Planta (1990)180:590-597
P l a n t a 9 Springer-Verlag1990
Partial characterization of the trait for enhanced K +-Na + discrimination in the D genome of wheat J. Gorham, R.G. Wyn Jones, and A. Bristol School of Biological Sciences, University College of North Wales, Bangor, Gwynedd, LL57 2UW, UK
Abstract. The long arm of chromosome 4D of wheat (Tritieum aestivum L.) contains a gene (or genes) which influences the ability of wheat plants to discriminate between N a + and K +. This discrimination most obviously affects transport from the roots to the shoots, in which less N a + and m o r e K + accumulate in those plants which contain the long arm of chromosome 4D. Concentrations o f N a + and K + in the roots, and C1- concentrations in the roots and shoots, are not significantly affected by this trait, but N a +, K + and C1- contents of the grain are reduced. The trait operates over a wide range o f salinities and appears to be constitutive. At the m o m e n t it is not possible to determine accurately the effect of this trait on growth or grain yield because the aneuploid lines which are available are much less vigorous and less fertile than their euploid parents.
Key words: Ion transport - Salinity (ion uptake) - Salt tolerance (genetic trait) - Triticum (salt tolerance)
chromosomes (from the hexaploid wheat Chinese Spring) had been substituted for their homoeologues in the A and B genomes of the tetraploid wheat cultivar L a n g d o n (Joppa 1987; Joppa and Williams 1988). The use of ditelosomic lines of the hexaploid wheat Chinese Spring enabled us to confirm that the enhanced trait for K + - N a + discrimination was located on chromosome 4D, and specifically on the long arm of this chromosome. This paper presents a more detailed examination of the effects of D-genome chromosomes on the salt tolerance and ion-uptake patterns in wheat, and brings together new ion-uptake and other data with some results which have been presented as parts o f contributions to a number of recent conferences (e.g. Wyn Jones and G o r h a m 1989). We suggest a possible site for the discrimination between N a + and K + and discuss the practical exploitation of this trait in improving the overall salt tolerance of tetraploid wheat.
Material and methods Introduction A number of studies have demonstrated that hexaploid bread wheat (Triticum aestivum L., genome B B A A D D ) is more salt-tolerant (in the broadest sense) than tetraploid (T. turgidum L., genome BBAA), or diploid wheats (T. monococcum L., genome AA) (Rana et al. 1980; Joshi et al. 1982; Francois etal. 1986; Weimberg 1987, 1988; but see also Sayed 1985). Our own studies have shown that the D genome (from Aegilops squarrosa L., genome D D ) contains a trait for enhanced K + - N a + discrimination, and that this trait is expressed in synthetic hexaploid wheats derived from hybrids between tetraploid wheat and Ae. squarrosa (Shah et al. 1987). We have been able to demonstrate that this trait is located on chromosome 4D ( G o r h a m et al. 1987) by examining Na + and K + concentrations in leaves of salt-treated plants of disomic substitution lines in which D-genome
Growth of plants. Triticum turgidum cv. Langdon, Triticum aestivum cv. Chinese Spring and synthetic wheats and their various aneuploid lines were obtained from Dr. L.R. Joppa (USDA-ARS, Fargo, N.D., USA) and Dr. C. Law (FPSR, Cambridge, UK) and grown in saline hydroponic culture as described by Gotham et al. (/987). The concentrations of the major nutrients were as follows: K +, 6.8mol-m-3; NO~-, 8.4mol-m-3; PO2, 1.7mol-m-3; C a 2+, 0.73mol-m-3; Mg2+, 0.92molm-3; Na +, 0.3molm -3. Salt (NaCI+CaC12 to give an Na+/Ca 2+ ratio of 20:1) was added to the culture solution at a rate of 25 mol.m 3.d-1 to give the final amounts stated in the legends. Production of the Langdon (tetraploid wheat) disomic substitution lines, which incorporate Dgenome chromosomes from Chinese Spring hexaploid wheat, has been described by Joppa (1987). The presence of monosomic or telosomic representatives of the substituted A- or B-genome chromosomes is indicated in Table 1. For experiments with low-salt-grown seedlings, grain was germinated in 0.5 mol-m 3 C a S O 4 alone in a growth cabinet maintained at 22~ C with 16 h of light daily supplied by a mixture of warm-white fluorescent tubes and tungsten light bulbs at 300 gmol photons, m- 2. s- 1 of photosynthetically active radiation (PAR).
