions (F=7.25, (LSD conditions planted both than pores ofthese (~0.2%) greaterfraction a significantly had control unplanted 5 pores between onthe effect had asignificant ‘genotype’ (F=6.78, between 15 the that controls) revealed unplanted the including (3 levels ‘genotype’ and (2levels) ‘moisture’ fixed effects two with the between 20 pores diameter had apore volume pore the ± 1.6%of 38.7 average The o Po e.g.Reps (see samples the insome of distance with oscillations of constants by decay bulk porosity minimum at constant remained (i) PVFeither observed. were patterns twodistinct root from surface, the away decrease in b all casessupported increased displayed almost uniformly curves The fitted treatments. between differences Table summarised in Figure 4, dashed lines (Figure some cases allofthe replicates almost of decrease the that showed replicates ofindividual Comparison (0. the root awayfrom Further reached. an (Figure average conditions, planted Within the re size distribution Accepted Article ofaverage decrease initial verall pore size distribu pore verall 4 P< P< ). Despite ). Despite orange orange

0.05), 0.05), the that indicating 0.05), - , which was reflected by corresponding decay constants of constants decay corresponding by reflected , whichwas

, average L2 norm = 0.0 L2norm average , 20 µm (F= 20 µm

was observed with minimum withminimum observed was

P< P< . (ii) PVF solid lines solid while the while - 0.05). The controls had also a 1.6% greater fraction of pores between between 25 pores fractionof greater a1.6% also had Thecontrols 0.05). between20 Pores 0.05). 25 µm, which amounted to28. whichamounted 25 µm, l arge 5 , blue blue , y the data thedata y 2

. 4.52, 4.52, I

ndividual fitting parameters were uncorrelated parameters fitting ndividual

variance between individual replicates individual between variance

increased increased , , average L2 norm = 0.0 norm = , average L2

circles

wet controls had a 2 had controls while the increase of theincrease while P< . It is worth noting that the measured measured the that worth noting . Itis tion within the imaged region was relatively narrow (Figure 6). On 6). (Figure narrow relatively was the imaged region within tion

0.05) and a 1.5% greater mean fraction of pores between 25 ofpores fraction greater mean a1.5% 0.05) and -

(see replicate 2 of WT wet ofWT replicate 2 (see dry within 0 to 0to within

).

F away from the root and tended towards towards tended root from and the away moisture treatment had a 2% smaller mean fraction of pores ofpores fraction mean smaller 2% hada moisture treatment 41 itting of the of itting wet )

yielded 3 - dry

to 1 mm) the average mm)the to 1 showed variation with distance from the root surface root surface the from distance with variation showed - % smaller fraction of these pores than the planted theplanted than pores these fraction of % smaller ~ 25 µm were also significantly affected by the ‘genotype’ the‘genotype’ by affected significantly were also 25 µm

treatment led to a broader pore size distribution. The distribution. poresize to abroader led treatment 0.

at a distance distance at a 3 poorer

mm from the root surface where a minimum aminimum where root surface the mm from

5 2

with

8 further away from the root was onlyseenin was the root awayfrom further ± 1.1% of the pore volume. pore the ± 1.1%of ) .

The resulting fitting parameters ar parameters resultingfitting The fits

Dexter’s model Dexter’s

at thesoil - - than dry in Figure Figure 4) dry in 10 µm (F=17.53, µm(F=17.53, 10 of approx. 0.25 mm 0.25 approx. of between between 15

,

4 fitting fitting

Figure 4 indicate 4 Figure

and

seems to to seems

near the root theroot was near -

root interface, which was not in not in whichwas interface, root displayed conspicuous conspicuous displayed 5 the

( from from and showed no showed and Equation 3, Equation

-

. 20 new model new Away from the root a fromthe root Away

incr P<

WT µm and

0.05), 0.05),

. At distances further further distances . At Two , which was reflected reflected was , which eas

