Waterlogging tolerance in the tribe Triticeae: the adventitious roots of Critesion marinum have a relatively high porosity and a barrier to radial oxygen loss

Authors


Correspondence: T. D. Colmer. E-mail: tdcolmer@cyllene.uwa.edu.au

Abstract

Nine species from the tribe Triticeae – three crop, three pasture and three ‘wild’ wetland species – were evaluated for tolerance to growth in stagnant deoxygenated nutrient solution and also for traits that enhance longitudinal O2 movement within the roots. Critesion marinum (syn. Hordeum marinum) was the only species evaluated that had a strong barrier to radial O2 loss (ROL) in the basal regions of its adventitious roots. Barriers to ROL have previously been documented in roots of several wetland species, although not in any close relatives of dryland crop species. Moreover, the porosity in adventitious roots of C. marinum was relatively high: 14% and 25% in plants grown in aerated and stagnant solutions, respectively. The porosity of C. marinum roots in the aerated solution was 1·8–5·4-fold greater, and in the stagnant solution 1·2–2·8-fold greater, than in the eight other species when grown under the same conditions. These traits presumably contributed to C. marinum having a 1·4–3 times greater adventitious root length than the other species when grown in deoxygenated stagnant nutrient solution or in waterlogged soil. The length of the adventitious roots and ROL profiles of C. marinum grown in waterlogged soil were comparable to those of the extremely waterlogging-tolerant species Echinochloa crus-galli L. (P. Beauv.). The superior tolerance of C. marinum, as compared to Hordeum vulgare (the closest cultivated relative), was confirmed in pots of soil waterlogged for 21 d; H. vulgare suffered severe reductions in shoot and adventitious root dry mass (81% and 67%, respectively), whereas C. marinum shoot mass was only reduced by 38% and adventitious root mass was not affected.

Introduction

The tribe Triticeae (nomenclature following Löve 1984) includes the crop species Triticum aestivum (bread wheat), Triticum durum (durum wheat), Hordeum vulgare (barley) and Secale cereale (rye). These crops are all intolerant to soil waterlogging (Musgrave 1994; McFarlane & Wheaton 1990; Bourget, Finn & Dow 1966). Differences in tolerance to soil waterlogging among cultivars of wheat have been reported (Sayre et al. 1994; Thomson et al. 1992), but the growth and/or yield of the ‘tolerant’ cultivars was still reduced by around 70–80% when waterlogged. Whilst this may be a superior level of tolerance in comparison to even more sensitive cultivars, such reductions in growth and yield are very large compared to those of tolerant species such as Oryza sativa (rice) (Thomson et al. 1992).

Undomesticated species within the Triticeae, known as ‘wild relatives’, are a potential source of genes for the improvement of stress tolerance (e.g. drought, salinity) in crop species from the tribe (Fedak 1985). Superior waterlogging tolerance in undomesticated species in the Triticeae compared to wheat has also been documented (Taeb, Koebner & Forster 1993; Davies & Hillman 1988), but the physiological basis of the tolerance has not been investigated.

Waterlogged soils are usually anaerobic and chemically reduced (Ponnamperuma 1984), as a result of the rapid consumption of O2 by soil microorganisms and the low diffusivity of gases in water preventing replacement of the consumed O2 (Laanbroek 1990). Root extension into a waterlogged soil is therefore largely determined by the movement of O2 from the atmosphere to the root apex via the root and shoot aerenchyma (Armstrong 1979). Effective internal aeration of the root systems contributes to the waterlogging tolerance of wetland species (Armstrong 1979; Justin & Armstrong 1987).

Features that enhance internal aeration of the adventitious roots of waterlogging-tolerant grass species such as O. sativa and Phragmites australis have been studied extensively (Armstrong 1971; Armstrong & Armstrong 1988; Gries, Kappen & Lösch 1990; Colmer et al. 1998; Jackson & Armstrong 1999). The features that they possess, such as extensive aerenchyma and low radial O2 loss (ROL) from the basal regions of adventitious roots, offer insight into the physiological traits that could be targeted to increase the waterlogging tolerance of dryland cereals such as wheat.

This study evaluated nine species in the tribe Triticeae for tolerance to growth in stagnant deoxygenated nutrient solution, root porosity, and patterns of ROL from adventitious roots. The species studied included three common in wetland habitats (Critesion marinum, Lophopyrum elongatum, Lophopyrum turcicum), three pasture species (Psathyrostachys juncea, Pascopyrum smithii, Elymus scabrus) and three crop species (T. aestivum, H. vulgare, S. cereale). Plants were first evaluated in aerated or stagnant deoxygenated nutrient solution, and then further experimentation on C. marinum (the only species found to have a strong barrier to ROL in its adventitious roots) and its closest cultivated relative, H. vulgare, was conducted in pots of drained or waterlogged soil. The waterlogging-tolerant Echinochloa crus-galli L. (P. Beauv.) was also included in the soil experiment as a ‘check’ species.

Materials and methods

Experiments on nine species in the Triticeae

Plant culture

Seeds of the species listed in Table 1 were washed in 0·4% (w/v) NaHClO3 solution for 45 s and rinsed thoroughly with deionized water before being placed on plastic mesh floating on 0·1-strength aerated nutrient solution in darkness in a 20/15 °C (day/night) phytotron. The composition of the nutrient solution at full strength was (mol m−3): K+ 3·95, Ca2+ 1·50, Mg2+ 0·40, NH4+ 0·625, NO3 4·375, SO42– 1·90, H2PO4 0·20, Na+ 0·20, H4SiO4 0·10; and the micronutrients (mmol m−3): Cl 50, B 25, Mn 2, Zn 2, Ni 1, Cu 0·5, Mo 0·5, ethylenediaminetetra-acetic acid ferric monosodium salt 50. The solution also contained2·5 mol m−3 2-[N-Morpholino]ethanesulfonic acid (MES) and the pH was adjusted to 6·5 with KOH (increasing the final K+ concentration to 5·60 mol m−3). All chemicals used were of analytical grade.