J. Gorham et al. : K+-Na § discrimination in wheat Two days after germination the solution was replaced with a mixture containing 2 m o l . m -3 Ca(NO3)2 and 1 mol.m 3 MgSO4, and the seedlings grown for a further 5 d prior to use in 22Nauptake experiments.
Sap extraction and analysis. Leaf saps were extracted by a freezethaw technique followed by mechanical disruption and centrifngation (Gorham et al., 1987). Inorganic-ion concentrations in the expressed saps were determined by ion chromatography, also as previously described. Roots were first washed for 10 min in 20 mol. m - 3 Ca-acetate solutions which were adjusted to the same osmotic pressures as the culture solutions by the addition of sorbitol, and then blotted with tissue paper to remove surface moisture. Measurement of photosynthesis and transpiration. Measurements of carbon-dioxide exchange rates (CER) and transpiration rates (E) were made in situ on plants growing in hydroponic culture in the greenhouse using an LCA-2 infra-red gas analyser, a DL1 data logger, an ASU/MF air supply unit (taking air from 3 m above the greenhouse) and a Parkinson narrow leaf chamber, all supplied by the Analytical Development Co., Hoddesdon, Hefts., UK. Measurements were made in bright sunlight ( > 1 mol photons.m 2. s- 1 PAR) and were taken within 30 s of applying the leaf chamber (i.e. before the start of stomatal closure). Studies of 22Naflux. Measurements of 22Na fluxes into intact, lowsalt-grown wheat seedlings were made essentially as described by Jeschke and Jambor (1981). The experimental vessels were 20-cm 2 plastic syringe barrels fitted with plastic taps. 22Na was measured in a well-type gamma counter. To concentrate the 22Na in efflux solutions an excess of Dowex 50 (H + form) ion-exchange resin was shaken with the solution in a test tube. Most of the activity was then found to be in the bottom 1-2 cm 3 of the tube and could be positioned inside the well of the counter. Statistical analysis. All results are expressed as means + standard errors. Means were compared using the t-test facility of the STATPACK (University of York, UK) statistical package.
Results Photosynthesis, transpiration and inorganic-ion accumulation in Langdon disomic substitution lines. C o n c e n t r a t i o n s
600 - -
591 o f i n o r g a n i c c a t i o n s in the s a p f r o m the y o u n g e s t fullye x p a n d e d leaves o f L a n g d o n , Aegilops squarrosa R L 5003, the s y n t h e t i c h e x a p l o i d ( L a n g d o n / R L 5003) a n d the full set o f D - g e n o m e d i s o m i c s u b s t i t u t i o n lines g r o w n for 21 d in the p r e s e n c e o f 1 5 0 m o l . m - 3 NaC1 a n d 7.5 m o l . m - 3 CaC12 a r e s h o w n in Fig. 1. T h e e q u i v a l e n t d a t a for i n o r g a n i c a n i o n s are p r e s e n t e d in Fig. 2. C o n c e n t r a t i o n s o f N a § were l o w e s t in the full s y n t h e t i c hexap l o i d a n d in the s u b s t i t u t i o n lines i n v o l v i n g c h r o m o s o m e 4 D (i.e. the 4 D ( 4 A ) a n d 4D(4B) s u b s t i t u t i o n lines). In a g r e e m e n t w i t h p r e v i o u s o b s e r v a t i o n s on s y n t h e t i c hexap l o i d s w h e a t s ( S h a h et al. 1987) all three lines h a d l o w e r N a + c o n c e n t r a t i o n s t h a n Ae. squarrosa. C o n c e n t r a t i o n s o f K + were h i g h e r in the synthetic, Ae. squarrosa a n d the t w o 4 D s u b s t i t u t i o n lines t h a n in L a n g d o n o r a n y o f the o t h e r s u b s t i t u t i o n lines. T h e 2D(2B) s u b s t i t u t i o n line h a d significantly ( 0 . 