, followed by the by , followed s wet

a trend e -

was equalto was way ANOVA ANOVA way

Figure Figure

slightly. slightly. while while in Figure 4). Figure in observ ( Equation 1, Equation

significant significant e

showing the the - 5 - 30 µm 30 µm , ed in ed in green

is

This article isThis article by protected copyright. All rightsreserved. m theseaggregates inbetween grow preferentially to were observed roots and more apparent are aggregated structures size, mm At 2 condi packing the resulting et al., 2006; (e.g. Feeney < to sieved used soil current study Whalley et al et (Feeney al., 2006; ofroots presence the dueto macroporosity to significant increases observed that studies many 2017) and etal., (Koebernick porosity detectable contrasti has This study Discussion theroot. near distribution (Figure8b) 1mmdistance 0and between exponentially decreased t from Genuchten (Figure 7).Van the roots from µm 0.85 18.02± and ± 0.60µm pore larger torelatively shifted distribution Genuchten onvan significant effect treatments smaller asignificantly had treatment control the that indicated treatme Plant p=0.23). wet decreased region scanned to used was approach 0.1) sizedistribution pore the Fitting of significantly increase was sizeclass pore smallest the while also treatments, planted relativetothe the controls in pores inlarger indicate resultsincreases these Overall, LSD (F=4.06, - Accepted3 Article , but other research used a looser 1.2 1.2 usedalooser research , but other

P< treatment compared to compared he root (Figure 8a) root (Figure he 0.0 P< 5). There were no significant differences between the two genotypes NRH and WT. WT. NRHand genotypes two the between differences significant no There were 5). by the moisture treatment (F=4.39, (F=4.39, treatment the moisture by 0.05; LSD LSD 0.05; ,

with no difference between the contrasting genotypes. Moisture treatment had no had treatment Moisture genotypes. thecontrasting between difference with no ,

s but

are summarised in Table 3. Table 3. in are summarised

the Brooks

was unaffected by the plant treatment treatment theplant by unaffected was P< analyse analyse nt had a significant effect onv effect asignificant nt had ng findings to our previous work where root hairs were observed to increase increase to observed hairswere whereroot work previous to our findings ng 30 between ofpores fraction greater a1.6% and 0.05) , Helliwell et al., 2017; Koebernick et al., 2017). Our smaller Our smaller 2017). etal., Koebernick al.,2017; Helliwell et

indicating relative indicating

tion produced a soil environment more typical of intraggregate structure typicalofintraggregate more environment asoil tion produced - Corey Corey the pore size distributions. size distributions. the pore

at 1 mm distance) distance) 1mm at 25 (Koebernick et al. 2017) et al. (Koebernick was better with the better with was formulation formulation

0

(F=2.7, p=0.11). Within the planted treatments, the pore size pore the treatments, planted Within the p=0.11). (F=2.7,  m, whereas M

s g m g

ly increased linearly between 0 between linearly increased The van Genuchten parameter parameter Genuchten van The (mean pore size at thesoil size pore at (mean

smaller P< - 3 (L2 norm norm = (L2 . 0.05)

and a wider pore size distribution with distance distance with sizedistribution pore awider and previous research used soil sieved to < 2mm to sieved used soil research previous an Genuchten an Genuchten

pores near the root, while van Genuchten whilevan the root, near pores ,

van Genuchten equation Genuchten van indicating a relatively smaller pore size inthe poresize relatively smaller a indicating R . including unplanted controls controls unplanted including

esulting fitting parameters parameters fitting esulting 0.36). T 0.36). Soil in the syringe barrels was at 1.3 at1.3 was barrels the in syringe Soil

( P< , herefore, the van Genuchten Genuchten the van herefore,

indicating a narrower pore size poresize narrower a indicating 0.05)

(F=5.33, (F=5.33, - root interface surface 16.74 interface surface root mm mm than the planted theplanted than

d.

was significantly significantly was - and 1 mm distance 1 mmdistance and P< ., 2005). However, However, 2005). .,

35 µm (F=672, µm 35

0.05). Fisher’s LSD LSD Fisher’s 0.05). (average L2 norm L2 (average particle for the entire entire the for (F=1.55, (F=1.55,

size and size P< 0.05;

M

the

g = .