Table 1.  Species (after Löve 1984; synonyms in parentheses after Wang et al. 1996) and accessions or cultivars used and the sources
SpeciesCultivar/AccessionSource
  1. Sources of seeds: (1) originally from The University of Manitoba, Winnipeg, Canada, courtesy of Prof. J Dvorak, University of California (Davis); (2) Australian Winter Cereals Collection; (3) Institute of Sustainable Irrigated Agriculture (Victoria); (4) Agriculture Western Australia. Superscripts a and b were used to indicate two different accessions of L. turcicum. Two were studied because Lophopyrum species are considered by others as one of the main ‘wetland’ groups in the Triticeae.

Lophopyrum elongatumD accession1
Triticum aestivumcv. Chinese Spring1
Lophopyrum turcicumaAUS 250602
Lophopyrum turcicumbAUS 250612
Critesion marinum (Hordeum marinum)AUS 4059622
Secale cerealecv. South Australian AUS 194362
Elymus scabrusAUS 242972
Psathyrostachys junceacv. Bozoisky-Select3
Pascopyrum smithiicv. Rosana3
Hordeum vulgarecv. Stirling4

Germination of the species was staggered to ensure that plants were at a similar developmental stage (2·0–2·2 leaves) on the same date, coinciding with the onset of root-zone O2 treatments. The first whorl of adventitious roots was starting to emerge from all species at this developmental stage. Lophopyrum elongatum and Pa. smithii were germinated 27 d before the onset of treat-ments; Ps. juncea, C. marinum, L. turcicum and E. scabrus 20 d beforehand; and T. aestivum, S. cereale and H. vulgare 13 d beforehand. The first and second groups of species were exposed to natural light and 0·25-strength nutrient solution 7 d after imbibition. The first group was transplanted into 4·5 dm3 plastic pots containing full-strength aerated nutrient solution 14 d after imbibition, while the second group was transplanted 10 d after imbibition. The third group of species were exposed to natural light and 0·25-strength nutrient solution 3 d after imbibition and were transplanted into the plastic pots 5 d after imbibition. For each species, four seedlings were transplanted into each of six pots and held into the lid using polystyrene foam holders that allowed expansion as the plants grew. The pots and lids were previously covered with aluminium foil to prevent light penetration into the solution.

In the root-zone O2 treatments, three of the pots of each genotype continued to be well aerated, while the other three were given a hypoxic pretreatment by flushing with N2 gas until the O2 concentration in the solution declined to approximately 0·03 mol m−3, after which they were left stagnant for 24 h. The solution in these pots was then replaced with deoxygenated nutrient solution containing 0·1% (w/v) agar to prevent convective movements in the solutions (‘stagnant’ treatment) (Wiengweera, Greenway & Thomson 1997). This treatment may mimic the changes in gas composition found in waterlogged soils (decreased O2, increased ethylene) better than other methods used to impose root-zone O2 deficiency in solution culture such as N2-flushed systems (Wiengweera et al. 1997). The sink for O2 loss from the roots to the agar would have been lower than that found in many waterlogged soils (which can become highly reduced); nevertheless, root growth into the deoxygenated stagnant agar would be dependant upon O2 diffusion within the roots. Solutions in the aerated pots were renewed and all solutions were then renewed every 7 d. In the middle of each 7 d period, the stagnant pots were flushed with N2 gas for 10–20 s to ensure that the 0·1% agar remained suspended and that the development of boundary layers adjacent to the roots was disrupted. Oxygen concentration in the bulk solution in the stagnant pots never rose above 0·003 mol m−3, whereas in aerated pots it averaged 0·28 mol m−3 (portable Syland O2 and temperature meter Model 610; Syland Scientific, Heppenheim, Germany). Oxygen concentration at the surface of the roots was presumably greater than that in the bulk solution, since ROL would have resulted in an aerated ‘rhizosphere’ (Wiengweera et al. 1997), even though microorganisms would have consumed a proportion of the O2 lost to the root medium. Pots were arranged in a randomized complete-block design with three replicates. Treatments were maintained for 21 d.

An initial harvest of one plant from each pot was taken at the time the treatments were imposed. Shoot and root dry mass were measured and plant developmental stages recorded. The respective shoot and root dry mass (mg) of each species at the onset of treatments were: T. aestivum 114  ±  9, 36  ±  4; H. vulgare 78  ±  7, 21  ±  4; S. cereale 88  ±  5, 20  ±  4; L. turcicuma 34  ±  2, 11  ±  2; L. turcicumb29 ± 2, 10 ± 1; E. scabrus 23 ± 2, 7 ± 1; L. elongatum 14 ± 1, 6 ± 0; C. marinum 32 ± 4, 11 ± 1; Pa. smithii 33 ± 3, 11 ± 1; Ps. juncea 16 ± 1, 6 ± 1.

Following the 21 d of treatment, the number of tillers and adventitious roots, the length of the longest adventitious root, and dry mass of shoots, seminal and adventitious roots were measured on two plants in each pot, with the mean value for these two plants forming a single replicate for statistical analyses. The relative growth rate (RGR) was calculated from the initial and final harvests on a dry-mass basis. In a separate experiment, maximum length of adventitious roots of plants of each species growing in stagnant deoxygenated solution was determined by taking weekly measurements of root length until no further increase in length of the longest adventitious root was found for two consecutive weeks. In this second experiment, plants were grown as described above and the stagnant treatments lasted 10 weeks.

