yamaguchi 2010_ root growthwaterstressprotein

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Complexity and coordination of root growth at low water

potentials: recent advances from transcriptomic and

proteomic analysespce_2064 590..603

MINEO YAMAGUCHI & ROBERT E. SHARP

Division of Plant Sciences, 1-31 Agriculture Building, University of Missouri, Columbia, MO 65211, USA

ABSTRACT

Progress in understanding root growth regulation and

adaptation under water-stressed conditions is reviewed,

with emphasis on recent advances from transcriptomic and

proteomic analyses of maize and soybean primary roots.

In both systems, kinematic characterization of the spatialpatterns of cell expansion within the root elongation zone

showed that at low water potentials, elongation rates are

preferentially maintained towards the root apex but are

progressively inhibited at more basal locations resulting in a

shortened growth zone. This characterization provided an

essential foundation for extensive research into the physi-

ological mechanisms of growth regulation in the maize

primary root at low water potentials. Recently, these studies

were expanded to include transcriptomic and cell wall pro-

teomic analyses of the maize primary root, and a proteomic

analysis of total soluble proteins in the soybean primary

root. This review focuses on findings related to protection

from oxidative damage, the potential roles of increased

apoplastic reactive oxygen species in regulation of wall

extension properties and other processes, region-specific

phenylpropanoid metabolism as related to accumulation of

(iso)flavonoids and wall phenolics and amino acid metabo-

lism. The results provide novel insights into the complexity

and coordination of the processes involved in root growth

at low water potentials.

Key-words: maize; soybean; proteomics, root growth, tran-scriptomics; water stress.

INTRODUCTIONUnder water stress conditions, some types of roots have theability to continue elongation at low water potentials thatcompletely inhibit shoot growth (Sharp & Davies 1979;Westgate & Boyer 1985; Sharp, Silk & Hsiao 1988). This isconsidered an important feature of plant adaptation towater-limited conditions that helps to maintain an adequateplant water supply (Sharp & Davies 1989; Ober & Sharp2007).The ability to maintain elongation at low water poten-tials is pronounced in the primary root of a range of species,

which helps seedling establishment under dry conditions byensuring a supply of water before shoot emergence (Sharpet al. 1988;Spollen et al. 1993;van der Weele et al. 2000);thisresponse is illustrated for maize and soybean seedlings inFig. 1. Detailed understanding of the physiological mecha-

nisms involved has been mostly limited to the primary rootof maize, which has been studied extensively by Sharp andco-workers using protocols that allow precise and reproduc-ible imposition of water deficits. Key findings in this systemwere reviewed by Sharp et al. (2004) and Ober & Sharp(2007) and, therefore, are summarized only briefly in thisreport. Recently, studies were expanded to the primary rootof soybean in order to compare and contrast responses towater stress in the roots of important monocotyledonousand dicotyledonous crop species (Yamaguchi et al. 2009).Inboth cases, the research has taken advantage of a kinematicapproach, that is, the study of spatial and temporal patternsof cell expansion within the root elongation zone (Erickson

& Silk 1980; Sharp et al. 1988; Walter, Silk & Schurr 2009),which greatly facilitated the discovery of mechanismsinvolved in the response of root growth to water stressconditions. This review highlights recent advances gainedfrom studies in which thekinematicapproach wascombinedwith transcriptomic and proteomic analyses both to build onthe existing physiological foundation and to reveal novelinsights into the complexity and coordination of metabolicprocesses involved in thegrowthresponses to water stressofthe primary roots of maize and soybean.

SPATIAL PATTERN OF THE RESPONSE OFELONGATION RATE TO WATER STRESS IN

MAIZE AND SOYBEAN PRIMARY ROOTS

Knowledge of the spatial pattern of growth rates withintissues can be a powerful tool to investigate the effectsof environmental variation on plant development (Erick-son & Silk 1980; Walter et al. 2009). The advantage of thisapproach is exemplified by studies of the growth responsesto water stress in primary roots of maize (Sharp et al. 1988;Liang, Sharp & Baskin 1997; Fan & Neumann 2004) andsoybean (Yamaguchi et al. 2009). As shown in Fig. 2, thespatial distribution of the response of elongation rate towater stress is very similar in the two species, with elonga-tion being preferentially maintained towards the root apex.

Correspondence:R.E.Sharp.Fax:+1 573 882 1469; e-mail: SharpR@

missouri.edu

Plant, Cell and Environment (2010) 33, 590603 doi: 10.1111/j.1365-3040.2009.02064.x

2010 Blackwell Publishing Ltd590


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Remarkably, local elongation rates are fully maintained inthe early ontogenetic phases of growth even under severewater stress (water potential of-1.6 MPa). However, decel-eration and cessation of expansion occur closer to the apexthan in well-watered roots, resulting in a shortened elonga-tion zone. These patterns allow the identification of three

contiguous regions with distinct elongation characteristics:region 1, which encompasses the apical region in whichelongation rates are maintained in water-stressed roots;region 2, in which elongation rates reach a maximum inwell-watered roots but are progressively inhibited underwater stress; and region 3, in which elongation deceleratesin well-watered roots and is completely inhibited in water-stressed roots.

This spatial characterization provides an essential foun-dation for investigation of the mechanisms of growth regu-lation in water-stressed roots. Clearly, different mechanismscan be expected to underlie the distinct responses to waterstress in the different regions, and investigation of mecha-

nisms involved in the maintenance of elongation need tofocus on the apical region. However, the inhibition of elon-gation in the basal region is also important to understand,because this is probably part of a coordinated response toallow the preferential utilization of limited resources (waterand growth substrates) in the apical region. In addition towithin-region comparisons, the kinematic analysis alsoallows consideration of effects that may be linked withdevelopmental changes associated with the shortening ofthe elongation zone rather than to specific responses towater stress. Accordingly, as illustrated in Fig. 2, changesthat are observed in region 2 of water-stressed rootscan also be compared with region 3 of well-watered roots,

which exhibits comparable elongation characteristics. Thisapproach has been effectively utilized in recent transcrip-tomic and proteomic analyses to help distinguish specificresponses of gene expression and protein abundance towater stress from changes that may merely reflect the occur-rence of growth deceleration and tissue maturation closer

to the apex (Fig. 3). These results are discussed in moredetail below.