This article isThis article by protected copyright. All rightsreserved. of Theoverestimation possible. simple as as the model larger than increased is porosity overwhich thearea Therefore, surface. plane bya represented was surface root µ radius the particle namely oversimplif This packing. cases interface, soil root atthe effect surface/wall universally. almost soil the volume at pore anincreased Incontrast, thereplicates. half of increas significant for evidence Compelling compaction. soil the degreeof for especially true This was roots. individual for distributions porosity different be therevery can soil relatively homogeneous patterns different very surface showed root interface thesoil at fraction porevolume ofincreased observations previous confirms study The present micrfrom 50.7mm density of hair root average an we found growth, restricted root hair this suggests 1.3 Mgm ha with compared (as root anchorage increase maize not did hairs thatroot found etal.(2016) Bengough ourstudy: in therhizosphere in macropores evidence. than contradictory 2005) Whalley et al. P (2005) Whalley et al. al.(2009) Hallett et inearlierstudies asreported microbiota, soil poro detectable the increased 2006), al., (Kutileket pores larger decreasing root bythe compression from likely resulted distribution poresize the narrowed Whereas sizedistribution. pore the andnarrowed volume fraction inc significantly roots of Presence impact. a major had roots of presence the formation, porosity on impact no observable having hairs ofroot the presence Despite lants were Acceptedm). Article We note that, for the sake of simplicity, the model used a Cartesian aCartesian themodel used sakeofsimplicity, forthe that, note We

(average L2 norm =0.028) L2norm (average es in soil density density soil es in - (Helliwell et al. 2017; Koebernick et al. 2017) et Koebernick al. 2017; et al. (Helliwell , as well as the well asthe amplitude, , as ographs. ographs. 3

or greater using soil (though sieved to <2mm) from the same field as our study. Although Although study. our as samefield the from <2mm) sieved to (though soil using greater or

also also

. I t may be argued t beargued t may significantly olderin significantly

The proposed The

the macropore size increased in the rhizosphere, similar to the the similarto rhizosphere, in the increased size macropore the , hence the differences may reflect temporal dynamics in the rhizosphe in dynamics maytemporal reflect thedifferences , hence

on rhizosheath soil aggregates. aggregates. soil rhizosheath on (corresponding to a decay constant of constant todecay a (corresponding ication is however reflected in reflected ishowever ication

, which It may also be that root hair extension was restricted by the lack of thelack by wasrestricted extension hair root bethat also It may , despite the oversimplification of using a mono a using of the oversimplification , despite hat this i this hat

formulation of porosity varia porosity of formulation

should only depend on depend on only should was about 4 about was

sity probably resulted from the combined activity of roots and ofroots activity thecombined from resulted probably sity previous s not true in the present case the present s not in true

for individual replicates suggesting that even ina even that suggesting replicates individual for fitted the experimental data well in the majority of majority inthe data well experimental the fitted (Feeney et al. 2006; Hallett et al.2009) Hallett et et2006; (Feeney al.

studies - fold greater than the aperture of the sieve ( ofthesieve the aperture than greater fold

irless mutants) when soil density increased to to increased density mutants) soil when irless

. The radial porosity distribution about the about distribution porosity radial . The (Feeney et al. 2006; Hallett et al. 2009; al. et 2009; Hallett al. 2006; et (Feeney somewhat somewhat

compared to the sieve aperture may the sieveaperture to compared

. The assumption is that is assumption . The

tion (Eq. 1) tion reased the total detectable pore pore detectable total reased the

empirical

) was only observed in roughly roughly in onlyobserved was ) - root interface was observed observed interface was root ,

but coordinate system, i.e.the system, coordinate , which accounts for the for accounts , which it was decided decided it was fitting parameters parameters fitting - sized sphere sized sphere findings of of findings . In the work bywork the . In

is much is much to keep to re rather rather re - 1

taken taken =125 - – root root

This article isThis article by protected copyright. All rightsreserved. in imaging,as such resolved Accepted a structure rhizosphere in change temporal result a be a of therefore may differences observed Some the of age). ofroot the variation tominimise syringe barrels note We artefacts. imaging by affected that was zone the into extended this region because be analysed not could This from the roots. furtheraway extended have may in samples these that compression constant compaction soil degree of beyond fromthe root, away Further model. present bythe be captured that cannot volume pore in variations produce which might be can organisation clayparticle that noted to be has It resolution. the limiting imaging of model because the present in theroots by unaffected theinitialsieved effect beyond soil then gel Moreno 2005; et al. the in formation aggregate rolein animportant play to thought mucilage are individu of aggregation of indication ~250 porosity minimum distance of and theradial porosity Article whil rigidparticles, term assumes surface packing 4 of replicate (e.g. compaction with appreciable to thecomputed isreached closer porosity minimum the andTable 2), in Fig.5 of replicate1 (e.g. is minimal When compaction 1. inEq. effect compaction packing and irregular shapes particles shaped regularly or packing spheres particles the nature of the spherical distan volume with oscillations of in soil.The occurrence to occur expected be would which packings, random 2014) mono therefore spheres, the modelled from which are experiment, particles the soil Undoubtedly, root surface. the near aggregates of formation the indicate that the age of the imaged roots was not controlled (beyond scanning at the same depth of the ofthe depth same atthe scanning (beyond notcontrolled was roots the imaged the ageof that µ , - sized spheres are theworst are sized spheres m in most m in although the model used model the although