Root porosity

Porosity (% gas volume root volume−1) was measured for adventitious and seminal roots of plants grown in aerated or stagnant nutrient solutions using the method of Raskin (1983) with equations as modified by Thomson et al. (1990). The root systems of two plants from each pot were separated into adventitious and seminal roots, cut into 40–50 mm segments, and a subsample of approximately 2 g fresh mass was used for the measurements.

ROL from intact adventitious roots in an O2-free medium

Plants grown in treatment solutions for 21–30 d were sealed into rubber lids fitted on 100 mm × 100 mm × 170 mm Perspex chambers filled with an O2-free solution containing 0·1% (w/v) agar and (mol m−3) Ca2+, SO42– (0·5) and K+, Cl (5). The intact root system was immersed in the O2-free solution while the shoot system remained in air. A cylindrical platinum electrode (height 5 mm; internal diameter 2·25 mm) (Armstrong 1994) was placed around a selected adventitious root with a length of 100–120 mm. The flux of O2 from the root to the electrode was measured (Armstrong & Wright 1975) with the centre of the electrode positioned either 80 mm or 5 mm behind the apex. The first measurements were taken at least 2 h after the plants were transferred into the system. All measurements were taken at 20 °C in a temperature-controlled room with a photon flux density at shoot height of 100 µmol m−2 s−1.

More detailed experiments on H. vulgare and C. marinum

Results from the experiments described above showed large differences between H. vulgare and C. marinum in characteristics that contribute to internal O2 movement in roots, and thus the maximum penetration of the roots into the stagnant deoxygenated nutrient solution. Therefore, further experimentation on these two species (same cultivar and accession as used above) was carried out.

Solution culture experiment

Plants of H. vulgare and C. marinum were grown in solution culture as described above; however, the root-zone O2 treatments were maintained for 35 d. At the end of the treatment period, the plants were transferred to the 20 °C room and ROL measurements (described above) were taken at 80, 60, 40, 20 and 5 mm behind the apex of 115–130-mm-long adventitious roots. Hand cross-sections were taken at 10 mm below the root–shoot junction and 50 mm behind the apex of 110–150-mm-long adventitious roots. One root from a plant in each pot was used as a replicate (i.e. three roots from three separate plants in three separate pots). The sections were photographed using a photo-microscope and the area of aerenchyma within the sections analysed using image analysis software (public domain NIH image program Version 1·61, US National Institutes of Health, Bethesda, MD, USA).

Soil experiment

Seeds of H. vulgare and C. marinum were washed in 0·4% NaHClO3 solution for 45 s and rinsed thoroughly with deionized water before being germinated in darkness on filter paper soaked with 0·5 mol m−3 CaSO4 solution for 24 h in a 20/15 °C (day/night) phytotron. Echinochloa crus-galli L. (P. Beauv.) var. mitis (Accession 108990, Australian Tropical Forages Genetic Resource Centre), was also included in this experiment to provide a waterlogging-tolerant ‘check’ species with which to compare the responses of H. vulgare and C. marinum. Seeds of E. crus-galli were germinated on filter paper soaked with 0·5 mol m−3 CaSO4 and 0·1 mol m−3 KNO3 in a 30 °C room with 8 h light (150 µmol m−2 s−1)/16 h dark cycles for 24 h. Seeds of C. marinum were imbibed and then planted 3 d before H. vulgare and E. crus-galli so that plants were at a similar developmental stage at the onset of treatments. Three seeds of one species were planted per pot with nine pots of each species established. The pots (height 450 mm; diameter 150 mm) contained approximately 9 kg of Kojonup sand. Nutrients mixed through the soil prior to planting were (mg kg−1): NH4NO3 (357), KH2PO4 (60), K2SO4 (160), CaCl2·H2O (178), MgSO4·7H2O (22), ZnSO4 (11), MnSO4 (11), CuSO4·5H2O (6), H3BO3 (0·8), Na2MoO4·7H20 (0·2). This soil type and nutrient composition was used in earlier work on waterlogging tolerance in wheat (Thomson et al. 1992). Platinum probes, to measure soil redox potential, were installed 150 mm below the soil surface. Pots were in a 20/15 °C (day/night) phytotron and watered daily to field capacity.

At the 2·0–3·0-leaf stage (13 and 10 d after imbibition for C. marinum and H. vulgare, and E. crus-galli, respectively), an initial harvest of three pots of each species was taken. The respective shoot and root dry mass at the onset of treatments were (mg): H. vulgare 32 ± 6, 17 ± 1; C. marinum 14 ± 5, 5 ± 1; E. crus-galli 3 ± 0, 2 ± 0. Waterlogging was imposed in three of the six remaining pots of each species by partially submerging them in tanks of deionized water, so that water entered the bottom of the pot and rose to the soil surface. A transparent plastic tube, attached to the drainage hole and placed vertically along the side of each pot, prevented water drainage and allowed monitoring of the water level within the soil column. During the treatment period, the waterlogging was 0–3 mm above the soil surface. Controls, in freely draining pots, were watered to field capacity each day. Pots were arranged in a completely randomized design with three replicates. The treatments were maintained for 21 d. Soil redox potential at the end of the treatment period was, on average, + 50 mV in waterlogged soil and + 450 mV in drained soil.