REGULATION OF MAIZE PRIMARY ROOTGROWTH AT LOW WATER POTENTIALS PHYSIOLOGICAL BACKGROUND

Physiological research on mechanisms involved in growthresponses of the maize primary root to water stress hasfocused on three primary areas: (1) the role of abscisicacid (ABA) accumulation; (2) the relationship of osmoticadjustment to root growth maintenance; and (3) modifica-tion of cell wall extension properties.

Growth-sustaining role of ABA accumulation

Although hormones are likely to play important roles inroot growth regulation under water-stressed conditions,the involvement of most of these compounds has not beenelucidated. The exception is the accumulation of ABA,which was investigated in maize primary roots by usingABA-deficient mutants and inhibitors of ABA synthesis todecrease endogenous ABA levels in seedlings growing atlow water potentials. The results have changed the viewof the role of ABA from the traditional idea that thehormone is generally involved in growth inhibition

Vermiculite water potential (MPa)

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Shoot

Figure 1. Elongation rates of the primary root (solid circles) and shoot (open triangles) of maize and soybean seedlings at various waterpotentials; the data are plotted as a percentage of the rate under well-watered conditions.After germination, seedlings were transplanted

into vermiculite at different water potentials (obtained by thorough mixing with different amounts of water) and grown at 29 C andnear-saturation humidity in the dark to minimize evaporative water loss. Accordingly, the seedlings were exposed to constant conditionsof water potential. For roots, data were evaluated when elongation rates were steady; for shoots, data represent maximum elongationrates obtained after transplanting. The transcriptomic and proteomic studies reviewed in this report were conducted at a water potentialof-1.6 MPa, which imposed a severe water stress treatment during which primary root but not shoot elongation occurred. Reproducedfrom Spollen et al. (1993) with permission from Bios Scientific Publishers.

Root growth at low water potentials 591

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(reviewed in Sharp 2002). Instead, it was discovered thataccumulation of ABA, which increases towards the rootapex (Saab, Sharp & Pritchard 1992), is required for themaintenance of elongation in the apical region of theelongation zone at low water potentials (Saab et al. 1990;Sharp et al. 1994), and this response was shown to involveprevention of excess ethylene production (Spollen et al.2000). In associated work, the action of ABA in maintain-ing root elongation was shown to involve changes in

electrophysiology (Ober & Sharp 2003). The results indi-cated that under water stress, set points for ion homeosta-sis shifted in association with the maintenance ofelongation rates in the apical region, and that ABA playsa role in regulating the ion transport processes involvedin this response. Additional studies suggested that ABAaccumulation might also play regulatory roles in theresponses of both osmotic adjustment and cell wall exten-sibility in water-stressed maize roots, as detailed below.

Well watered

(WW)

Water stressed

(WS, 1.6MPa)

1 cm

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(b)

Figure 2. (a) Displacement of marks away from the apex of maize primary roots during a 3.5 h period following marking for rootsgrowing under well-watered (WW) or water-stressed (WS, water potential of -1.6 MPa) conditions (grown as described in Figure 1). Thewhite lines indicate the vertical displacement of the root apices and of marks originally located at 5 and 10 mm from the apices duringthe growth period. Separation of marks, and hence tissue expansion, is apparent throughout the apical cm in the WW root. In the rootgrowing at low water potential, in contrast, separation between marks occurred only in the apical region, illustrating that water stressresulted in a shortened elongation zone. Reproduced from Sharp et al. (1988) with permission from the American Society of PlantBiologists. (b) Displacement velocity as a function of distance from the root cap junction of primary roots of WW and WS maize seedlings(cv. FR697). The inset shows the profile of displacement velocity for WW and WS soybean primary roots (cv. Magellan). Relativeelongation rates (h-1) are obtained from the derivative of velocity with respect to position. The maize and soybean velocity curves arereproduced from Sharp et al. (2004) & Yamaguchi et al. (2009) with permission from Oxford University Press and Wiley-Blackwell,respectively; the original data were calculated from root elongation rates and cell length profiles as described by Silk, Lord & Eckard(1989). Regions 1 to 3, as described in the text, are indicated. The green arrows indicate the comparisons between treatments withinregions 1 and 2 and the comparison of WS region 2 with WW region 3 which were made in the transcriptomic and proteomic analyses

reviewed in this article.

592 M. Yamaguchi & R. E. Sharp



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Relationship of osmotic adjustment to rootgrowth maintenance

The elongation zone of the maize primary root exhibits asubstantial capacity for osmotic adjustment at low waterpotentials (Sharp, Hsiao & Silk 1990). By applying kine-matic principles to characterize the deposition rates ofwater and solutes within the elongation zone, it was dem-onstrated that the rate of proline deposition (nmolesproline mm-1 length h-1) increased dramatically in theapical region under water stress; the resulting increase inproline concentration was responsible for as much as 45%

(in the apical mm) of the total osmotic adjustment (seeFig. 7, inset; Voetberg & Sharp 1991). In contrast, in thebasal region, the decrease in osmotic potential in water-stressed roots compared with well-watered roots could beaccounted for by decreased rates of dilution of othersolutes, particularly hexoses, as a result of growth inhibition(Sharp et al. 1990). The proline results provided the firstdemonstration of a major role for increased rates of solutedeposition in the osmotic adjustment of growing regions inhigher plants exposed to low water potentials.This responseis more dynamic than would be expected if the osmoticadjustment were merely an inevitable accumulation ofunused solute when growth is inhibited (Munns 1988).

Accordingly, it was concluded that osmotic adjustmentin the maize primary root elongation zone is likely to bea highly regulated process and an important adaptiveresponse that helps to maintain root growth under waterstress conditions.

To address the metabolic basis of the increase in proline

deposition in the root elongation zone under water stress,proline synthesis, catabolism and transport rates weremeasured using radiolabelling flux analysis techniques(Verslues & Sharp 1999). The results indicated that theincrease in proline deposition was primarily attributable toincreased proline transport to the root tip.Additional workshowed that ABA accumulation is required for the increasein proline deposition (Ober & Sharp 1994), suggesting thatABA may play a role in regulating proline transport to theapical region.