due to the assumption of continuum deformation. continuum of the assumption due to –

at least least within at (Naveed et al. 2017a; Naveed al. 2017a; et 2017b) et (Naveed al.

(Roblee etal.1958) (Roblee

cases, which is about twice as large as the sieve aperture radius. This may This apertureradius. large thesieve as twice which isabout cases, ce from the root ce from - Espíndola et al. 2007; Vidal et al. 2018) Vidalet etal.2007; Espíndola non the analysed the analysed closer tothe - spherical, polydispersed, and randomly packed andrandomly polydispersed, spherical, Helliwell et al.(2017) et Helliwell soil size was likely due to aggregation. to likelyaggregation. was due size soil packing - packing amo objects packing

. found al particles at the root plane. Root and microbial derived derived microbial Root and plane. root the particles at al was afairassumption was

The

, porosity increased in some of the replicates, indicating some some indicating the replicates, of some in increased , porosity

1

ro

-

on a regular lattice and porosities porosities and lattice aregular on is only to some extent to some only is

mm distance from the root. One possible explanation is explanation Onepossible the root. from mm distance large values of of large values ot, whileot, inother

in some of the data sets, indicate thesets, of data some in

affected by roots by roots affected . Figure 5 shows that minimum porosity was reached at reached was porosity minimum that shows 5 . Figure (Mueller 2010) (Mueller WT wet WT e the Dexter type compaction compaction type Dexter e the ,

showed that porosity at thesoil that porosity showed ng all ng all , so the observed extension of the ofthe extension theobserved , so -

dry

.

Such oscillations are observed when when observed are oscillations Such are partially a result of the combined the combined aresultof are partially . replicates This

, Barley exudates initially disperse and disperse initially exudates Barley in Fig. 5 and Table 2). In our model, the model, 2). Inour Table Fig.5and in convex (Dorioz et al. 1993; V al. (Dorioz et 1993; but are notobs but are

a lead physical s shapes in 3D in shapes long the roots. Temporally Temporally the roots. long

the porosity remained remained porosity the The mixed phase was assumed assumed was phase mixed The to the discrepancy between between discrepancy the

parameter , would

s deviate significantly significantly deviate

erved for highly highly for erved that the assumption of theassumption that rhizosphere rhizosphere

decreases

be greater for for greater be value than in cases in than value (Baule and Makse and (Baule - idal et al. 2018) idal et al. root interface root . Theoretically, Theoretically, WT wet WT packing (Caravaca

of in the in the the

be an be an

pore pore

- dry

This article isThis article by protected copyright. All rightsreserved. compression. and coalescence aggregate to most due likely rhizosphere, the within observed Accepted no provides pores shifti paths and that fractions pore smaller ofthe the frequency systems increased macroporosity to increase beenshown have systems root coarse While morphologies. root variability different with substantial field canshow (Kutíl compression soil indicates pores smaller of frequency increased An root at the surface. larger pores of expense atthe smallerpores of frequency anincreased showed clearly distribution size the pore theair analysed for only was which size distribution, by study theprevious In contrast, etal.2014) sizes(Haling arange textural of of soils sieve coarsely themore seen in than shorter substantially explain soils coarser in root hairs longer produce (2017) Koebernick et al. hair putative that show differences These soil finer textured Article study. the present in porestructure on genotype effect lack thea of therefore isdifference main The genotypes. both of near the surface root fraction increased volume an indeed showed mixed pha the were of part pores, thesmaller particularly study, thepresent in pore classified as were that voxels ofthe proportion A phases. and pore) solid water three phases the into soil While two studies. inthe approaches segmentation the different air mutant, NRH air of fraction increased showed genotypes used study,which previous similar ina the observations to different quite study was properties example for through roots, the from away conceivablefurther isalso age relatedeffect an age. Such with changed significantly a ek et al. 2006; Leij et al. 2002) Leijet 2006; etal. ek