After 21 d of treatment, plants were carefully washed free of soil and the roots placed in a container of 0·5 mol m−3 CaSO4. One plant from each pot was used for measuring ROL from intact adventitious roots (as described above). Plants were transferred into the O2-free medium used for the ROL measurements, a maximum of 20 min after being washed from the soil, and measurements commenced after a 2 h ‘equilibration’ period. Measure-ments for H. vulgare (drained soil only), C. marinum and E. crus-galli were taken at 80, 60, 40, 20 and 5 mm behind the apex of 100–140 mm roots. As a result of the short length and many laterals on adventitious roots of H. vulgare grown in waterlogged soil, ROL measurements for previously waterlogged plants of this species were taken at 70, 60, 40, 20 and 5 mm behind the apex of 80–100 mm roots.

The number of tillers and adventitious roots, length of the longest adventitious roots and dry mass of shoots, adventitious and seminal roots were recorded for the other two plants from each pot. Shoot RGR between the initial and final harvests was calculated on a dry-mass basis.

Statistical analyses

Evaluations of stress tolerance in plants are often based on measurements of the proportional reduction in growth under stressed (when compared to non-stressed) conditions. However, inherent traits (i.e. those in non-stressed conditions), such as small size and slower rate of development, may contribute to the perceived tolerance when assessments are based on proportional reductions. However, tolerance resulting from these traits is of no use in an agronomic context and may confound screening procedures based on phenotype. Studies to identify physiological traits may overcome some of the inherent limitations that exist in tolerance rankings based only on phenotype by identifying traits that enhance tolerance independent of habit (Yeo & Flowers 1986). In addition, statistical procedures that account for any inherent relationship between habit and response to stress may also facilitate comparisons of stress tolerance in diverse species. Differences in the growth habit among the ‘wild’ and cultivated species used in the present study may cause the reduction in response to stress to vary systematically between, for example, the large and small species. This problem can be overcome by calculating the line of best fit that relates growth in the presence of stress to that in the absence of stress (Beebe et al. 1997; Pagel & Harvey 1988). The deviation from this line is then taken as the measure of the performance of the species under stress. A positive deviation indicates that a species performed better than expected (i.e. was more tolerant than expected) under stress when compared to the overall relationship that exists between performance under stress and performance in the absence of stress within the data set of the species studied.

A regression line was fitted to the relationship between a variable (e.g. shoot dry mass) measured for plants in the stagnant treatment (used as the response variable) and the same variable measured for plants in the aerated treatment (used as the explanatory variable). If the line had a positive slope, this indicated that the pattern of variation over species was broadly similar in the two treatments. However, some species showed positive deviation from the regression line and some showed negative deviation. The deviation from the regression line was calculated separately for each replicate of the waterlogged treatment, and an anova was performed on the deviations from the regression lines to determine whether this variation among species was significant relative to the variation among observations in a single species. Mean deviations were compared by using the least significant difference (LSD) (P= 0·05). The aspects of root and shoot growth in the aerated solution and the percentage deviations from unity [(value in stagnant solution/value in aerated solution) − 1] were analysed using anova to examine the effect of species. Means were compared by using the LSD (P= 0·05).

The data on the length of adventitious roots were subjected to an anova to examine the effect of species on root length in aerated or stagnant solutions. The porosity data were subjected to an anova to examine the effects of species, root type, treatment and the interactions between the three. Data on ROL are presented throughout with standard errors as a measure of the variation. The cross-sectional area of aerenchyma in the adventitious roots of H. vulgare and C. marinum were analysed using a split-plot anova to examine the effect of species, treatment and position of the section along the root. In experimentation involving H. vulgare, C. marinum and E. crus-galli, an anova was used to analyse the effect of species, treatment and their interaction on aspects of shoot and root growth.

Results

Plant growth

The shoot, seminal root and adventitious root dry mass of the cereal species T. aestivum, H. vulgare and S. cereale in the aerated solution were around 3–30-fold greater than those of the other species (Table 2). Shoot RGR of H. vulgare was 1·5-fold greater than that of L. elongatum, but variation among species other than these two was less substantial. Reductions in the values achieved by the three cereal species when grown in the stagnant solution were 55–69% for shoot dry mass, 22–29% for shoot RGR, 93–96% for seminal root dry mass and 47–72% for adventitious root dry mass. Among the other species, the effect of growth in the stagnant solution ranged from a 6–54% decrease in shoot dry mass, 3–20% decrease in shoot RGR, 64–69% decrease in seminal root mass and from a 41% decrease to a 38% increase in adventitious root dry mass. When grown in the aerated solution, the numbers of stems and adventitious roots of the cereal species were around 1·5–6·2-fold greater than those of the other species, with the exception of C. marinum, which had more or no fewer stems than the cereal species and did not differ substantially from T. aestivum and S. cereale in number of adventitious roots (Table 3). Reductions in the number of stems among cereals grown in the stagnant solution ranged from 43 to 65%, and from 4 to 35% among the others. Reductions in the number of adventitious roots among cereals grown in the stagnant solution ranged from 0 to 32% and from a 19% decrease to a 41% increase among the others.

Table 2.  Aspects of shoot and root growth for a range of species from the tribe Triticeae after being raised to the 2·0–2·2-leaf stage in the aerated solution and exposed to aerated or stagnant deoxygenated nutrient solution for a further 21 d. Values in the aerated solution, percentage deviations from values in the aerated solution for plants raised in the stagnant solution (% Dev) and deviations from the regression line relating a variable measured in the stagnant solution (‘response’ variable) to the same variable measured in the aerated solution (‘explanatory’ variable) (Fig. 1) (Reg dev) are given. Values are the means of three replicates; each replicate is the mean of two plants from an individual pot. See Materials and methods for an explanation of the statistical analyses
SpeciesShoot dry mass (g)Shoot RGR (mg g−1d−1)Root dry mass (g)
SeminalAdventitious
Aerated% DevReg. devAerated% DevReg devAerated% DevReg devAerated% DevReg dev
  • ns

    not significant;