Modification of cell wall extension properties

The distinct responses of elongation rate in the apical andbasal regions of the elongation zone under water stressconditions were shown to be associated with differentialresponses of cell wall extension properties (Wu et al.1996). The possibility that an enhancement of cell wallextensibility may contribute to the maintenance of elon-gation in the apical region of water-stressed roots wasinitially suggested from the study of osmotic adjustment.Although the extent of osmotic adjustment was substan-tial, it was insufficient to maintain turgor at well-wateredlevels in roots growing under severe water stress. This wasassessed directly by measuring the spatial distribution ofturgor using a cell pressure probe, which showed that

turgor was decreased by over 50% throughout the elon-gation zone of roots growing at a water potential of-1.6 MPa compared with well-watered controls (Spollen& Sharp 1991). In the apical region, the maintenance ofelongation, despite the decrease in turgor, suggested thatlongitudinal cell wall extensibility increased under waterstress, which was confirmed by direct assessment of cellwall extension properties. In the 05 mm region, acid-induced cell wall extension was markedly increased inwater-stressed roots compared with well-watered roots,whereas in the 510 mm region, acid-induced extensionwas greatly inhibited under water stress (Wu et al. 1996).Further evidence that cell wall extension properties are

inhibited in the basal region of the elongation zone ofwater-stressed maize roots was provided by Fan &Neumann (2004) and Fan et al. (2006).

Additional studies showed that extractable activitiesof two cell wall proteins with known or proposed wallloosening properties, expansins and xyloglucanendotransglycosylase/hydrolase (XTH), were substantiallygreater in the apical region of the elongation zone in water-stressed roots compared with well-watered roots (Wu et al.1994, 1996). In addition, the susceptibility of the walls toexogenous expansins increased in this region of water-stressed roots, indicating that modifications of cell wallstructure or chemistry that facilitated expansin accessibility

Figure 3. Numbers of water stress-responsive maize transcripts(Spollen et al. 2008), maize cell wall proteins (Zhu et al. 2007)and soybean proteins (Yamaguchi et al. 2009) in region 1 (R1)and region 2 (R2) of the primary root elongation zone. Thenumbers of responsive transcripts and proteins in R2 whichexhibited similar and significant responses when R2 ofwater-stressed (WS) roots was compared with region 3 (R3) ofwell-watered (WW) roots are indicated in parentheses andmarked with asterisks; these changes are considered to beindependent of developmental changes associated with thestress-induced shortening of the elongation zone and, therefore,are likely to be specific responses to water stress.




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or action also play an important role in the increase inextensibility. On the other hand, expansin activity alsoincreased in the basal region of the elongation zone inwater-stressed roots compared with well-watered roots(Wu et al. 1996), which was unexpected given the greatlydecreased acid-induced extension in this region. This dis-

crepancy is, presumably, also explained by modifications ofcell wall structural composition that cause wall stiffening.This could not be confirmed by tests of expansin suscepti-bility, however, because the cell walls in this region did notrespond to exogenous expansins in either well-watered orwater-stressed roots.

Additional work showed that ABA accumulation wasnecessary for the increase in XTH activity in the apicalregion of the elongation zone in water-stressed roots (Wuet al. 1994), although in contrast, alterations of expansingene expression at low water potentials did not appear to beABA dependent (Wu et al. 2001).

KINEMATIC APPROACH TO TRANSCRIPTOMICAND PROTEOMIC ANALYSES OF ROOTGROWTH RESPONSES TO WATER STRESS

The spatial characterization of the response of elongationrate to water stress in maize and soybean primary roots(Fig. 2) and the physiological knowledge gained to dateprovide a firm underpinning for functional genomics studies(Sharp et al. 2004; Poroyko et al. 2005). Transcriptomic andproteomic analyses provide powerful tools to investigatethe molecular basis of plant responses to stress. However,when whole organs (or the entire plant) are analysed, theresults provide only an averaged depiction of responses that

may be highly variable in different regions of the plant.Thisconcern is particularly important when growth responsesare of primary interest because of the developmental gra-dient that occurs within growing regions. The shortening ofthe elongation zone in water-stressed roots highlights thisissue; in addition to the normal developmental gradientthat exists within the elongation zone (encompassing zonesof both cell division and elongation, elongation only andmaturation), the response of elongation rate to low waterpotential varies with distance from the root apex. Accord-ingly, it is not surprising that in several recent studies, waterstress-induced changes in transcript expression and proteinabundance were reported to be highly divergent in different

regions of the elongation zone (Bassani, Neumann & Gep-stein 2004; Sharp et al. 2004; Poroyko et al. 2007; Zhu et al.2007; Spollen et al. 2008; Yamaguchi et al. 2009).

Figure 3 summarizes the numbers of water stress-responsive transcripts and proteins in three studies of rootgrowth responses to water stress, the results of which aresynthesized in Figs 4 to 7: (1) a transcriptomic analysisof the maize primary root (Spollen et al. 2008); (2) aproteomic analysis of cell wall proteins (water-soluble andlightly ionically bound fraction) in the maize primary root(Zhu et al. 2007); and (3) a proteomic analysis of totalsoluble proteins in the soybean primary root (Yamaguchiet al. 2009). The maize studies revealed that the changes in

gene expression and cell wall protein abundance in regions1 and 2 of the elongation zone in response to water stresswere largely region specific. This finding emphasizes theimportance and effectiveness of the kinematic approach totranscript and protein profiling studies. Furthermore, thecomparison of water-stressed region 2 with well-watered

region 3 suggested that a large proportion of the stress-induced changes in region 2 might have been attributableto growth deceleration/tissue maturation associated withthe shortening of the elongation zone, rather than to spe-cific responses to water stress. Thus, although region 2showed a greater total number of differentially expressedgenes, the analyses suggested that region 1 exhibits agreater number of genes and cell wall proteins that aredirectly involved in regulating the water stress response.This result is consistent with the induction of a network ofadaptive processes in both regions, but, in particular, forthe maintenance of elongation in region 1. Figures 4 and5 provide schematic representations of the predicted

functions and interactions of several of the water stress-responsive proteins/genes and associated metabolites inthe cell wall and cytosol in regions 1 and 2 of the elongationzone of the maize primary root.