(Bodner et2014) al. (Bodner filled pores andsub pores filled finer root systems can use the available pore space more effectively as preferential growth growth aspreferential effectively more porespace the available use root systemscan finer ing

, the lack of a genotype effect in the present present study inthe effect lackofagenotype the (Naveed etal. (Naveed 2017a)

but a more heterogeneous soil structure soil moreheterogeneous but a large large ng the pore ng the - filled porosity decreased near th near decreased filled porosity

macropores as preferential growth paths, a shift towardssmalle ashift paths, growth preferential as macropores use d here d

are influenced by the soil by the are influenced – - . Here, we show that in a much finer textured growth medium, which which medium, growth textured finer in amuch that show Here, we . resolution soil particles, the present study only distinguishes two (i.e. two distinguishes only thepresent study particles, soil resolution

-

microbial degradation of root exudates, which may change soil physical physical may change soil which exudates, root of degradation microbial solid mineral particles, air particles, mineral solid size distribution towards smaller pores via in pores smaller towards size distribution , root hairs seemed to have no significant effect on pore structure. structure. pore on effect nosignificant to have seemed , root hairs - . The variation of pore volume with distance from the root in this in the root from withdistance volume ofpore . Thevariation filled pores at the root surface for the surface for attheroot filled pores Koebernick et al. (2017) etal. Koebernick . Shifts in pore size distribution as affected by root growth in the inthe root growth by affected as distribution size inpore Shifts (Haling et al. 2014) (Haling et al. - induced changes changes induced (Bodner etal (Bodner e root. The differences are partially explained by by explained partially are differences root. The e characteristics . -

(Koebernick et et al. 2017) (Koebernick filled pores, with distance from the root. Here, fromthe root. withdistance filled pores, - filled pores, and a mixed phase comprising comprising phase mixed anda filled pores, d soil (Brown et al. 2012) and in model inmodel and etal.2012) (Brown d soil se in se

. 2014; Holtham et al. 2007) etal. Holtham . 2014; did not show a significant change of pore of change asignificant show not did to

, which may , which where the root hair length is lengthis theroot hair where

soil structure structure soil (Bodner et al. 2014) etal. (Bodner Koebernick et al. (2017) et Koebernick al. . Barley roots havebee roots Barley . Koebernick et (2017) Koebernick et al. WT - also also growth into the larger intothelarger growth

genotype, while for the the for while genotype, as observed in observed as . The previous study previousstudy . The go some way to some way go r pore sizes can be sizescan r pore . It was suggested suggested . Itwas

the same same the , fine root root , fine , which , which n shown to n shown

segmented segmented For theFor

This article isThis article by protected copyright. All rightsreserved. Accepted beamtime ofthe preparation in Sinclair advice for like thank toalso would We beamtime. our helpduring considerable whoprovided Silvia Cipiccia, Oxfo LightSource, at I13Diamond the beamline of theuse acknowledge Theauthors NE/L00237/1. by funded TR isalso EP/M020355/1. TRare and IS Government. Scottish the of Division Services Analytical & Science & Environment Rural the from support receivesfinancial Institute Hutton The James BB/L025825/1. by funded AGB is and BB/J00868/1 by BBSRC MN AR are funded TSG,and PDH, LKB, by ERC646809DIMR KRD isfunded BB/L025620/1. SARISA byBBSRC arefunded TR andIS NK, LJC, Acknowledgments Article at particularly rhizosphere, the increase pores might smaller towards and theshift imp have could changes These fraction. volume pore intotal increase by an accompanied surface root affect inter weconclude porestructure intragreggate rhizosphere. the structurein pore affectthe didnot morphology hair root contrasting with genotypes work previous to In contrast activity. root by altered significantly are parameters structural soil evidencethat provide Our results root. growing by the lar the of packing incomplete that causes effect by awall which isexplained surface, theroot near fraction islarger volume pore this, Despite ortant implications on water and solute uptake by roots, as the enhanced total volume of pores pores volumeof total theenhanced as roots, by uptake solute water and on implications ortant rdshire, UK (Experiment UK(Experiment rdshire, - aggregate pore structure. structure. pore aggregate In line with our earlier argument that the structure in the present study resembled resembled present study inthe structure the that earlierargument linewithour In