  • *

    P < 0·05;

  • **

    P < 0·01;

  • ***

    P < 0·001

T. aestivum2·570− 550·205178− 22− 6·80·295− 930·0050·420− 470·028
H. vulgare3·131− 67− 0·069206− 270·40·347− 930·0030·606− 580·018
S. cereale2·640− 69− 0·150192− 29− 13·20·374− 96− 0·0070·475− 72− 0·069
L. turcicuma0·700− 370·064175− 136·60·088− 890·0000·099140·029
L. turcicumb0·697− 320·079182− 1114·60·084− 860·0020·108− 10·021
E. scabrus0·364− 30− 0·026162− 110·40·025− 74− 0·0010·073− 26− 0·016
L. elongatum0·151− 6− 0·081141− 3− 5·30·017− 640·0000·02038− 0·030
C. marinum0·648− 260·103171− 910·20·022− 75− 0·0010·154160·072
Pa. smithii0·742− 54− 0·046175− 20− 6·70·047− 86− 0·0010·112− 38− 0·016
Ps. juncea0·346− 40− 0·081172− 15− 0·40·035− 810·0000·070− 41− 0·037
LSD0·327***29**0·230 ns18***11**14·8*0·109***15**0·007 ns0·112***47***0·045***
Table 3.  The number of stems and adventitious roots for a range of species from the tribe Tritceae after being raised to the 2·0–2·2-leaf stage in the aerated solution and exposed to aerated or stagnant deoxygenated nutrient solution for a further 21 d. Values in the aerated solution, percentage deviations from values in the aerated solution for plants raised in the stagnant solution (% Dev) and deviations from the regression line relating a variable measured in the stagnant treatment (‘response’ variable) to the same variable measured in the aerated treatment (‘explanatory’ variable) (Fig. 1) (Reg dev) are given. Values are the means of three replicates; each replicate is the mean of two plants from an individual pot. See Materials and methods for an explanation of the statistical analyses
SpeciesStem numberAdventitious root number
Aerated% DevReg devAerated% DevReg dev
  • **

    P < 0·01;

  • ***

    P < 0·001

T. aestivum11·2− 43− 0·3734·304·89
H. vulgare13·3− 65− 3·1451·2− 32− 6·63
S. cereale14·2− 55− 1·6731·0− 14− 0·83
L. turcicuma7·5− 290·2310·3410·98
L. turcicumb6·8− 230·3912·514− 0·50
E. scabrus7·3− 61·88·827− 0·83
L. elongatum3·8− 40·168·318− 1·99
C. marinum15·8− 183·6727·23510·79
Pa. smithii6·7− 35− 0·4014·83− 1·60
Ps. juncea5·0− 31− 0·6812·2− 19− 4·28
LSD2·5***26**2·08***6·2***37**3·69***

Regression of each of the growth variables for plants in the stagnant solution on the same variable for control plants in the aerated solution produced significant relationships, with the exception on shoot RGR. For the other five parameters measured, the slope was less than one and the intercept was positive: that is, the species proportionately less affected by growth in the stagnant solution were those with few stems (tillers + the mainstem) and adventitious roots, and lower total dry mass of shoot, adventitious and seminal roots (data for shoot mass, tiller number and seminal root mass in Fig. 1, data for other parameters not shown).

Figure 1.

The regression relationships of selected aspects of growth in a range of species from the tribe Triticeae when grown in stagnant deoxygenated nutrient solution against the same variable when grown in aerated nutrient solution. Plants were raised in the aerated solution until the 2·0–2·2-leaf stage and then exposed to treatments for the final 21 d. Hordeum vulgare is represented by the letter H and C. marinum by the letter C. Each data point is the mean of three replicates (with the mean of two plants from an individual pot per replicate) and the regression analyses were carried out as described in Materials and methods. Regression equations (values in parentheses are standard errors of the preceding coefficients, P-value is for the null hypothesis that the slope is zero) are: (a) shoot dry mass (g) y = 0·297 (± 0·034)x + 0·170 (± 0·055) P < 0·001, r2 = 0·90; (b) stem number y = 0·44 (± 0·16)x + 1·83 (± 1·57) P = 0·025, r2 = 0·49; (c) seminal root dry mass (g) y = 0·038 (± 0·007)x + 0·006 (± 0·001) P < 0·001, r2 = 0·77. Regression relationships for all the other aspects of growth and development measured are not shown for reasons of brevity

None of the species showed significant deviation from the regression line for shoot dry mass or seminal root dry mass (i.e. none of the species grew better than would have been expected given the relationship that existed between the value of a variable in the aerated solution and that in the stagnant solution) (Table2). However, both L. turcicumb and C. marinum displayed positive deviation from the regression line for shoot RGR. Critesion marinum also displayed greater positive deviation from the regression line than most, or all, of the other species for stem number, adventitious root number and adventitious root dry mass (Tables2 and 3).

Root porosity of plants grown in aerated or stagnant nutrient solutions

There was no difference in the porosity of the seminal roots among the species when grown in the aerated nutrient solution (Table4). The seminal root porosity of L. turcicum, L. elongatum, C. marinum and Ps. juncea was 1·8–3·3-fold greater in stagnant than in aerated solution, whereas in the other species, seminal root porosity did not differ between the two treatments. The adventitious root porosity of all the species was 1·8–4·6-fold greater for plants grown in stagnant compared to aerated solution. Critesion marinum had a 1·8–5·4-fold higher adventitious root porosity than any other species when plants were grown in the aerated solution and 1·2–2·8-fold higher when grown in the stagnant solution. Triticum aestivum had the second-highest adventitious root porosity of all the species when grown in the stagnant solution; T. aestivum and C. marinum were the only two species in which porosity exceeded 20%(Table4).