In the total soluble protein analysis in soybean, in con-trast, the largest proportion of water stress-induced changesin protein abundance were common to regions 1 and 2(Fig. 3). Moreover, only three of the total of 31 changes inregion 2 did not exhibit similar and significant responseswhen region 2 of water-stressed roots was compared withregion 3 of well-watered roots. Accordingly, the majority ofthe stress-induced changes in protein abundance in region 2were likely to be specific responses to water stress. The

differences in the soybean proteomic analysis to the resultsdescribed for the maize transcriptome and cell wall sub-proteome analyses are probably the result of the compara-tive lack of resolution of the total soluble protein analysis(using two-dimensional gel electrophoresis), which prima-rily detected only the most abundant cytosolic proteins thatwere regulated under water stress. These proteins maymostly be required for processes of stress adaptationthroughout the elongation zone.

Protection from oxidative damage animportant response throughout theelongation zone

The largest functional category of stress-responsive proteinsin the soybean study (most of which were increased inabundance) was the control of reactive oxygen species(ROS) metabolism (Yamaguchi et al. 2009). In the maizeanalysis, consistently, the expression of transcripts for pro-teins involvedin ROS metabolism tendedto beup-regulatedin both regions 1 and 2, in comparison to the predominantlyregion-specific patterns of expression of transcripts in otherfunctional categories (Spollen et al. 2008). Abiotic stressesincluding water stress often result in increased ROS produc-tion, which may cause oxidative damage (Iturbe-Ormaetxeet al. 1998; Apel & Hirt 2004; Moller, Jensen & Hansson




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2007).Accordingly, the commonalityof the results related tocontrol of ROS in the maize and soybean systems indicatesthat protection from oxidative damage is probably of

general importance in the adaptation of the root elongationzone to water stressconditions, ratherthan being specificallyassociated with growth maintenance in region 1 or growthinhibition in region 2 (Figs 4 & 5).

Water stress-regulated transcripts and proteins related tocontrol of ROS metabolism in the maize and/or soybeanstudies included several ROS-scavenging proteins, forexample, catalase, glutathione transferase and peroxidases.In addition, several metal-chelating proteins includingmetallothioneins and ferritins were up-regulated in bothregions 1 and 2, indicating that control of free metal ions isalso important for adaptation. Metallothioneins chelateheavy metal ions such as iron and copper, whereas ferritin

exclusively sequesters iron. Free iron and copper ions catal-yse the Fenton reaction that generates hydroxyl radicals,the most harmful ROS, from hydrogen peroxide. In water-

stressed soybean roots, the increased abundance of ferritinproteins was shown to effectively sequester more iron andthereby prevent excess free iron throughout the elongationzone (Yamaguchi et al. 2009).

Excess ROS can oxidize cell components including pro-teins, lipids and sugars, and can also trigger apoptosis-likecell death (Solomon et al. 1999;Gechev et al. 2006).Accord-ingly, the up-regulation of several proteinase inhibitors inregions 1 and/or 2 of both maize and soybean [includingcysteine proteinase inhibitor (maize and soybean),subtilisinchymotrypsin inhibitor (maize) and trypsininhibitors (soybean)] is likely to help prevent the degrada-tion of oxidized proteins, allowing time for recovery

Focal planesEpidermal

cell wall

Figure 4. Schematic representation of predicted functions and interactions of water stress-responsive proteins/genes and associatedmetabolites in the cell wall and cytosol in region 1 of the elongation zone of the maize primary root. Major differences in responsebetween regions 1 and 2 are indicated by shaded text. Non-italicized text and solid connecting arrows indicate responses that have beendemonstrated in published studies; italicized text and dashed connecting arrows indicate responses that are hypothesized to occur buthave not yet been demonstrated. Up-regulation under water stress is indicated by the short upward arrows; for ABA, proline and oxalateoxidase (OxO), the double arrows indicate that accumulation or activity in region 1 was greater than in region 2. *Flavonoidaccumulation has not been determined in water-stressed (WS) maize roots; however, isoflavonoids were shown to accumulate in region 1of the soybean primary root under water stress (see Figure 6; Yamaguchi et al. 2009). The inset shows increased apoplastic reactive oxygenspecies (ROS) in the epidermis of region 1 in WS compared to well-watered (WW) roots, as indicated by confocal microscopy of rootsstained with H2DCF (2,7-dichlorodihydrofluorescein, green fluorescence), a membrane-impermeable ROS indicator. The right-handdiagram illustrates a transverse view of the root surface and focal planes. The increase in apoplastic ROS in WS roots is consistent withthe increases in abundance of superoxide dismutase (SOD) and oxalate oxidase, which contribute to H 2O2 production, and peroxidases(POX), which can also contribute to ROS production including the generation of hydroxyl radicals ( OH) from H2O2 in the presence ofsuperoxide (O2-) and/or reductant (e.g. NADH).The ROS images are modified from Zhu et al. (2007) and reproduced with permissionfrom The American Society of Plant Biologists.




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[possibly by glutathionation involving the up-regulation ofglutathione transferase activity; Moons (2005)]. Cysteineproteinase inhibitor activity may be particularly importantin the inhibition of apoptosis (Solomon et al. 1999;Belenghiet al. 2003).

The results also suggested that supplemental protectionfrom excess ROS occurs preferentially in region 1 of water-stressed roots. Both isoflavonoids in soybean (Fig. 6) and

proline in maize (Fig. 7) were shown to accumulate substan-tially and preferentially towards the apex of roots growingat low water potentials. Maize does not have the enzymesfor isoflavonoid synthesis that were up-regulated insoybean, but up-regulation of enzymes for flavonoid syn-thesis (Fig. 6) suggests that flavonoids probably accumulatein the elongation zone of water-stressed maize roots; thishas not yet been assessed. Both (iso)flavonoids and prolinehave antioxidant activity (Jovanovic et al. 1994; Nerya et al.2004; Kruk et al. 2005; Kaul, Sharma & Mehta 2008) inaddition to other potential functions in stress adaptation(discussed in the following sections). Accordingly, the pref-erential accumulation of these compounds in the apical

region of water-stressed roots is likely to help in protectionfrom excess ROS, thereby contributing to the maintenanceof elongation in this region.

