Jun-bang Wang?Xiu-juan Zhang?Chu Wu

Advances in experimental methods for root system architecture and root development

Jun-bang Wang?Xiu-juan Zhang?Chu Wu

Plant roots play important roles in acquisition of water and nutrients,storage,anchoring,transport,and symbiosis with soil microorganisms,thus quantitative researches on rootdevelopmentalprocesses are essentialto understand rootfunctions and rootturnover in ecosystems, and atthe same time such researches are the mostdifficult because roots are hidden underground.Therefore,how to investigate efficiently root functions and root dynamics is the core aspect in underground ecology.In this article,we reviewed some experimental methods used in root researches on rootdevelopmentand rootsystem architecture,and summarized the advantages and shortages of these methods.Based on the analyses,we proposed three new ways to more understand root processes:(1)new experimental materials for root development;(2)a new observatory system comprised ofmultiple components,including many observatory windows installed in field,analysis software,and automatic data transport devices;(3)new techniques used to analyze quantitatively functional roots.

Rootsystem analysisFractal geometryNovel materials

Introduction

There are greatdifferences in developmentbetween aboveand below-ground parts of plants.The development of above-ground plantparts is less affected by environmental factors,thus branching patterns of above-ground parts are similar among individuals of the same species.In contrast, the developmentof root systems is greatly affected by the soilenvironmentalfactors,such as soilnutrients(especially the availability of nitrogen and phosphate)(Zhang and Forde 1998;Williamson et al.2001;Linkohr et al.2002; Wu etal.2005;Hilletal.2006;Jain etal.2007;Vidaletal. 2010;Peretetal.2011;Gan etal.2012;Gruber etal.2013; White et al.2013),soil water(Berntson and Woodward 1992;Manschadi et al.2006,2008;Henry et al.2011; Bengough et al.2011),soil temperature(Mackay and Barber 1984;Kaspar and Bland 1992;Clausnitzer and Hopmans 1994),soil pH(Walter et al.2000),soil compaction(Bushamuka and Zobel1998;Busscher etal.2000; Tracy et al.2010a),and slope gradients(Di Iorio et al. 2005;Pierret et al.2007;Sun et al.2008),thus the morphology and physiology of roots show great plasticity. Because of the relationships between above-and belowground parts of plants,it is necessary to maximize root/ rhizosphere efficiency to improve plant productivity and nutrient efficiency(Shen et al.2013).Root traits can be used to create phosphate efficientcrop varieties in response to limited global phosphate resources(Gahoonia andNielsen 2004).Therefore,root development,root system architecture,and root dynamics have been the focuses in botany and ecology.An obstacle to the study ofrootsystem architecture is to measure and quantify the three-dimensional configuration of root systems in soil.At the same, root dynamics is not monitored efficiently because of hidden roots in dark medium.Researchers have attempted to overcome the two obstacles in order to understand root functions and rootresponses to soilenvironmentalfactors. In this paper,we review some advantages and shortages in experimental methods for study of root development,root system architecture,and root dynamics,and propose some new ways to enhance root research.

The theoretic bases for analysis of root system

Root system architecture,as defined by Lynch(1995), refers to the spatialconfiguration ofthe rootsystem,i.e.the explicit geometric deployment of the root axes.The root system architecture of plant seedlings is relatively simple but adult plants,especially adult trees,such as Sequoia sempervirens,have complex root system architecture (Nadezhdina and Cˇerma′k 2003;DiIorio etal.2005)thatis difficult to study.

Although plant root systems show plasticity and complexity,within a given species root systems are similar because of shared genetic characteristics.The similarity provides the basis for their analysis.

Mandelbrot(1975)coined the term‘‘fractal’’.A fractalis generally a rough or fragmented geometric shape that can be splitinto parts,each ofwhich is(atleastapproximately) a reduced-size copy ofthe whole shape,i.e.self-similarity. A fractal often has traits including:(1)self-similarity;(2) fine or detailed structure at arbitrarily small scales;(3) simple and recursive definition;(4)irregularity which is difficultto describe by Euclidean geometry;(5)Hausdorff dimension which is greater than its topological dimension. Based these traits of fractals,smaller parts of roots are fractal representations of larger parts of roots,thus fractal analysis is an appropriate tool for study of root systems. Therefore,fractal analysis was used to describe root systems of many plant species(Table 1).Fractal analysis has also been used to compare the responses of root system architecture to nutrient deficiency(Eghball et al.1993; Nielsen etal.1999),droughtstress(Wang etal.2009),and efficiency of soil exploration by roots(Walk et al.2004).