1 % ) h i g h e r l e a f N § c o n c e n t r a tions t h a n L a n g d o n . C o n c e n t r a t i o n s o f C a 2 § a n d M g 2§ in l e a f s a p were n o t s i g n i f i c a n t l y d i f f e r e n t a m o n g the v a r i o u s lines (Fig. 1), b e i n g r e d u c e d b y salinity in all cases ( d a t a n o t shown). C h l o r i d e c o n c e n t r a t i o n s in the leaves were n o t g r e a t ly affected b y the s u b s t i t u t i o n o f c h r o m o s o m e 4 D for 4 A o r 4B (Fig. 2), b u t were slightly l o w e r in these two lines, a n d in Ae. squarrosa a n d the s y n t h e t i c h e x a p l o i d , t h a n in L a n g d o n . T h e 2D(2B), 3D(3B) a n d 7D(7B) L a n g d o n s u b s t i t u t i o n lines h a d l e a f C1- c o n c e n t r a t i o n s considerably higher than those of Langdon. Leaf NO~ c o n c e n t r a t i o n s were h i g h e r in Ae. squarrosa, the s y n t h e t ic h e x a p l o i d , a n d the 4 D ( 4 B ) a n d I D ( 1 A ) s u b s t i t u t i o n lines t h a n in L a n g d o n , b u t l o w e r in m o s t o f the o t h e r s u b s t i t u t i o n lines. A clear effect o f c h r o m o s o m e 5 D c a n be seen in the l e a f S O ] - c o n c e n t r a t i o n s . T h e s e were h i g h e r in the 5 D ( 5 A ) a n d 5D(5B) s u b s t i t u t i o n lines t h a n in L a n g d o n o r the o t h e r s u b s t i t u t i o n lines, w h e t h e r the p l a n t s were g r o w n in the presence (Fig. 2) o r a b s e n c e ( d a t a n o t s h o w n ) o f salt. E l e v a t e d S O l - c o n c e n t r a t i o n s were n o t f o u n d in Ae. squarrosa o r the s y n t h e t i c hexa-
iiiiiLiiiiiiii N a +
500
400
300 Fig. 1. Inorganic cations (mol-m-3 expressed sap) in leaves of wheats grown for 21 d in nutrient solution plus 150 mol. m 3 NaC1 (+7.5 mol-m -3 CaCI2). LANG = Langdon tetraploid wheat; AE.SQ. = Aegilops squarrosa; SYN= synthetic hexaploid wheat; 1D (IA) etc. = Langdon disomic substitution lines
200 100
ID 2D 3D 4D 5D 6D 7D 1D 2D 3D 4D 5D .6D 7D LANG AE. SYN (IA) (2A) (3A) (4A) (5A) (6A) (7A) (1B) (2B) (3B) (4B) (5B) (6B) (7B) SQ.
592
J. Gorham et al. : K+-Na + discrimination in wheat Ol-
700 600 500
400 300 Fig. 2. Inorganic anions (mol-m 3 expressed sap) in leaves of wheats grown for 21 d in nutrient solution plus 150 molto -3 NaC1 (+7.5 mol-m -3 CaClz). L A N G = Langdon tetraploid wheat; AE.SQ. = Aegilops squarrosa; S Y N = synthetic hexaploid wheat; 1D (1A) etc. = Longdon disomic substitution lines
200 100
1D 2D 3D 4D 5D 6D 7D 1D 2D 3D 4D 5D 6D 7D LANGAE. SYN (IA) (2A) (3A) (4A) (5A) (6A) (7A) (1B) (2B) (3B) (4B) (5B) (6B) (7B) SQ.
root cation concentrations (mol-m-3 in expressed sap) in the tetraploid wheat Langdon, Aegilops squarrosa, and a synthetic hexaploid wheat, and in disomic substitution lines of D-genome chromosomes in Langdon. Values are means of four (Langdon, Aegilops squarrosa and the synthetic hexaploid) or six replicates -+ SEs