Table 4.  Porosity of the seminal and adventitious roots of a range of species from the tribe Triticeae when grown in aerated or stagnant deoxygenated nutrient solution for the final 21 d. Plants were grown to the 2·0–2·2-leaf stage in the aerated solutions prior to the onset of treatments. Values are the means of three replicates; each replicate is the mean of two plants from an individual pot. The LSD is for genotype × aeration treatment × root type combination
SpeciesPorosity (% gas volume/root volume)
Seminal rootsAdventitious roots
AeratedStagnantAeratedStagnant
  • ***

    P < 0·001

T. aestivum2·092·675·7521·62
H. vulgare2·514·496·7115·84
S. cereale1·461·362·649·01
L. turcicuma2·724·976·9315·67
L. turcicumb2·795·785·0116·07
E. scabrus0·861·302·8713·15
L. elongatum3·057·677·6413·47
C. marinum3·887·0413·8325·44
Pa. smithii2·373·567·4816·47
Ps. juncea1·474·852·5511·77
LSD   2·86***

Length of adventitious roots of plants grown in aerated or stagnant nutrient solutions

The three cereal species, T. aestivum, H. vulgare and S. cereale, had the longest adventitious roots of the species studied when grown continuously in aerated nutrient solution: they were 220–530 mm longer than the roots of the other species (Table5). However, when grown in the stagnant solution for the final 21 d, C. marinum had the longest adventitious roots of all nine species examined (Table5). Longer treatment periods imposed in a second experiment enabled the maximum length of adventitious roots when dependent on internal O2 movement to supply the apex to be determined. Under these conditions, adventitious roots of C. marinum were about 90 mm longer than those of L. turcicumb, which formed the next-longest roots, and S. cereale had the shortest roots at only 33% of the length of the roots of C. marinum. The longest adventitious roots of H. vulgare and T. aestivum were, respectively, only 49 and 59% of the length of those of C. marinum.

Table 5.  The length of the longest adventitious roots of a range of species from the tribe Triticeae after being grown for the final 21 d in aerated or stagnant deoxygenated nutrient solution. Plants were raised to the 2·0–2·2-leaf stage in the aerated solution prior to the onset of treatments. The maximum root length in stagnant deoxygenated nutrient solution is the root length beyond which no further root elongation was observed for a period of 14 d (determined in a separate experiment). Values are the means of three replicates; each replicate is the mean of two plants from an individual pot. The LSDs refer to differences within the corresponding column
SpeciesAdventitious root length (mm)
AeratedStagnantStagnant (max.)
  • ***

    P < 0·001

T. aestivum684184192
H. vulgare652164160
S. cereale65597108
L. turcicuma275167225
L. turcicumb268166237
E. scabrus265115126
L. elongatum155124215
C. marinum402271325
Pa. smithii459155198
Ps. juncea312105135
LSD92***15***27***

ROL from intact adventitious roots in an O2-free root medium

Rates of ROL from any given position along a root in an O2-free root environment partially reflect two factors: (i) the O2 concentration within the root (determined by the amount of aerenchyma, distance from the root–shoot junction, rate of O2 consumption), and (ii) the resistance to O2 movement from the aerenchyma and through the cell layers near the root exterior to the root surface. For intact adventitious roots of plants raised in the aerated solution, rates of ROL at 80 mm behind the apex were 10–150-fold faster than those at 5 mm behind the apex (Table6). For plants raised in the stagnant solution, rates of ROL at 80 mm behind the apex were faster than, or equal to, those at 5 mm behind the apex for all species with the exception of C. marinum. ROL from the adventitious roots of C. marinum was higher at a location close to the apex than in the more basal position (Table6). Thus, C. marinum was the only species tested in which the basal zones of adventitious roots had a strong barrier to ROL when plants were grown in the stagnant solution.

Table 6.  Rates of radial O2 loss at 80 mm and 5 mm behind the apex of 100–120 mm adventitious roots from intact plants of a range of species from the tribe Triticeae. Plants were raised to the 2·0–2·2-leaf stage in the aerated solution and then grown in aerated or stagnant deoxygenated nutrient solution for the final 21 d prior to being transferred to an O2-free root medium for the measurements, taken at 20 °C. Values are the means of three replicates ± SE
SpeciesRadial O2 loss (nmol m−2 s−1)
80 mm behind root apex5 mm behind root apex
AeratedStagnantAeratedStagnant
T. aestivum135·7 ± 17·1179·1 ± 28·01·0 ± 1·081·3 ± 22·3
H. vulgare102·2 ± 25·9217·8 ± 49·60·0 ± 0·0103·8 ± 44·9
S. cereale52·8 ± 48·1101·3 ± 40·44·8 ± 0·8103·7 ± 23·8
L. turcicuma207·5 ± 29·7206·6 ± 39·73·6 ± 2·9168·8 ± 6·7
L. turcicumb241·6 ± 37·1198·8 ± 33·18·5 ± 6·2115·0 ± 69·1
E. scabrus184·6 ± 12·7207·1 ± 105·11·3 ± 0·7101·9 ± 55·5
L. elongatum465·1 ± 48·3219·7 ± 62·325·2 ± 19·7246·3 ± 52·5
C. marinum329·3 ± 47·924·2 ± 22·133·9 ± 13·5272·7 ± 56·3
Pa. smithii305·1 ± 23·3221·4 ± 62·118·2 ± 8·9229·2 ± 12·4
Ps. juncea188·0 ± 83·4167·4 ± 59·72·8 ± 3·8111·6 ± 40·6

A more detailed spatial analysis of ROL from the adventitious roots of H. vulgare and C. marinum was undertaken in a separate experiment. ROL from adventitious roots of H. vulgare grown in the aerated solution was substantial only at 80 mm behind the apex, decreasing to values close to zero near the apex (Fig.2). Critesion marinum had substantial, although highly variable, rates of ROL from the basal regions of adventitious roots of plants raised in the aerated solution, decreasing to values close to zero at 5 mm behind the apex. Hordeum vulgare grown in the stagnant solution prior to the measurements had substantial ROL along the entire root length. In contrast, adventitious roots of C. marinum raised in the stagnant solution had high rates of ROL close to the apex but very low rates at more basal positions (Fig.2). These data confirmed that C. marinum had a strong barrier to ROL induced in adventitious roots of plants grown in the stagnant solution.