These applications offractalanalysis are deficientbecause they were only involved in two-dimension structures of root systems,and spatial configurations of root systems are not taken into consideration.In natural ecosystems,most soil nutrients are in the soil layer of 10-20 cm deep,thus root spatial configurations are related to nutrition uptake. Therefore,some parameters involved in description for root spatialconfiguration,such asthe anglesbetween primary root and the firstlateralrootsand the anglesbetween rootlinksin a root system,should be analyzed in fractal analysis.Data obtained from a complex root system are great,thus new software willbe developed foranalyzing them.

Roottopology isanotherterm used fordelineation ofroot systems.Asdefined by Lynch(1995),the term refersto how individual root axes are connected to each other through branching.Unlike branching of above-ground parts,root branching shows greatspatiotemporalchanges,forexample, first-orderfine roots can be connected with second-orderand the third-order roots(Pregitzer et al.2002),and even with fourth-order roots(Wang etal.2006),leading to the complexity ofrootbranching.Regardlessofits complexity,root branching links can be classified into three types,i.e.exterior–exterior(EE),exterior–interior(EI),and interior–interior(II)(Fig.1,Fitter and Stickland 1991).

Three topological parameters can be calculated to describe the relationships between links:

Magnitude(a):number of exterior links.The total number of root tips categorized as EI and EE(Fig.1a)or total numbers of root tips categorized a EE(Fig.1b).

Altitude(l):number of links in the longest unique path from the base link to an exterior link.In Fig.1a,from an exterior link labeled EE to the base link,the number of root links is 10,i.e.l=10.In Fig.1b,from any exterior link labeled EE to the base link,the number ofrootlinks is 4,i.e. l=4.

Total exterior pathlength(pe):sum of numbers of links in all possible unique paths from the base link to all exterior links.In Fig.1a,pe=64;in Fig.1b,pe=32.

Two topological indices can be used

Altitude-slope:the slope of the regression of log10a on log10l for a given root system.In the plot with log10l as the x axis and log10a as the y axis,the slope of the regression is the altitude-slope for a set of root system.

Pathlength-slope:the slope of the regression of log10peon log10l fora given rootsystem.In the plotwith log10l as the x axis and log10peas the y axis,the slope of the regression is the pathlength-slope for a given rootsystem.

In addition,some parameters are used to describe the morphology ofrootsystems,such as roothairdensity,root diameter,the occurrence pattern of lateral roots,the distance from the tip to the lattermostlateralroot.Anatomical traits such as the sizes of epidermal cells in the elongation zone,the cell density of the meristem zone,and the size of meristem zone are not usually involved in the description of root systems.

Similar to fractal analysis,root topology does not take root spatial configuration into consideration.

Table 1 The plant species which were analyzed using fractal analysis

Fig.1 Diagram showing the distinction between extreme branching patterns(a:herringbone;b:dichotomous).Exterior–exterior(EE); exterior–interior(EI);interior–interior(II)[e.g.a link between the two dashed lines in(b)cited from(Fitter and Stickland 1991]

Experimentalmethods for measurementofrootsystems

The complexity of rootsystem architecture raises obstacles to data acquisition that are addressed by advances in technology.Digital images help to quickly acquire data about root system architecture.Meanwhile,related software is helpful for processing these data.

Root system analysis technologies based on scanners were developed,including WinRhizo and RootSnap.Specific root analysis software was also developed,including Multi-ADAPT(Ishikawa and Evans 1997),AMAPmod (Danjon et al.1999a,b,2008),Delta-T Scan and Win-RHIZO(Boumma etal.2000),REGR(Walter etal.2002), RootFlow(van der Weele et al.2003,KineRoot(Basu et al.2007),SmartRoot(International Atomic Energy Agency 2006;Draye 2008),RootLM(Qi et al.2007), Phytomorph(Miller etal.2007;Spalding,2009),RootNav (Pound et al.2013),RootTrace(French et al.2009),and Growth Tracer(Iwamoto etal.2013).RootFlow and REGR made use of optic flow-based techniques(Barron et al. 1994;Barron and Liptay 1994).All of these applications have their strengths and weaknesses.The combination of scanner and software was used to measure some root parameters using three-dimension techniques that provided more configuration information about root system architecture(Danjon and Reubens 2008).For entire root systems,some morphologicaltraits can be analyzed,including total root length,root average diameter,totalroot surface, project surface,root tips,root volume.Some parameters related to rootlinks can be measured,such as totalnumber of links in a rootsystem,their average length and diameter, volume,and branching angles.Topological parameters can also be measured,such as altitude,magnitude,and exterior path length.