Taken together, the co-regulation of ROS-scavengingproteins, proteinase inhibitors and metabolites with anti-oxidant activities suggests that a complex and coordinatedset of proteins is regulated under water stress to protectthe root elongation zone from oxidative damage under

water stress conditions (Figs 4 & 5). It is interesting tonote that flavonoid synthesis (Ithal & Reddy 2004),proline accumulation (Ober & Sharp 1994) and theexpression of several ROS-scavenging proteins (Seki et al.2002) including catalase (Guan, Zhao & Scandalios 2000)and ferritins (Lobreaux, Hardy & Briat 1993) have beenreported to be regulated by ABA, suggesting that ABAaccumulation may play an important role in cellular pro-tection from ROS-induced oxidative damage in water-stressed roots of both maize and soybean. Consistent withthis hypothesis, the maize vp14 mutant, in which ABAaccumulation under water stress is restricted (Sharp 2002),exhibits increased cytosolic ROS levels in the primary

Figure 5. Schematic representation of predicted functions and interactions of water stress-responsive proteins/genes and associatedmetabolites in the cell wall and cytosol in region 2 of the elongation zone of the maize primary root. Major differences in responsebetween regions 1 and 2 are indicated by shaded text. Non-italicized text and solid connecting arrows indicate responses that have beendemonstrated in published studies; italicized text and dashed connecting arrows indicate responses that are hypothesized to occur buthave not yet been demonstrated. Up-regulation under water stress is indicated by the short upward arrows. Apoplastic reactive oxygenspecies (ROS) signalling (as shown for region 1 in Figure 4) may also occur in region 2, but for clarity, this is not shown. The inset showsincreased autofluorescence from lignin and other phenolic compounds under ultraviolet excitation in transverse sections from the centre

of region 2 of water-stressed (WS) compared to well-watered (WW) roots (M. Yamaguchi & R.E. Sharp, unpublished data). OxO, oxalateoxidase; POX, peroxidase.




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root elongation zone under water-stressed conditions

(I.J. Cho, M. Sivaguru & R.E. Sharp, unpublished data).As detailed above, accumulation of ABA is essential forthe maintenance of elongation in the apical region of themaize primary root at low water potentials; whether ABAis similarly required for the adaptation of soybean primaryroots to water stress has not been investigated. Therelationship of ABAs potential involvement in ROSmetabolism in water-stressed roots to its above-mentionedrole in preventing excess ethylene production (Spollenet al. 2000) is under investigation; notably, ROS produc-tion and ethylene synthesis have been shown to be inter-related in other systems (Overmyer et al. 2000; Moederet al. 2002).

Increased apoplastic ROS in water-stressed

roots potential roles in spatialgrowth regulation

In contrast to the oxidative damage that can occur if ROSlevels are excessive, ROS may also play positive roles inregulating the response of root growth to water stress.Thus,it is of particular interest that the maize cell wall proteomicstudy revealed that in region 1 of water-stressed roots,several proteins which increased in abundance were relatedto ROS generation (Fig. 4; Zhu et al. 2007).These includedtwo putative oxalate oxidase/germin proteins and a super-oxide dismutase [Cu-Zn], which contribute to H2O2 pro-duction (Fig. 4). Notably, an oxalate oxidase was also the

Figure 6. Outline of phenylpropanoid metabolic pathways and water stress-responsive genes/proteins involved in those pathways.Enzymes which were differentially expressed in region 1 (R1) and/or region 2 (R2) of the primary root elongation zone in the soybeantotal protein analysis (Yamaguchi et al. 2009) and the maize transcriptome analysis (Spollen et al. 2008) are indicated in green and purple,respectively. Blue or red boxes indicate the fold-change response (up- or down-regulated, respectively) of changes in protein abundanceor transcript expression; white boxes indicate no statistically significant regulation under water stress. It should be noted that severalO-methyltransferases (OMT) were regulated under water stress in the maize transcriptome analysis; based on sequence, the fold-change

values shown are likely to represent ZRP4, which is thought to be involved in both lignin and ferulate synthesis (Barrire et al. 2007;Riboulet et al. 2009). Dashed arrows indicate multiple steps. The metabolic map is based on Winkel-Shirley (2001) & Barrire et al.(2007). Insets A and B show, respectively, that isoflavones accumulate towards the apex, whereas lignin staining (Mule stain) increasestowards the base of the elongation zone in water-stressed (WS) compared to well-watered (WW) soybean primary roots. Lignins arestained red-purple (guaiacyl-syringyl lignin) or brown (guaiacyl lignin).Asterisks in inset A denote significant differences between WSand WW values (P< 0.05). These profiles are consistent with the up-regulation of enzymes involved in isoflavonoid biosynthesis in region1 and the high level of up-regulation of CCoAOMT in region 2 of water-stressed roots. The figures are modified from Yamaguchi et al.(2009) and reproduced with permission from Wiley-Blackwell. CCoAOMT, caffeoyl-CoA O-methyltransferase; CHI, chalcone isomerase;CHR, chalcone reductase; CHS, chalcone synthase; C3H, C3-hydroxylase; F3H, flavone 3-hydroxylase; GT, glucosyl transferase; HCT,hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferase; IFR, isoflavone reductase; IFS, isoflavone synthase.