Figure 2.

Rates of radial O2 loss at selected positions behind the apex of intact adventitious roots of C. marinum, H. vulgare and E. crus-galli in an O2-free medium. Plants in the solution culture experiment (‘solution’) were raised until the 2·0–2·2 leaf stage in aerated conditions, and then either in aerated (bsl00000) or stagnant (▪) deoxygenated nutrient solution for the final 35 d. Echinochloa crus-galli was not included in the solution culture experiment. Measurements were taken for roots 115–130 mm in length. Plants in the soil experiment (‘soil’) were raised in drained conditions until the 2·0–3·0-leaf stage, and then either in drained (○) or waterlogged (●) soil for the final 21 d. Measurements were taken for roots 100–140 mm in length, except roots of waterlogged H. vulgare, which were only 80–100 mm in length. Measurements were taken at 20 °C. Values given are the means of three replicates ± SE

Cross-sectional area of aerenchyma in adventitious roots of H. vulgare and C. marinum grown in aerated or stagnant nutrient solutions

The proportional cross-sectional areas of aerenchyma in adventitious roots of H. vulgare grown in the aerated solution were 0·6 and 0% at 10 mm below the root–shoot junction and 50 mm behind the apex, respectively. In contrast, aerenchyma occupied 20·2 and 4·0% at the same respective positions in adventitious roots of C. marinum grown in the aerated solution. When grown in the stagnant solution, the area of aerenchyma in adventitious roots of H. vulgare increased to 33·4 and 24·3%, and in C. marinum to 30·4 and 31·3% at the same respective positions (P < 0·001; LSD 7·1). The cells in the root cortex of both species were arranged in a hexagonal fashion.

Growth of H. vulgare, C. marinum and E. crus-galli in pots of drained or waterlogged soil

Shoot RGR and final dry mass in E. crus-galli were not affected by 21 d of soil waterlogging, whereas waterlogging reduced the shoot RGR of C. marinum by 12% and that of H. vulgare by 43%(Table7). The final shoot dry mass of C. marinum and H. vulgare decreased by 38 and 81%, respectively, in response to soil waterlogging. Critesion marinum had more stems than the other two species, and soil waterlogging decreased the stem number of C. marinum and H. vulgare by 44 and 86%, respectively, whereas stem number in E. crus-galli was not affected.

Table 7.  Aspects of growth and development of H. vulgare, C. marinum and E. crus-galli grown in drained soil to the 2·0–3·0-leaf stage and then in either drained or waterlogged soil for the final 21 d. Values are the means of three replicates; each replicate is the mean of two plants from an individual pot. The LSDs refer to the species × treatment combination
SpeciesShoot RGR
(mg g−1 d−1)
Shoot dry
mass (g)
Stem
number
Seminal root
dry mass (g)
Adventitious rootMaximum
length (mm)
Dry
mass (g)
Number
  • *

    P < 0·05;

  • **

    P < 0·01;

  • ***

    P < 0·001

Drained
H. vulgare1741·4810·80·3210·2124·5445
C. marinum1880·8918·80·0760·1827·0493
E. crus-galli2270·464·20·0520·069·5366
Waterlogged
H.vulgare1000·291·50·0200·0716·0127
C. marinum1650·5510·50·0200·1532·0210
E. crus-galli2230·433·80·0230·1415·0237
LSD12***0·22***2·6***0·020***0·07**7·5*49***

Hordeum vulgare produced a 4·2–6·2-fold greater seminal root dry mass than the other two species when grown in drained soil, whereas in waterlogged soil there was no significant difference in the seminal root dry mass among the three species (Table7). The adventitious root dry mass of E. crus-galli was around 70% of that of C. marinum or H. vulgare in drained soil. Growth in waterlogged soil caused no change in the adventitious root dry mass of C. marinum, a 3-fold decrease in H. vulgare and a 2·3-fold increase in E. crus-galli. The number of adventitious roots increased (although not significantly) in response to soil waterlogging in C. marinum and E. crus-galli, whereas it decreased in H. vulgare. The length of the longest adventitious roots of all species was shorter for plants grown in waterlogged soil than for those raised in drained soil, but the longest root lengths of C. marinum and E. crus-galli in waterlogged soil were almost double those of H. vulgare.

ROL from roots of H. vulgare, C. marinum and E. crus-galli grown in drained or waterlogged soil prior to the measurements

ROL from adventitious roots of C. marinum grown in either drained or waterlogged soil was slower at the two most basal positions tested than at the more apical positions (Fig.2). In contrast, ROL from adventitious roots of H. vulgare grown in waterlogged soil was substantial at all positions tested, with little difference between the basal and apical positions tested along the root. For H. vulgare raised in drained soil, the rates of ROL were substantially less than those from roots of plants raised in waterlogged soil; ROL from the roots of drained plants decreased to very low rates towards the apex. Echinochloa crus-galli had similar profiles of ROL along adventitious roots of plants raised in either drained or waterlogged soil: ROL from the basal regions was much less than that from the apical regions.