Table 2. Effect of 150 mol.m 3 (+7.5 mol.m 3 CaClJ NaC1 on photosynthesis (COz exchange rate, CER) and transpiration (E) in the tetraploid wheat Langdon, Aegilops squarrosa, and a synthetic hexaploid wheat, and in disomic substitution lines of D-genome chromosomes in Langdom Values are the means of six replicates _ SEs
NaC1 added (mol. m - 3)
NaC1 added (mol- m - 3)
Table 1. Effect of 150 mol.m -3 NaC1 (+7.5 mol-m-3CaC12) on
Potassium 0
Langdon (BBAA) 81.0_+ 6.7 Aegilops 64.0_+ 8.0 squarrosa (DD) Synthetic 102.0-+ 8.1 (BBAADD)
Sodium 150
0
150
30.3-+3.7 63.3-+3.1
1.8-+0.6 1.4-+0.3
37.5-+3.2 36.8-+1.2
47.5-+0.9
2.0-+0.8
37.5-+4.8
Langdon disomic substitution lines 1D(1A) 77.8-+ 7.5 35.7_+7.5 2D (2A) 75,8_+ 7.0 29.2_+4.0 3D(3A) 79.5_+ 9.2 37.5-+7.3 4D (4A) 73.2-+ 8.8 29.8_+4.1 5D(5A) a 68.7_+11.9 34.7-+3.5 6D (6A) 97.0-+11.2 19.7-+3.6 7D (7A) 51.7__. 9.2 12.8-+3.1
2.6-+0.9 1.2_+0.5 2.1-+0.3 3.5-+0.9 5.9_+3.3 2.4-+0.6 2.8-+0.4
28.0-+6.5 36.0-+5.2 15.5-+3,1 47.5-+8.8 31.0-+4.0 23.3+4,6 21.7___6.3
1D (1B) 2D (2B) 3D (3B) a 4D (4B) a 5D (5B) ~ 6D (6B) ~ 7D(7B)
3.8-+1.7 2.0-+0.4 3.3+1.6 6.5-+4.3 1.8-+0.4 2.4-+0,8 0.3_+0.3
30.3-+6.2 35.8-+5.1 25.0-+4.6 27.7__6.8 23.3-+5.3 22.0+3.1 19.2+5.3
77.7-+ 7.4 68.7-+ 6.1 81.3-+ 7.5 60.0-+ 6.9 62.3_+ 6.6 66.7-+ 7.7 21.2--+11.3
32.7-+7.5 26.3-+4.8 23.7-+4,9 24,5-+8.3 22.5-+4.5 21,7_+ 1,5 8,8_+1,7
Langdon (BBAA) Aegilops squarrosa (DD) Synthetic (BBAADD)
Photosynthesis CER (~tmol.m-2.s -1)
Transpiration E (mmol.m-2.s -t)
0
150
0
150
10.4-+1.1 11.2-+1.2
5.3 -+0.5 7.9-+1.8
8.7-+1.4 10.5-+0.8
3.8-+0.5 5.4-+0.9
13.6 -+ 1.4
8.9 • 2.0
9.7-+0.6
6.1-+1.4
7.6_+1.2 6.3_+1.0 8.2_+0.7 6.5__.1.0 6.1_+1.2 8.6_+0.9 7.2_+1.4
3.3_+1.2 3.1_+0.5 2.3_+0.8 4.1___0.6 4.2_+1.5 4.3_+0.6 3.6_+0.9
Langdon disomic substutition lines 1D (1A) 8.7_+1.4 3.9_+1.4 2D (2A) 7.9_+1.1 4.1_+0.7 3D (3A) 11.6_+1.5 2.6_+1.0 4D (4A) 10.6_+1.7 4.5_+1.9 5D (5A) 8.3_+1.2 4.6_+0.7 6D (6A) 12.4-+1.0 5.4-+0.4 7D (7A) 10.1_+2.0 3.8_+0.7
" Mono- or telosomic for the replaced A or B chromosome
1D 2D 3D 4D 5D 6D 7D
(1B) (2B) (3B) (4B) (5B) (6B) (7B)
11.9_+2.2 12.7_+0.8 11.1 _+1,4 9.8-+1.7 9.6_+1.4 10.0_+0.9 9.3_+0.9
5.6_+1.0 10.4_+1.6 3.8_+0.4 2.6_+0.8 10.4_+1.1 3.0+_1.0 3,5 _+0.9 9.5 _+1.3 3.1 +0,6 6.2_+1.6 7.9-+1.3 4,8--+0.7 4.9_+1.6 8.0-+1.2 3.1 _+IA 5.2_+0.6 8.5_+0.8 3.9_+0.3 2.3-+1.0 8.0+_1.0 2.4-+ 0.9
ploid. I n o r g a n i c P O 2 c o n c e n t r a t i o n s in the s u b s t i t u t i o n lines were n o t significantly different f r o m those in L a n g don. C o n c e n t r a t i o n s o f N a § a n d K + in the r o o t s o f these p l a n t s (Table 1) w e r e n o t affected by the p r e s e n c e o f
the 4 D c h r o m o s o m e to the s a m e e x t e n t as were l e a f c o n c e n t r a t i o n s . A t 150 m o l - m a NaC1 the r o o t N a + conc e n t r a t i o n s were a l m o s t identical in L a n g d o n , A e . s q u a r r o s a a n d the synthetic h e x a p l o i d . S o d i u m c o n c e n t r a t i o n s in the L a n g d o n s u b s t i t u t i o n lines were generally lower,
J. Gorham et al. : K+-Na + discrimination in wheat
593