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second-highest up-regulated transcript in region 1 of water-stressed maize roots (Spollen et al. 2008), and histochemicalanalysis indicated that oxalate oxidase activity increasesparticularly in the apical region under water-stressed con-ditions (J. Zhu & R.E. Sharp, unpublished data). Oxalate,the substrate of oxalate oxidase, might be generated fromascorbate or glycolate (Davies & Asker 1983; Green & Fry2005). Consistent with these results, increased apoplastic

ROS levels were demonstrated in region 1 of water-stressedroots by quantification of H2O2 content in apoplastic fluidand by in situ imaging (Fig. 4, inset). The increased ROSlevels were sustained during growth under steady condi-tions (as described in Fig. 1) rather than a transient eventfollowing stress imposition, suggesting that the responsemay be associated with a continuing process of stress adap-tation. Clearly, given the maintenance of elongation underwater stress in region 1 (Fig. 2), the increase in apoplasticROS in this region seems likely to be associated with posi-tive rather than negative consequences. Oxalate oxidaseproteins also increased in abundance in regions 2 and 3 ofwater-stressed roots, again predicting increased production

of H2O2; however, this could not be confirmed because oftechnical limitations of the assay procedures in theseregions.

To test if enhanced oxalate oxidase activity and apoplasticROS affect root elongation, a transgenic maize line con-stitutively expressing a wheat oxalate oxidase (Ramputhet al. 2002) is being evaluated. Initial results show thatunder well-watered conditions, primary root elongation

rate increased by as much as 30% compared with the segre-gated transgene null line (J. Zhu, J. Simmonds & R.E. Sharp,unpublisheddata).Interestingly,oxalateoxidaseactivitywasincreased particularly in the apical region of the elongationzone, simulating the effect of water stress. Taken together,these results support the hypothesis that the increase inoxalate oxidase activity and apoplastic ROS in the apicalregion of water-stressed roots is positively associated withthemaintenanceof elongation in this region. Experiments todetermine the mechanisms of root growth regulation byoxalate oxidase/apoplastic ROS are in progress.

At least two possible mechanisms can be postulated forpromotion of root elongation by apoplastic ROS (Fig. 4).

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Figure 7. Outline of glutamate and proline metabolic pathways and water stress-responsive genes involved in those pathways. Enzymeswhich were differentially expressed in region 1 (R1) and/or region 2 (R2) of the primary root elongation zone in the maize transcriptomeanalysis (Spollen et al. 2008) are indicated. Blue or red boxes indicate the fold-change response (up- or down-regulated, respectively) ofchanges in transcript expression; white boxes indicate no statistically significant regulation under water stress. Dashed arrows indicatemultiple steps.The metabolic map is based on Miflin & Habash (2002) and the KEGG Database (http://www.genome.jp/kegg/pathway.html). The inset shows that proline accumulates dramatically towards the apex of water-stressed (WS) compared to

well-watered (WW) maize primary roots; the data are reproduced from Voetberg & Sharp (1991) with permission from the AmericanSociety of Plant Biologists. AlaAT, alanine aminotransferase; AS, asparagine synthetase; GAD, glutamate decarboxylase; GOGAT,glutamate synthase; GS, glutamine synthetase; PO, proline oxidase; P5CS, pyrroline-5-carboxylate synthetase; SSD, succinic semialdehydedehydrogenase; KG, a-ketoglutarate.




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Firstly, generation of hydroxyl radicals from H2O2,by eitherthe Fenton reaction or peroxidase activity in the presence ofsuperoxide and/or reductant (e.g. NADH),can play a directrole in cell wall loosening via polysaccharide cleavage (Fry1998; Liszkay, Kenk & Schopfer 2003; Passardi, Penel &Dunand2004;Schopfer&Liszkay2006;Kukavica et al. 2009;

Mller et al. 2009). This possibility is of particular interestbecause of the above-described increase in longitudinal cellwall extensibility in region1 of themaize primary root underwater-stressed conditions. Importantly, there is evidence forthis mechanism of wall loosening in the elongation zone ofwell-watered maize primary roots (Liszkay, van der Zalm &Schopfer 2004), although the possible modification of thisactivity in theresponse of root growthto water stresshas notbeen investigated. Secondly, ROShave been proposed to actas signalling molecules in various processes (Moller et al.2007); for example, apoplastic hydroxyl radicals have beenshown to activate Ca channels that are necessary for growthof root hairs (Foreman et al. 2003).

There is evidence that apoplastic superoxide is localizedpreferentially in the apical region of the maize primary rootunder well-watered and osmotically stressed conditions(Liszkay et al. 2004; Bustos et al. 2008). In addition to beinga potential source of the increase in H2O2 production inregion 1 of water-stressed roots (together with the increasein oxalate oxidase activity), superoxide may also interactwith peroxidases to convert the H2O2 to hydroxyl radicals,as noted above. Superoxide can also reduce Fe3+ back toFe2+ to sustain the Fenton reaction, thereby increasinghydroxyl radical generation. Accordingly, the apical local-ization of superoxide may be an important factor in deter-mining the specificity of growth maintenance in the apical

region of water-stressed roots. Plasma membrane NADPHoxidase is an important source of apoplastic superoxide(Foreman et al. 2003), and up-regulation of NADPHoxidase by both water stress and ABA treatment wasreported in maize leaves (Jiang & Zhang 2002). However,four NADPH oxidase-related sequences that were includedin the maize transcriptomic analysis were not differentiallyexpressed in either region 1 or 2 of water-stressed maizeroots (Spollen et al. 2008).

Apoplastic ROS can also have wall tightening effects,involving oxidative cross-linking of cell wall phenolics(Fig. 5). As noted above, apoplastic ROS generation prob-ably also increased in region 2 of water-stressed maize

roots (in this regard, the transcriptomic analysis suggestedthat glycolate oxidase, which was up-regulated in thisregion, might contribute to production of oxalate, the sub-strate of oxalate oxidase). Notably, transcripts for bothO-methyltransferase and extensin-like protein were up-regulated in region 2 under water-stressed conditions(Spollen et al. 2008); O-methyltransferase is probablyinvolved in the synthesis of wall phenolics (Riboulet et al.2009). Peroxidase catalyses the polymerization of phenoliccompounds as well as the tyrosine residues of extensin usinghydrogen peroxide (Passardi et al. 2004), thereby givingmechanical strength to cell walls. Consistent with theseresults, increased levels of wall phenolics were observed in

the basal region of the elongation zone in water-stressedmaize roots (Fig. 5, inset), as shown previously by Fan et al.(2006).This response may play an important role in decreas-ing longitudinal wall extensibility in region 2 under water-stressed conditions. Apoplastic ROS signalling may alsooccur in region 2, but for clarity, this is not shown in Fig. 5.