drier drier (Koebernick etal.2017) (Koebernick MT12525

water potentials when the soi potentials when water ERC 646809DIMR, BBSRC SARIC BB/P004180/1 and NERC andNERC BB/P004180/1 SARIC BBSRC 646809DIMR, ERC Roots increased the frequency of smaller pores close to the closeto pores smaller of thefrequency increased Roots ) . We would like to than to like We would ger, incompressible mineral particles which are displaced displaced which are particles ger, incompressible mineral

that presence of hairs of presence that

and Mike Ogden for help at the beamline helpfor at the MikeOgden and

that used that unsaturated unsaturated

l is most limiting. most limiting. l is k Dr. Shashidara Marathe and Dr. Dr. and Marathe Shashidara Dr. k a more more a o n barley hydraulic conductivity in the in conductivity hydraulic

also funded by EPSRC EPSRC fundedby also heterogeneous soil, different different soil, heterogeneous

roots

is more likely to is morelikely .

. Ian Ian

This article isThis article by protected copyright. All rightsreserved. aremean Data Table 1 Tables Accepted Article wet NRH WT wet wet NRH WT wet Treatment

Plant -

dry -

dry

(

Hordeum vulgare Hordeum ± SD Shoot mass Shoot 0.181±0.04 0.129±0.02 0.194±0.03 0.127±0.04 ( (fresh) g ) .

WT,

wild

- t ype plant ype

cv. Optic) 161.7±29.3 7.0 118.2± 165.9±12.1 104.0±34.9 ( length Shoot mm )

;

NRH performance measurements at the time of scanning. scanning. of thetime at measurements performance

, hairless plant. hairless

This article isThis article by protected copyright. All rightsreserved. Table 2

Accepted wet NRH Article Condition NRH wet NRH

WT wet Fitted parameters of the modified model (Eq model ofthemodified parameters Fitted - dry

SD Mean 5 4 3 2 1 SD Mean 5 4 3 2 1 SD Mean 5 4 3 2 1 Replicate

0.012 0.406 0.398 0.395 0.429 0.402 0.407 0.012 0.389 0.373 0.381 0.408 0.392 0.389 0.010 0.390 0.402 0.386 0.388 0.373 0.399

1.88 0.60 1.28 1.26 1.95 1.85 1.06 0.29 0.96 1.21 1.63 0.22 2.87 0.51 0.81 0.60 0.73 0.79 0.35 0.31 0.29

n 2). 341.4 26.2 326.6 306.5 342.3 371.0 306.7 306.4 19.2 329.4 306.6 306.4 340.5 341.2 352.2 14.0 313.4 306.4 306.4 306.4 306.4

381.9 259.6 609.0 1120.9 435.4 430.1 523.0 535.4 833.3 873.2 2521.9 618.3 260.7 547.2 417.8 129.9 541.1 758.4 579.6 439.6 546.2

0.052 0.137 0.267 0.214 0.221 0.061 0.427 0.413 0.124 0.291 0.396 0.129 0.414 0.363 0.151 0.125 0.273 0.352 0.373 0.373 0.214

This article isThis article by protected copyright. All rightsreserved. Plant ( C WT wet wet NRH Control WT wet wet NRH Condition conditions. different forthe interest of regions scanned the CT Table 3 plant. hairless and ontrol wet ontrol

Accepted Article is the bulk porosity, porosity, bulk is the WT wet

Hordeum vulgare

-

Average wet is the mixed phase porosity. Plant ( Plant porosity. phase is themixed dry -

dry

-

-

dry dry

( ± SD Mean 4 3 2 1

18.64 18.64 18.08 18.27 18.05 17.87 18.04 SD

)

modified van Genuchten parameters fitted to the pore size distributions of of size distributions the pore to fitted parameters Genuchten van modified

± ± 0 ± ± ± ± is the Dexter decay constant, constant, Dexterdecay is the

cv Optic).

0.43 0.32 0.4 0.46 0.43

.37

0.028 0.394 0.418 0.412 0.400 0.347

WT,

6.72 7.08 6.83 6.24 6.85 6.9 wild Hordeum Hordeum 0.95 1.57 0.21 2.86 1.79 1.40 ± ± ± ± ± ± 0.32 - 0.34 0.17 0.46 0.16 0.2 type plant; NRH, plant;NRH, type hairless plant.

vulgare

16.6 323.8 306.4 308.7 345.0 334.9

is the root radius, is theroot

cv Optic). cv Optic).

