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436 M. IIJIMA ET AL.N2 flowMixedRice/Pearl milletFanN2 outPearl milletRiceCO2 trap columnShadingPumpRoot chamberWatre bathWatercoolerPumpPumpMagnetic stirrerFigure 2. Schematic diagram of the gas exchange chamber. The water temperature inside the root chamber was precisely controlledusing a water bath, and the water was circulated using four mini-pumps at 4 L min 1. Air in the gas exchange chamber was mixedcontinuously by two fans.Table 1. Air temperature in a gas exchange chamber and water temperature in a root chamber.Phase ITemperature ( C)AirWaterPhase IIMaximumMinimumMeanMaximumMinimumMean32.3 .129.6 .031.9 .129.0 .032.1 .129.3 .031.6 .129.6 .131.1 .129.1 .131.3 .129.3 .1Notes: Values are means standard error.Germany) and a temperature sensor (K320, TakeyamaKagaku, Japan) which was placed at about 5 mm belowthe root system. Further, an air-bubbling inlet tube and anexhaust tube were placed to relieve the pressure duringair-bubbling. The distilled water was continuously stirredusing a magnetic stirrer. The water temperature inside theroot chamber was precisely controlled using a water bathequipped with a cooler connected to a pump (9 L min 1),and the water was circulated using four mini-pumps at4 L min 1 (Table 1). Air in the gas exchange chamber wasmixed continuously by two fans (Table 1). The CO2 concentration was monitored using a portable photosynthesisanalyzer (LCpro SD, ADC BioScientific, UK). The O2 releasefrom the plant roots was evaluated during the followingtwo continuous phases (Lee, 2003; Maricle & Lee, 2007):Phase I–Light (photosynthetically active radiation of168.9 1.6 μmol m 2 s 1) and ambient air conditions weremaintained in a normal state to allow photosynthesis for30 min. Distilled water inside the root chamber was continuously air-bubbled, starting from setting up the rootsystem until the beginning of phase I, and the dissolved O2concentration was then measured continuously for 30 min(Figure 3). In all of the measurements, phase I started at1300 h of the day. Phase II–The gas exchange chamber O2level was reduced to less than .8% by pumping N2 intothe chamber at 50 L min 1 for 10 min before the start ofphase II at 1400 h of the day (Figure 3); subsequently, theflow rate was reduced to 5 L min 1 for the entire phase.Photosynthesis was stopped by creating darkness by covering the chamber with plastic shedding (photosynthetically active radiation of .1 .0 μmol m 2 s 1), and CO2 wasremoved continuously from the column. Other conditionswere the same as in phase I. After the O2 transfer measurements, fresh weight of shoots and roots, number ofleaves, and tillering number of the plants were measured.Several investigators including Colmer et al. (1998) usedroot surface area-based concentration; however, they usedonly single root axis not whole root system for the estimation of root surface area. The majorities of studies dealingwith whole root system used root fresh/dry weight-basedconcentrations, and thus, the root surface area was notmeasured. All statistical analyses were performed usingExcel Statistics Version 2012 software (SSRI).Results and discussionO2 transfer between mixed plantsAwala et al. (2016) reported that the productivity indrought-adapted crops of close mixed planting with ricewas higher than that of normal single planting under

PLANT PRODUCTION SCIENCE flooding condition in the field experiment. The mitigationeffects of rice on flood stress for drought-adapted cropswould most probably be attributed to the relatively O2 richenvironments and/or O2 transfer when pearl millet wasclosely grown with rice (Iijima et al., 2016). To confirm thehypothesis, O2 transfer was evaluated under a water culturecondition without soil. Figure 4 illustrates the phenomenaof O2 absorption and release by plants in a water culture.O2 normal diffusion (nD) from aboveground to water, O2reverse diffusion (rD) from water to aboveground, and rootrespiration (R) are the factors that affect O2 concentrationin a water culture. In addition to these factors, O2 transfer(T) between different neighboring species can be considered to exist under mixed planting conditions. Rice rootsare known to release O2 from the shoot system via theaerenchyma (Yamauchi et al., 2013). Upland crop speciesalso release O2 (Maricle & Lee, 2007), although the amountis smaller (Figure 4, Single pearl millet) compared to rice.The O2 released from rice roots is most probably absorbedby the neighboring upland species due to their higherO2 demand caused by a lower O2 supply from shoots. O2released by pearl millet may be absorbed by rice as well.In order to accurately compare O2 absorption/releasebetween single planting and mixed planting, we combined the two single-species crops of rice and pearl millet immediately before the experiment (defined as mixedplanting in this study). The only difference between single planting and mixed planting was whether the roots ofthe two different species stayed together during the shortperiod of measurement or