Discussion

Nine species from the tribe Triticeae (three considered to be from wet- and six from dry-land habitats) were evaluated for root porosity and spatial patterns of ROL from adventitious roots. Of these, C. marinum[an adventive (weedy) species common to saline meadows and marshes (von Bothmer et al. 1991)] was the only species with a relatively high porosity and a strong barrier to ROL in the basal root zones. These two traits also occur in the waterlogging-tolerant O. sativa (Armstrong 1971; Colmer et al. 1998), P. australis (Armstrong & Armstrong 1988; Jackson & Armstrong 1999) and E. crus-galli (this study). The combination of high porosity and a barrier to ROL enhance the longitudinal movement of O2 within the root towards the apex, thus increasing root penetration into anaerobic soils (Armstrong 1979). The presence of these traits associated with waterlogging tolerance in C. marinum are highly encouraging with respect to the prospect of enhancing these traits in cereal crops, since wide-crosses between hexaploid wheat and C. marinum are possible (Jiang & Dajun 1987).

Comparisons of the growth of C. marinum and H. vulgare in drained and waterlogged soil confirmed the superior tolerance of C. marinum to soil waterlogging. Shoot RGR and tiller production were maintained at higher levels in C. marinum than in H. vulgare(Table7). However, soil waterlogging still reduced tiller production by 50% in C. marinum. Tillering is a component of growth that is severely affected by waterlogging in sensitive cereals (Musgrave 1994; Taeb et al. 1993; Cannell et al. 1984). The reductions in shoot growth in H. vulgare and C. marinum contrasted with the small impact of soil waterlogging on E. crus-galli when grown as a waterlogging-tolerant check species in the same experiment (Table7). However, E. crus-galli produced 4–5 times fewer tillers in drained soil than C. marinum, and this may have contributed to the differences in proportionate responses to waterlogging of this parameter in the two species. Given the high porosity, barrier to ROL and extensive adventitious root deve-lopment in C. marinum, reductions in its shoot growth in response to stagnant deoxygenated nutrient solution and soil waterlogging presumably result from inadequacies in other traits.

Growth in stagnant deoxygenated solution has been shown to induce a barrier to ROL in three genotypes of O. sativa (Colmer et al. 1998). Decreases in ROL from the basal zones of adventitious roots of C. marinum when grown in the stagnant solution were found in the present study (Table6; Fig. 2). However, when C. marinum was grown in drained or waterlogged soil, the barrier to ROL in the basal regions of the adventitious roots was present in plants from both treatments. Whilst the induction of the barrier to ROL may be of adaptive value (Colmer et al. 1998), a study of a wider variety of species, grown in a range of soil types and moisture contents (waterlogged to water-deficit), is needed before the adaptive significance of changes in radial permeability to O2– and perhaps other substances – can be fully evaluated. Moreover, although barriers to ROL have been documented in several wetland species (Visser et al. 2000; Armstrong 1964), knowledge on the anatomical basis (and chemical composition) of such barriers is scant. Lignification of the epidermal–hypodermal cylinder may be the anatomical basis of the barrier to ROL in the adventitious roots of P. australis (Armstrong et al. 2000). In addition, suberin deposits in walls of cells near the root exterior (Armstrong & Armstrong 1988) and densely packed hexagonal cell arrangements of the epidermal–hypodermal cylinder giving low tissue porosity (Connell, Colmer & Walker 1999) have been suggested to contribute to the ‘barrier’ to ROL in some species. Additional studies are required to identify the mechanisms resulting in low rates of ROL from the basal root zones of C. marinum and other species.

The porosity of the adventitious roots of C. marinum was 1·2–2·8-fold greater than that of the eight other species of the Triticeae evaluated in the present study. Nevertheless, the porosity of the roots of C. marinum was only moderate (aerated 14%, stagnant 25%) in comparison to values in other wetland grass species such as O. sativa (Armstrong 1971; Colmer et al. 1998), Glyceria maxima (Smirnoff & Crawford 1983), Brachiaria mutica and Echinochloa polystachya (Baruch & Merida 1995), which typically range from 20–25% under O2-sufficient conditions to 40–50% in O2-deficient conditions. The similarity in porosity between C. marinum and T. aestivum yet far superior adventitious root lengths of the former when grown in stagnant deoxygenated nutrient solution illus-trates that the enhanced internal aeration, and thus root penetration, of C. marinum results from a combination of traits (e.g. porosity, a barrier to ROL, and possibly others). Armstrong (1979) has used mathematical models to assess the influence of porosity, barriers to ROL and root tissue O2 consumption rates (the latter was not determined in the present work) on maximum length of roots solely dependent on an internal supply of O2 for the apex. The models show the importance of having a favourable combination of the above factors for root penetration into anaerobic substrates.

An eco-geographical survey of the genus Hordeum revealed that 25 of the 32 species were known to occupy areas likely to experience episodes of waterlogging (coastal, saline or freshwater marshes; along ditches; in saline and freshwater meadows; on the shores of lakes and ponds) (von Bothmer et al. 1991). Thus other species from the Hordeum–Critestion complex may possess similar traits associated with waterlogging tolerance as described here for C. marinum, and some may be even better adapted to waterlogged conditions than the accession of C. marinum used in the present study. On this basis, further investigation of species within the Hordeum–Critesion complex for traits associated with waterlogging tolerance is warranted.

Acknowledgments

The Grains Research and Development Corporation is acknowledged for financial support of this project and a student scholarship for M.P.M. We thank the various sources of germplasm used in this study.

Received 30 October 2000;received inrevised form 28 January 2001;accepted for publication 20 February 2001

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