Some root analysis systems have their shortcomings, one of which is the fact that roots must be excavated and washed from soil cores before they can be scanned (Gregory 2006;Smit et al.2000).Excavation and washing must break some parts of the root system,leading to the underestimation of fine roots through breakage and loss of information on the spatial distribution and root system architecture.More seriously,data obtained laterare lack of the information about root spatialconfiguration.

More advanced devices were developed.More than ten years ago,X-ray computed tomography(CT)was firstused to analyze rootsystems(Heeraman etal.1997;Pierretetal. 1999).Because the scanning process of X-ray CT is nondestructive,the technique can track root growth and changes in root system architecture.Based on these advantages,the technique has been developed and widely used(Gregory et al.2003;Lontoc-Roy et al.2005;Perret etal.2007;Tracy etal.2010a,b;2012a,b;Mooney etal. 2012;Mairhofer etal.2012,2013).

X-ray CT can visualize the internal structure of opaque objects.X-ray CT scanners acquire a series of projections from different facets of a vessel that contains plants, measuring the attenuation of ionizing radiation passing through the vessel.These projections are combined to reconstruct a three-dimensional data set.Data values recorded at each voxel reflect the density of the imaged material and are usually mapped to greyscale intensity values for visualization purposes(Mooney 2002).But variations in the x-ray attenuation values of root material and the overlap in attenuation values between roots and soil caused by water and organic materials(especially nondecomposed litter)represent major challenges to data recovery.Software(such as RooTrack)has been developed to separate root systems from their soil environment in images obtained by X-ray CT and recover root system architecture traits(Mairhofer etal.2012,2013).

In addition,as a complementary toolto other predictive techniques,gelgrowth systems with superior opticalclarity have been introduced to facilitate noninvasive twodimensional(2D;Iyer-Pascuzzi et al.2010)and threedimensional(3D;Fang et al.2009;Clark et al.2010) imaging.These methods are only used for rootsystems of seedlings,thus its applications are limited.

At present,there are not available techniques used in field for in situ dynamic observation.Ground penetrating radar(GPR)should be usefulto investigate rootsystems of forest trees on forest ecosystem level(Nadezhdina and Cˇerma′k 2003;Butnor et al.2003;Barton and Montagu 2004;al Hagrey 2007).GPRs have not only the lowest wave length and resolution to detectsmalltargets butalso the highest wave attenuation that limits resolution and penetration in wet conducting media,thus they have been used to estimate single root segments or biomass with a relatively low precision(Butnor et al.2003;Barton and Montagu 2004;al Hagrey 2007).But,at present,the accuracy and resolution of GPRs are not enough for fine roots(rootdiameter1 mm),and more advanced GPRs are needed.Software applications have also been developed to analyze information collected by GRP,providing detailed investigation on rootsystems atthe ecosystem level.

Based on the analyses mentioned above,two new techniques will be developed:one is the combination of continuous three-dimensionalimaging and fractalanalysis/ topology used in laboratories;another is in situ continuous multiple-site observatory root systems with automatic data transport used in ecosystem level.

Novel plant materials used in root development

Root development includes three main aspects,i.e.elongation of roots,branching(including proteoid roots),and differentiation of root epidermal cells(i.e.root hair formation,including dauciform roots;Playsted et al.2006; Shane et al.2006).Lateral root formation is the base of developmentof the whole rootsystem in which rootelongation enlarges the volume of soil explored by roots,and root hairs greatly increase the absorptive surface area for water and nutrients in soil.Of the three aspects,lateralroot formation seems more important,thus investigation on lateral root formation has been the focus of the root development research.While various types of advanced microscopes are used in studies of root development,new plant materials are also used.Many mutants related to specific genes that affect root development and many transgenic Arabidopsis lines with GFP or GUS in roottissues showed noveltraits,i.e.specific occurrence in a tissue in roots.Thus these transgenic plants are often used in studies of root development.Lateral root formation starts with a series of anticlinal and periclinal divisions of pericycle cells,a layerofroottissue surrounding the vasculature (Malamy and Benfey 1997;Kurup et al.2005).Thus, detection of the activity of pericycle cells near the xylem poles is important for lateral root formation.Quiescent centers(QC)in roottips maintain the activities and sizes of the meristemsin roots,so detection ofQC isalso important.