Taken together, the cell wall proteomic and transcrip-tomic results suggest that regulation of apoplastic ROS hascritical roles in determining both the maintenance of elon-gation in the apical region and the inhibition of elongationin the basal region of water-stressed maize roots.

Phenylpropanoid metabolism region-specificaccumulation of (iso)flavonoids andwall phenolics

The transcriptomic and proteomic analyses indicated thatthe synthesis of phenylpropanoids including (iso)flavonoidsandwallphenolicsintherootelongationzoneisregulatedin

a complex and region-specific manner in response to waterstress. The total protein analysis of water-stressed soybeanroots (Yamaguchi et al. 2009)showed thatchalcone synthase7 increasedin abundance in region1 and chalcone reductaseand two isoflavone reductases increased in both regions 1and 2 (Fig. 6). These three enzymes are involved in isofla-vonoid biosynthesis; in particular, chalcone synthase 7 hasbeen reported to play a critical role in isoflavonoid synthesisin soybean seeds (Dhaubhadel et al. 2007). Similarly, themaize transcriptome analysis showed that enzymes involvedin flavonoid synthesis were up-regulated under water stress;putative flavone hydroxylase and chalcone isomerase wereup-regulatedinregions1and2,respectively(Fig. 6).Accord-

ingly, the results suggested that (iso)flavonoids accumulatein the root elongation zone under water stress, perhapsespecially in region 1 in the case of soybean. As alreadymentioned, this prediction was confirmed by analysing thespatial profile of isoflavone composition in the elongationzone of soybean roots, which showed that isoflavonoidsaccumulated towards the root apex in roots growing at lowwater potentials (Fig. 6, inset).

As discussed above, both flavonoids and isoflavonoids arepotent scavengers of ROS and, therefore, the preferentialaccumulation of these compounds in the apical region ofwater-stressed roots is likely to help in protection fromoxidative damage. In addition,these compounds have other

functions in plant cells that can be postulated to have rolesin water stress tolerance mechanisms. Of particular interestis evidence of their ability to influence auxin transport byinhibiting PIN (PIN-FORMED) proteins, thereby causingthe accumulation of auxin in nearby tissues (Mathesiuset al. 1998; Subramanian, Stacey & Yu 2006). Interestingly,water stress has been shown to cause auxin accumulation inthe elongation zone of maize primary roots (Ribaut & Pilet1994), and several auxin-inducible genes/proteins wereup-regulated in the elongation zone of both maize andsoybean roots in response to water stress (Poroyko et al.2007; Spollen et al. 2008; Yamaguchi et al. 2009). Further-more, applied auxin was shown to cause shortening of the




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elongation zone in well-watered roots of several species in amanner similar to the effect of water stress in maize andsoybean (Fig. 2; Hejnovicz 1961; Goodwin 1972; Ishikawa &Evans 1993). Taken together, these observations suggestthat auxin, and its regulation by (iso)flavonoids, may beimportant for controlling the growth response of primary

roots to water stress, although this possibility has not yetbeen assessed. In this regard, it is interesting that, as men-tioned above,ABA has been reported to regulate flavonoidsynthesis (Ithal & Reddy 2004); accordingly, ABA mightplay a role in modulating auxin localization in the elonga-tion zone of water-stressed roots.

Whereas the soybean results showed that water stress-induced isoflavonoid accumulation occurs preferentially inthe apical region where elongation rates are maintained,the results from both maize and soybean roots indicate thatsynthesis of wall phenolics including lignin and ferulates,which constitute other major branch pathways in phenyl-propanoid metabolism, is enhanced in region 2 where

elongation is inhibited (Fig. 6). As already noted, phenoliccomponents can result in cross-linking between wall poly-mers, and accumulate in the basal region of the elongationzone in water-stressed maize primary roots (Fig. 5, inset).Fan et al. (2006) showed that this response occurred inassociation with up-regulation of cinnamoyl-CoA reduc-tase, a key enzyme in lignin synthesis. Consistently insoybean, the recent proteomic analysis (Yamaguchi et al.2009) showed that caffeoyl-CoA O-methyltransferase(CCoAOMT), which catalyses the fourth step in lignin bio-synthesis from p-coumaroyl-CoA, was highly up-regulatedin region 2 under water stress, and accumulation of ligninswas demonstrated in the basal region of the elongation

zone (Fig. 6, inset).Interestingly, in maize, the cell wall proteomic analysisshowed that several b-d-glucosidases markedly increasedin abundance in regions 2 and 3 of the elongation zoneunder water-stressed conditions (Fig. 5; Zhu et al. 2007).Apoplastic b-d-glucosidases have been implicated in ligninsynthesis, via the cleavage of monolignol glucosides thatare synthesized inside the cell and secreted into the cellwall (Whetten, Mackay & Sederoff 1998). Therefore, theincreased abundance ofb-d-glucosidases may play a rolein the accumulation of lignin in the basal region of theelongation zone under water stress. It should be notedthat most of these b-d-glucosidases were also highly

up-regulated in region 1 of water-stressed roots. Theirfunction in this region is not known; an intriguing possi-bility, as discussed by Zhu et al. (2007), is that they mightfunction in release of ABA and/or other hormones fromconjugated forms.

In maize, additionally, O-methyltransferase (ZRP4) tran-script expression was highly down-regulated in region 1 andup-regulated in region 2 (different isoforms) under waterstress (Fig. 6). This enzyme is closely associated with pro-duction of both monolignols and ferulates (Barrire et al.2007; Riboulet et al. 2009). These results suggest that feru-late synthesis may be decreased and increased, respectively,in regions 1 and 2 of water-stressed roots. Ferulates are

abundant in the cell walls of monocotyledonous plants andhave a role in cross-linking wall polysaccharides. Increasesin wall-bound ferulates have been shown to correlateclosely with decreases in wall extensibility with tissue aging(Kamisaka et al. 1990). Interestingly, in wheat coleoptiles,both osmotic stress and ABA treatment suppressed the

increase of ferulate and diferulate in the cell walls whichoccurs during normal development, and this was associatedwith suppression of cell wall stiffening (Wakabayashi,Hoson & Kamisaka 1997a,b).These results suggest the pos-sibility that the pronounced accumulation of ABA in theapical region of water-stressed roots (Saab et al. 1992) mayrestrict ferulate synthesis and, thereby, contribute to theenhancement of wall extensibility and maintenance of elon-gation in this region.