98.8 500.6 604.8 485.0 346.8 565.8 WT

, w

ild

is the particle radius, radius, is theparticle - t

ype plant ype 0.095 0.138 0.066 0.267 0.188 0.031

;

NRH , This article isThis article by protected copyright. All rightsreserved. wild to right. left thesamelinefrom plottedon are condition Dexter’s fraction volume pore of Comparison Figure 5: eachcondition. for wet conditions different Figure PVF. effecton had nosignificant ( rhizosphere Figure black. in is shown root structure out masked and the particles Solid µm. in porediameter indicate local space. Colours particles soil solid blackisbackground structure, root whitethedetected is root, segmented steps. processing image the main grow barleyroot aCTscanned slice of Horizontal Figure 2: right model the while is 2D, drawing schematic the that Note the model. porosity thebulk approaches porosity phase themixed equals porosity the root, the to adjacent Immediately surface. theroot at particles spherical the of packing geometry distance Ata surface. theroot adjacentto porosity insoil the variation of drawing Schematic Figure 1: Figure captions AcceptedNRH Article - - dry watering treatments watering dry type ) consider 3D sphere packing. 3Dsphere consider

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This article isThis article by protected copyright. All rightsreserved. Accepted surface the root within eachreplicate for parameters Genuchten panel) Articlebarley Figure 8: from plot areobtained distances different indicate colours Different equation. distribution poresize Cumulative Figure 7: (WT) the bottom. barsat white conditions different the within sizeclass pore into 5 Figure 6 genotypes with both wet and wet wet and both with genotypes - µm wide wide classes µm root surface. Average van Genuchten parameters parameters Genuchten Average van surface. root over the entire imaged imaged the entire over : Pore size distribution distribution size Pore : Modified

while the model curve curve model the while van Genuchten parameters parameters Genuchten van . Each bar represents the relative fraction of the total pore volume bye volume pore ofthetotal fraction therelative represents bar . Each the wild the

Treatments include unplanted control, control, unplanted include Treatments region are shown in Table 3. inTable shown are region of the barley rhizosphere rhizosphere barley the of - type ( type WT - i dry watering tr dry watering s represented by the red dashed dashed line. the red by s represented )

wet in the barley rhizosphere rhizosphere thebarley in

± - of the pore size distribution over distance from the fromthe distance over distribution size the pore of dry SD 32 .

treatment - Pore size classes are indicated by the black and and the black by areindicated sizeclasses Pore µm eatments

(<1 wide annuli with increasing distance from from withincreasing distance annuli wide in the CT scanned region ofinterest region scanned theCT in ( 

Data points represent points Data mm) mm) on the left panel and left and panel the on , replicate 3. 3. , replicate

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no root hair (NRH) and wild (NRH) and hair no root fitted with the van Genuchten thevan with fitted e

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- binned type type ach This article isThis article by protected copyright. All rightsreserved. Accepted pen root anatomical Root predict phenes JP(2015) Lynch KW, JG,Loades Chimungu H Vogel Weller U, Blaser N, S, Koebernick Vetterlein D, Carminati A, microbial type rhizospheric mediates Plant A(2005) Roldán P, Torres MM, Alguacil Caravaca F, Astructured R(2004) Waugh DF, GJ,Marshall Muehlbauer P, Shaw McCallum N, Caldwell DG, Backscattered JC (1996) DuvalO, Bégon B, Nicoullaud I, A, Cousin Bruand W JA, Thompson TS, George LK, Brown H D, Kaul G, Leitner Bodner to inresponse roots barley wheat of and plasticity Morphological AG(2003) Bengough Bingham IJ, and waterstress, elongation, Root (2011) TA Valentine HallettPD, BM, AG,McKenzie Bengough soil aid Root p hairs (2016) BM K,McKenzie AG,Loades Bengough non sphericalto problems: from packing in challenges Fundamental HA(2014) Makse Baule A, Article coupled Quantifying SW (2014) Tyler TA, Ghezzehei SuárezF, S, Ruiz Berli M, Aravena JE, root of (2011)Effects SW Tyler GhezzeheiTA, Berli M, Aravena JE, in rootexudation and between drying soil Interplay TA(2014) AA, Ghezzehei Albalasmeh References Experiment publication and submission model. the NKdeveloped and KRD themanuscript. anddrafted data the analysed data. NK collected the AGB TSG, NK, SDK,PDH, Author contribu NK 3162. doi: 10.1093/jxb/erv121. doi: 3162. maize ( propertiesin biomechanical and 651 367: andSoil Plant gap? doi: 382. 375 marsh. 124: Geoderma salt asemiaridin Mediterranean aggregation soil and activities 143 40: Journal Plant ( Barley in reverse genetics forwardand for population mutant 895 60: America Journal i 319 110: ( barley water stressin with in combination deficiency phosphorus toleranceto on length inroot hair variation of implications arethe What 380: 133 Soil Plant and differently. 10.1023/A:1022891519039. Plant an soil strength. in spatial variation 59 62: Botany Experimental li review of a impedance: mechanical 10.1093/jxb/erv560. 1071 67: Botany Experimental of walls.Journal pore surface to 4423 10: Matter Soft spherical particles. Plant simulations. X using rhizosphere the in waterflow and deformation simulation properties hydraulic rhizosphere rhizosh mages