In the MRC Laboratory of Molecular Biology,led by Jim Haseloff(http://data.plantsci.cam.ac.uk/Haseloff/),GAL4-GFP lines were produced by Agrobacterium-mediated transformation of the C24 Arabidopsis line using the binary vector pBIN m-gfp5-ER.The m-gfp5-ER gene is GAL4 responsive and is linked to a GAL4 gene,a foreign transcription activatorderived from the yeastGAL4 gene in the GAL4-GFP lines.Because ofthe traits of GFP,the patterns of GAL4 expression in the GAL4-GFP lines can be immediately and directly detected.Some lines in the GAL4-GFP setshow specific patternsofGAL4 expression in roots, thus they are widely used in studies on root development.

Of allthe GAL4-GFP lines,J0121 seems to be the most useful for studies on root development.In J0121, GAL4::GFP expression is specific to 2–3 rootpericycle cell files adjacent to the xylem poles(Fig.2;Laplaze et al. 2005),thus the line can be used for detection of the first cell division of cells at the initial stage of lateral root formation.In J0192,GAL4::GFP is expressed in young lateralrootprimordia(Fig.2;Laplaze etal.2005),thus itis very useful for detecting the development of young lateral root primordial.After these two lines were produced,they have often been used in studies of root systems(Table 2).

Mostrecently,a GAL4-based targeted activation tagging system in Arabidopsis has been established(Waki et al. 2013).Host plants that expressed a synthetic transcription activator GAL4:VP16(GV)in an organ-or tissue-specific mannerwere transformed with a T-DNA harboring tandem copies of UAS,a GAL4-binding sequence.Thus the activation tagging system is a powerful tool for discovering novel genes that affect root development.

QC maintains the activity of root meristem and specific lines were produced to observe QC status(Table 2).GFP only occurs in QC25,thus QC25 is used in root development,especially used in monitoring the activity of QC and root meristem of plants treated with phytohormones and environmental stresses.

In other GAL4-GFP lines,GFP specifically occurs in columella(Q1630),root epidermis(J2301,J0631,and J2921),lateralrootcap(J0951),and stele(J2501)(Table 2).

Lateral roots originate from the pericycle cells opposite to xylem poles.These pericycle cells are distant from the root meristem zone and stop division.Therefore,the first step of lateral root formation is the recovery of the cell cycle ofsome pericycle cells.CYCB1;1 is a mitotic cyclin in Arabidopsis,and is expressed only around the G2/M transition in the cellcycle(Doerneretal.1996;Shauletal. 1996),therefore,CycB1;1::uid A reporterconstruct(Fig.3) is often used as the marker to detect the process of the cell cycle during lateralrootformation(Table 2;Casimiro etal. 2001;Aida et al.2004;Hutchison et al.2006;Ioio et al. 2007).

Fig.2 GFP expression in the enhancer trap lines J0121(a–c)and J0192(d–g).(a,b,d,e,f,g)Longitudinalconfocalsections of roots and(c)projected confocal view of the top part of a hypocotyl of living plants showing GFP fluorescence(green)and counterstained with propidium iodide(red).a GFP expression starts in the pericycle cells of the elongation zone.No GFP fluorescence is visible in the root meristem.b Expression in pericycle cells adjacent to a xylem strand. c Expression in the hypocotyl epidermis.d Expression in a stage I primordium.e Expression in a stage IIprimordium.f Expression in a stage III primordium.g Expression in a stage V primordium.Cited from Laplaze et al.2005

Table 2 Experimental materials often used in studies on root development

Rootdevelopmentis affected by environmentalfactors, thus lateral root initiation is a probabilistic event,but its frequency is set by fluctuating levels of auxin response (Laskowski 2013).In Arabidopsis,the process of lateral root formation consists of two major stages:cell cycle reactivation in the xylem pericycle and establishment of a new meristem(Himanen etal.2002).Pericycle reactivation depends on local concentration of auxin in the roots, whereas the outgrowth of lateral roots is regulated by shoot-derived auxin(Bhalerao et al.2002).Therefore, detection of local concentration of auxin in roots is very important for lateral root formation and root system development.