not. For this purpose, plantswith similar agronomical traits (fresh weight of shoot androot and the number of leaf and tiller) were selected (Table2) for both single- and mixed- planting crops. Thus, themorphological characteristics [e.g. ROL barrier formationnear the root tips (Shiono et al., 2011)] of mixed plantingshould be equivalent to those of single planting.The CO2 level was adjusted to 527 6 μmol mol 1 byusing a soda lime absorbent column at the start of phaseI (Table 3). O2 transfer between the two species can beevaluated by measuring O2 changes in the water culturewhen shoots were grown under O2-rich (20.0 .0% conc. inphase I) and O2-free dark (.8 .0% conc. in phase II) conditions (Figure 5). In phase I, the plants absorb dissolved O2 inthe water for root respiration and the roots simultaneouslyrelease O2 as normal diffusion from aboveground to waterdue to the O2-rich condition in the shoot system (Figure4). Thus, O2 changes in phase I in the cultured water canbe expressed as follows;O2 changes (phase I) nD RFigure 3. Dynamics of temperature and gas concentrations in agas exchange chamber (1) and a root chamber (2).rDrDrDrDnDTRO2 fromwaterSingle ricephase InD’TnDRootchamber(1)In phase II, the plants absorb dissolved O2 from the wateras was noted in phase I, and the shoots simultaneouslyrelease O2 as reverse diffusion from water to abovegrounddue to the O2-poor conditions in the shoot system (FigureO2 fromabove-groundR437RRRiceSingle pearl milletnD’Pearl milletMixedphase IIFigure 4. Illustration of O2 absorption and release by rice and pearl millet roots in the root chamber. The solid line and broken linerepresent the O2 movement in phase I and phase II, respectively. R, respiration; nD, normal diffusion; rD, reverse diffusion; T, transfer.

438 M. IIJIMA ET AL.Table 2. Growth traits of plants used for O2 transfer measurements.Fresh weight (g plant 1)RiceSingleMixedShoot1.40 .081.34 .08Pearl milletSingleMixed6.41 .376.30 .26Root.57 .03.57 .03nsnsLeaf no.7.6 .17.6 .1ns1.73 .131.74 .11ns9.4 .19.6 .1nsnsTiller no.2.3 .22.5 .3.0 .0.1 .1nsnsn21212121Notes: Values are means standard error. ns, no significant difference by Student’s t-test.n represents the sample size.Table 3. O2 and CO2 gas concentrations in a gas exchange chamber.Phase IStartGas concentrationO2 (%)20.0 .0CO2 (ppm)527 6Phase IIEndStartEnd20.0 .0474 16.8 .020 1.6 .011 1Notes: Values are means standard error.Phase II (-O2)O2 concentration (µM)220200180160RicePearl MilletJust-mixMixedR140120PM010Air bubbling2030where r is rice and p is pearl millet.mix O2 changes (phase I - II) nD (mix) rD (mix)(5)Phase I ( O2)240Equation (3) can be expressed by the total single plantingof rice and pearl millet (Total single) and mixed planting(Mix), respectively, as follows;Total-single-O2 changes (phase I II)(4) nD (r p) rD (r p)0102030where mix is mixed planting of rice and pearl millet.As mentioned above, in this experiment, morphologicalcharacteristics of mixed planting during the short period ofmeasurement were not quantitatively different from thatof single planting. Because the two plants were combinedjust before the experiment, rD was assumed to be the species characteristic value, and the total rD of single plantingrice and pearl millet {rD (r p)} was not different from thatof mixed planting {rD (mix)}. Therefore, (Equations (4) (5))can be expressed as follows;O2 changes (phase I - II) (Total single - mix)(6) nD (r p) nD (mix)Measuring time (min.)Figure 5. Dissolved O2 concentration of the distilled water in theroot chamber (Figure 1) where plant roots were situated (n 21)during the two 30-min consecutive phases: O2-rich light phase(Phase I) and O2-poor dark phase (Phase II) shoot environment.In order to accurately compare O2 absorption/release betweensingle and mixed, the two single plants of rice and pearl milletwere combined immediately before the experiment to measureO2 transfer.4). According to Lee (2003), the root respiration (R) duringPhase I can be assumed to be similar to that in phase II.Thus, the O2 changes in phase II in the cultured water canbe described as,O2 changes (phase II) rD R(2)nD (r p) nD (mix) T (mix)(7)Equation (7) is substituted into (6) as follows;Therefore, (Equations (1)–(2)) is,O2 changes (phase I II) nD rDSimilarly, the total amount of released O2 was also assumedto be the species characteristic value. The O2 released fromthe roots by mixed planting can be classified into twocategories: that remaining in the cultured water (residualcomponent, indicated as nD’ in Figure 4) and that transferred to different neighboring species (O2 transfer component, indicated as T in Figure 4). On the other hand, the O2released from the roots by single planting is representedonly by normal diffusion. Thus, the sum of O2 released bythe two single-species seedlings {nD (r p)} will be equivalent to the total O2 released by the mixed two seedlings{nD’ (mix) T (mix)} as follows;(3)The difference in O2 changes between phase I and phase IIindicates the sum of normal diffusion and reverse diffusion.O2 changes (phase I - II) (single total - Mix) {nD’ (mix) T (mix)} nD’ (mix) T (mix)(8)