wrote the manuscript and all other other authors all and manuscript wrote the MT12525 eath development. Plant and Soil 374: 739 Soil 374: and Plant eath development. of of

- http://dx.doi.org/10.1016/j.geoderm 328. doi: 10.1093 doi: 328. s s. Environ Sci Technol 45: 425 45: SciTechnol s. Environ oil oil .

p tions orosity for orosity , and TR designed the study. NK, SDK, AR, PDH, AGB, TSG, LKB, AGB, TSG,LKB, PDH, NK, SDK,AR, the study. designed TR and and Soil 376: 95 Soil 376: and .

- PDH was PI on funding from the BBSRC and the Diamond Light Light andthe Diamond the BBSRC from PIonfunding was PDH - P (2014) Coarse and fine root plants affect pore size distributions distributions affectsize plants pore root Coarsefine and P (2014) 150. 10.1111 150. doi:

- 901. doi: 901. a /aob/mcs085. nalyzing nalyzing

- - 68. doi: doi: 10.1093/jxb/erq350. 68. 661. doi:10.1007/s11104 661.

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d Soil 250: 273 250: Soil d 4429. c - 431. doi:10.1021/es102566j. 431. ompaction ompaction mays a.2004.05.010

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- - This article isThis article by protected copyright. All rightsreserved. pore soil dynamicsthe Modelingthe of D (2002) Or TA, Ghezzehei Leij FJ, pore upon compression uniaxial Theof influence KP (2006) Panayiotopoulos L, Jendele M, Kutílek Ve FR, Boone D, Schoonderbeek MJ, Kooistra LJ, Nav Cooper Raffan A, LK, Brown TS, SD,George Keyes KR, Daly N, Koebernick Marchant I, Sinclair R, BR,Boardman Pinzer P, DL,Talboys NJ, Jones Gostling KR, SD,Daly Keyes van C, Scotson M, Daniel Fletcher McKay Simon J, N, Duncan Koebernick Cooper L, S, Keyes do (2013) How WR Whalley MAJ, IC, Parry RW, Dodd J, Ashton K, Shen Jin Higuc Iijima M, Schol Matthews GP, Holtham DAL, the of Transactions Philosophical root growth. and plant compaction Soil DRP (1990) Hettiaratchi SJ Mooney WR, MillerRW, AJ, Whalley Ashton J, Craigon S, CJ,Mairhofer Sturrock Helliwell JR, Disentangling IM (2009) Young Scrimgeour CM, MC, AG,Rillig Bengough DS, Feeney Hallett PD, (2014) Root TS George IM, Young PJ, White TA, AG,Valentine Bengough LK, Brown RE, Haling (2006) IM Young M, Rivers RitzK, N, Nunan PD, Hallett T, Daniell JW, DS, Crawford Feeney andba fungi roots, of role The ChenuC(1993) M, Robert JM, Dorioz 401 Soil 97: roots. and Plant around soil Compressionof Dexter AR(1987) (2000)Root IM AG,Young Bengough PD, Hallett Czarnes S, enhance and roots macropores Artificialattractcrop A (2017) Walter KellerT, S, Braun Colombi T, AcceptedDOI: Article

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This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article

This article isThis article by protected copyright. All rightsreserved. Accepted Article