There are some auxin-responsive elements(AuxRE)in the promoters of auxin-responsive genes,such as IAA5 and IAA19 genes(Nakamura et al.2003),and AuxRE contains the TGTCTC sequence.ARF proteins can bind to AuxRE after Aux/IAA proteins are degraded and ARF proteins are released from Aux/IAA proteins,promoting auxin responses.A highly active synthetic AuxRE,referred to as DR5,was created by performing site-directed mutations in a natural composite AuxRE found in the soybean GH3 promoter(Ulmasov et al.1997).Because DR5 showed greater auxin responsiveness than a natural composite AuxRE(as shown in Fig.4),it became a useful reporter gene(acting as DR5::GFP or DR5::GUS)for studying auxin responses in wild-type plants and various mutants (Sabatini et al.1999;Friml et al.2002a,b;Ottenschla¨geret al.2003;Willemsen et al.2003;Laplaze et al.2007; Ohashi-Ito and Bergmann,2007;Petersson et al.2009).

Fig.3 Schematic of the CycB1;1::uid A reporter construct(pCDG). The upper panel highlights functionalmotifs of cyclin CycB1;1.The lower panel shows the 1.8 kb genomic fragment,including approximately 1.2 kb of promoter sequence and the first three exons of CycB1;1 coding sequence up to amino acid 116,translationally fused to the E.coliuid A gene.Thick lines representpromoter sequences or introns,boxes representexons.Cited from Colo′n-Carmona etal.1999

Fig.4 Histochemical staining for GUS activity in Arabidopsis seedlings transformed with DR5(97)-GUS.Seedlings were treated for24 h with 50 l M 1-NAA(right)or H2O(left)and then stained for GUS activity for*8 h(top)or 16 h(bottom).Cited from Ulmasov et al.(1997)

SCARECROW(SCR)gene encodes a putative transcription factor(DiLaurenzio etal.1996)thatis firstexpressed in QC precursor cells during embryogenesis,after which it extends to the initial cells for the ground tissue(cortex and endodermis)(Wysocka-Diller et al.2000).Because SCR gene regulates an asymmetric celldivision thatis essential for generating the radial organization of the Arabidopsis roots(Di Laurenzio et al.1996)and is involved in positioning the stem cellniche in the Arabidopsis rootmeristem (Sabatinietal.2003),its fusion constructs with GFP or GUS are widely used to study rootdevelopment(Table 2).

All the novel plant experimental materials provide us with information about root developmental processes, especially initiation of lateral roots and QC activity.Since root brain hypothesis have been accepted gradually (Balusˇka etal.2009,2010),some ofthe plantexperimental materials will be useful for investigation on the functions of root tips.In view of complexity of root brain,new plant experimental materials will be developed,especially those involved in neurotransmitters.

Additionally,since root development is affected by other phytohormones,such as ethylene(Rahman et al. 2001;Stepanova et al.2007),cytokinin(Aloni et al.2006; Laplaze et al.2007),and nitric oxide(Correa-Aragunde et al.2004,2006;Sto¨hr and Stremlau 2006),novel transgenic plants,like the above-mentioned transgenic lines with DR::GFP and DR5::GUS,are needed for investigation on the effects of ethylene,cytokinin and nitric oxide on root development,especially their effects on lateral root formation.New techniques and experimentalmaterials are needed to promote study of rootdevelopment.

Concluding remarks

In view of multiple functions of roots,we have to face crypticity and high plasticity of rootsystems.Development of new techniques for data acquisition and analysis and experimental materials will help us overcome difficulties and understand more details about root functions and root dynamics.

AcknowledgmentsWe thank Dr.Soo-Un Kim in Seoul National University for reviewing the manuscript.This work is supported by the projectof public benefits in China(No.201503221)and the open fund in the Institute of Root Biology,Yangtze University.

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17 September 2013/Accepted:15 April 2014/Published online:15 January 2015

Project funding:This work is supported by the project of public benefits in China(No.201503221)and the open fund in the Institute of Root Biology,Yangtze University.

The online version is available athttp://www.springerlink.com

Corresponding editor:Chai Ruihai

J.Wang

Key Laboratory of Ecosystem Network Observation and Modeling,Institute of Geographic Sciences and Natural Resources Research,Chinese Academy of Sciences, Beijing 100101,China

e-mail:jbwang@igsnrr.ac.cn

College of Horticulture and Gardening,Yangtze University, Jingzhou 434025,Hubei,China

e-mail:wuchuchu2001@126.com