PLANT PRODUCTION SCIENCE Rice3060PearlMilletSingleTotalMixed*Changes in O2 concentration(µM g fresh root weight 1 h 1)1020-10-20-30-60-50-100-70-140phase Iphase II-90-180phase I IIFigure 6. Changes in O2 concentration in the distilled water inthe root chamber (Figure 1) where rice and pearl millet rootsin single planting or mixed planting were situated (n 21). Anasterisk indicates a significant difference by a Student’s t-test atthe p 0.01 level. The difference between phase I and phase IIindicates the sum of diffusion (nD rD), and those betweensingle total and mixed indicates the oxygen transfer between thetwo species {T (mix)}.O2 transfer between rice and pearl millet was representedby the difference of O2 changes (phase I II) between single planting of rice and pearl millet and mixed planting.In this study, the sum of the O2 changes (phase I II) insingle planting of rice and pearl millet was significantlyhigher than that of mixed planting of rice and pearl millet(Figure 6; p .01), which indicated that O2 was transferredbetween rice and pearl millet under a water culture condition, at least in our experimental system. These results indicated that approximately 7 μM O2 g fresh root weight 1 h 1was transferred between the two plant species, and mostof them should be transferred from rice to pearl millet.Technical evaluation for O2 transferThe flood mitigation effects observed in previous studies(Awala et al., 2016; Iijima et al., 2016) for drought-adaptedupland crops could be attributed to the O2 transferredfrom the roots of flood-adapted to drought-adaptedcrops. To date, O2 release can be analyzed using severalmethods (Colmer, 2003; Wu et al., 2014). This study useda fiber optic O2-sensing probe (Maricle & Lee, 2007) tomeasure the O2 net flux from the whole root system tothe O2-rich (228 .4 μM) water environment, which hasnot been used before. So far, low O2 concentrations, i.e.15 μM (Sorrell & Armstrong, 1994), 60–70 μM (Lee, 2003),or 120 μM (Maricle & Lee, 2007), were used at the start439of the experiments; however, the root respiration rates ofthe drought-adapted upland species used in this studywere significantly reduced under such a low O2 concentration range within 30 min. Thus, estimating O2 releaseat low O2 concentrations was considerably difficult. In thisstudy, seedlings grown in the water culture without a soilmedium were used for the estimation of O2 transfer; thismight be different from the actual O2 released by a tangledroot system grown in soil. In the case of mixed planting, theroot surface sorption zone was partly disturbed by the mixing of different species, whereas that of the tangled rootsystems of the close mixed seedlings grown in soil wouldbe significantly disturbed by the mixed species duringgrowth. For example, the root hairs of epidermal cells of thetwo species may become entangled with each other andwith soil particles. Thus, the living sloughed root cap cellson the root surface during root penetration (Iijima et al.,2000, 2004) will also interact with those from the mixedneighboring species. Further study using a real soil systemis required to confirm this phenomenon in fields.AcknowledgementsWe thank the members of the projects entitled ‘Flood- andDrought-adaptive Cropping Systems to Conserve Water Environments in Semi-arid Regions’ by the framework of the ‘Science and Technology Research Partnership. We also thank allthe students of Kindai University for their assistance.Disclosure statementNo potential conflict of interest was reported by the authors.FundingThis work was supported by the Japan Science and Technology(JST) and the Japan International Cooperation Agency (JICA).ReferencesArmstrong, W. (1979). Aeration in higher plants. Advancesin Botanical Research, 7, 225–332. doi:10.1016/S00652296(08)60089-0Armstrong, W., Cousins, D., Armstrong, J., Turner, D. W., & Beckett,P. M. (2000). Oxygen distribution in wetland plant roots andpermeability barriers to gas-exchange with the rhizosphere:A microelectrode and modelling study with Phragmitesaustralis. Annals of Botany, 86, 687–703. doi:10.1006/anbo.2000.1236Awala, S. K., Yamane, K., Izumi, Y., Fujioka, Y., Watanabe, Y., Wada,K. C., Iijima, M. (2016). Field evaluation of mixed-seedlingswith rice to alleviate flood stress for semi-arid cereals.European Journal of Agronomy, 80, 105–112. doi:10.1016/j.eja.2016.07.003Brooker, R. W., Bennett, A. E., Cong, W. F., Daniell, T. J., George, T.S., Hallet, P. D., White, P. J. (2015). Improving intercropping:A synthesis of research in agronomy, plant physiology andecology. New Phytologist, 206, 107–117. doi:10.1111/nph.13132

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mixed cropping technique that enhances the flood stress tolerance of co-growing drought-adapted upland cereal crops (Awala et al., 2016; Iijima et al., 2016). Growing flood- and drought-adapted crops at the same time in the same field, as in close mixed planting, will be a new global chal-le