Importantly, the (iso)flavonoid and wall phenolic branchpathways of phenylpropanoid metabolism share a commonprecursor, p-coumarate-CoA (Fig. 6). Evidence for sub-strate competition between lignin synthesis and flavonoid

synthesis was provided by Besseau et al. (2007) in a study ofArabidopsis; inhibition of lignin biosynthesis (by genesilencing) induced accumulation of flavonoids in the leaves.Similarly, coordinated balance of metabolite flux between(iso)flavonoid and wall phenolics synthesis could play acritical role in determining the spatial regulation of rootelongation under water stress (Yamaguchi et al. 2009).

Glutamate and proline metabolism

As detailed above, previous studies showed that prolineaccumulates to high concentrations towards the apex ofwater-stressedmaizeprimary roots(Fig. 7, inset;Voetberg&

Sharp 1991), and the radiolabelling flux analysis results ofVerslues & Sharp (1999) suggested that this response isprimarily attributable to increased proline transport to theroot tip rather than local changes in proline metabolism.Nevertheless, the maize transcriptomic analysis providedadditionalinformationonchangesinaminoacidmetabolismthatmay supplementthis response(Fig. 7).Firstly,pyrroline-5-carboxylate synthetase, which catalyses the rate-limitingstepin proline synthesis fromglutamate, was up-regulated inregion 1 under water stress, indicating that local prolinesynthesis may have increased to some extent. Secondly, aputative proline oxidase was down-regulated in region 1.Asdiscussed byVerslues & Sharp (1999),although their results

suggested that proline catabolism in the apical region wasnot suppressed relative to well-watered roots, this does notimply a lack of regulation; it is unlikely that proline couldaccumulate to such high levels if proline catabolismincreases in proportion to proline concentration.

In region 2, pyrroline-5-carboxylate synthetase andproline oxidase were further up- and down-regulated,respectively, in response to water stress relative to theirresponses in region 1 (Fig. 7). In addition, several otherenzymes involved in glutamate biosynthesis were up-regulated, indicating increased production of glutamatethrough the glutamate synthase cycle and from a-ketoglutarate in the tricarboxylic acid (TCA) cycle. The




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evidence for activated proline synthesis, takentogether withthefact that proline deposition rates decreased inregion2inwater-stressed roots compared with well-watered roots(Voetberg & Sharp 1991), suggests that the basal region ofthe elongation zone may serve as a source of proline fortransport to region 1. Indeed, proline deposition rates in

region 2 were negative under water stress in the study ofVerslues&Sharp(1999),indicatinganetlossofprolinefromthis region. Nevertheless, as analysed in detail in that paper,the amount of proline transported from the basal to theapical region may be limited relative to that from the seed orother parts of the plant. In addition to its roles in osmoticadjustment and protection from excess ROS, the prolineproduced in region 2 could also be utilized for synthesis ofextensin, a hydroxyproline-rich protein; as noted above,extensin-like protein was up-regulated in region 2 underwater-stressed conditions.

Glutamate may also be used for g-aminobutyrate acid(GABA) synthesis (Fig. 7), and glutamate decarboxylase,

which catalyses the conversion of glutamate to GABA, washighly up-regulated in region 2 of water-stressed roots. Theproduction of GABA may have at least two roles in waterstress tolerance, the GABA shunt and pH regulation. Suc-cinic semialdehyde dehydrogenase, which catalyses the laststep in the GABA shunt, was also up-regulated in region 2under water stress (Fig. 7). The GABA shunt provides analternate path for the TCA cycle, bypassing mitochondrialenzymes including a-ketoglutarate dehydrogenase thatmay be degraded during oxidative stress. Therefore, underwater-stressed conditions, the shunt may have importantroles to prevent uncoupling of mitochondrial electrontransport and, thereby, limit the generation of ROS, and to

maintain the TCA cycle (Bouch & Fromm 2004). GABA,together with other polyamines, can also help to maintain astable cytosolic pH because of its H+ buffering properties.Consistently, lysine decarboxylase and arginine decarboxy-lase, which produce polyamines from lysine and arginine,were also up-regulated in region 2 under water stress (notshown in Fig. 7).

CONCLUSION

The recent transcriptomic and proteomic studies reviewedhere have built on previous physiological knowledge togain novel insights into the coordination of root growth

regulation and adaptation under water-stressed conditions.Understanding has been greatly facilitated by taking advan-tage of a kinematic approach to transcript and proteinprofiling. Many of the processes involved in root growthadaptation, and the underlying coordination of gene net-works, proteins and metabolites, are controlled in a region-specific manner in association with the distinct regionspecificity of growth regulation. In this review, we havefocused on selected processes for which the available infor-mation allows a framework of understanding. Clearly, muchadditional research is needed to more fully explore thecomplexity of root growth adaptation to water stress; forexample, the roles of hormones other than ABA, and the

interactions between different hormones, remain poorlyunderstood. Continued progress in understanding of rootgrowth regulation under water stress will lead to novelapproaches for improving drought tolerance throughgenetic and metabolic engineering of root function.

ACKNOWLEDGMENTS

Preparation of this review was supported by a grant fromMonsanto to R.E.S. and by the University of Missouri Foodfor the 21st Century Program. We thank Dr. Dale Blevins(University of Missouri) and Dr. Mel Oliver (USDA,Columbia, Missouri) for useful discussions and helpful com-ments on the manuscript, and Dr. Mayandi Sivaguru (Uni-versity of Illinois at Urbana-Champaign) for the diagram inthe inset to Fig. 4.

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Received 25 August 2009; received in revised form 6 October 2009;

accepted for publication 8 October 2009



yamaguchi 2010_ root growthwaterstressprotein

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