with the exception o f the 4 D ( 4 A ) s u b s t i t u t i o n . I n the p l a n t s g r o w n in the presence of salt, r o o t K + c o n c e n t r a tions were higher in Ae. squarrosa a n d the h e x a p l o i d t h a n in L a n g d o n , b u t lower i n the 7 D ( 7 A ) a n d 7D(7B) s u b s t i t u t i o n lines. T h e synthetic h e x a p l o i d h a d a slightly higher p h o t o synthetic rate per u n i t leaf area in the absence o f salt t h a n the diploid or t e t r a p l o i d wheats (Table 2). This difference was m a i n t a i n e d a n d e n h a n c e d w h e n the p l a n t s were g r o w n in 150 m o l . m - 3 NaC1. T h e relative reductions in the rates o f p h o t o s y n t h e s i s a n d t r a n s p i r a t i o n caused by salt were greatest in L a n g d o n a n d least in the synthetic hexaploid. I n the absence o f salt the rates of p h o t o s y n t h e s i s a n d t r a n s p i r a t i o n exhibited b y the
L a n g d o n s u b s t i t u t i o n lines were n o t greatly different f r o m those o f L a n g d o n itself. I n the presence o f salt the rates for the 4 D ( 4 A ) a n d 4D(4B) s u b s t i t u t i o n lines were a m o n g the highest for the s u b s t i t u t i o n lines, b u t were n o t significantly different f r o m the rates exhibited by L a n g d o n . These two lines exhibited a m o n g the smallest decreases in p h o t o s y n t h e s i s a n d t r a n s p i r a t i o n in the presence o f salt, especially a m o n g the B - g e n o m e substit u t i o n lines. Values for some o f the other s u b s t i t u t i o n lines (e.g. 2D(2B)) were significantly lower t h a n those for L a n g d o n .
Ion accumulation in Chinese Spring aneuploids. Sap osm o t i c pressures a n d i n o r g a n i c - i o n c o n t e n t s o f the y o u n -
Table 3. Sap osmotic pressure (mOsmol.kg 1) and inorganic solute concentrations (mol-m 3 in expressed sap) in aneuploid lines of Chinese Spring hexaploid wheat grown for three weeks in nutrient solution plus 125 mol.m-3 NaC1 plus 6.25 mol.m -a CaC12. Values are the means of at least five replicates_+ SEs. Data for lines lacking the long arm or the whole of chromosome 4D are shown in bold type
Sap osmotic pressure Sodium Potassium Calcium Magnesium Chloride Nitrate Orphosphosphate Sulphate K++Na + K+/Na +
Euploid chinese
Ditelosomic 4D short
Ditelosomic 4D long
Tetra 4D
Nullisomic 4B tetrasomic 4A
Nullisomic 4B tetrasomic 4D
Nullisomic 4D tetrasomic 4A
541 _+37 85 _+16 223 _+ 6 27 _+ 3 28 _+ 3 180 _+15 30 _+ 4 28 _+ 3 8 -+ 1 329 _+25 3.6_+ 0.6
594 _+61 261 _+26 71 _+ 8 13 -+ 3 17 _+ 5 230 -+41 17 _+ 4 32 _+ 3 10 _+ 8 332 +29 0.6_+ 0.4
550 _+58 103 +25 232 +19 24 _+ 5 26 _+ 5 210 _+34 29 • 8 24 _+ 2 8 -+ 2 335 _+34 4.0_+ 1.4
673 _+101 78 -+ 12 232 _+ 16 24 _+ 3 37 _+ 5 195 _+ 33 35 _+ 6 37 -+ 5 13 -+ 2 301 _+ 13 3.5_+ 0.7
552 _+36 95 _+17 206 _+12 25 _+ 3 29 _+ 5 184 +19 29 • 6 26 _+ 4 10 _+ 1 333 _+36 3.4_+ 0.6
495 61 231 20 27 165
592 _+77 274 _+31 124 _+38 16 _+ 1 19 + 4 161 _+12 30 _+ 6 44 -+ 3 12 _+ 1 408 _+54 0.5_+ 0.2
_+23 -+11 -+12 _+ 2 _+ 3 _+15 55 _+12 20 -+ 2 7 _+ I 292 -+10 5.5_+ 1.4
Table 4. Growth and yield data for Chinese Spring and Langdon aneuploid lines of wheat grown in hydroponic culture with nutrient solution plus 0, 75 or 125 mol-m -3 NaC1 (+0, 3.75 or 6.25 mol.m -a CaC12). Values are the means_+SEs of data from at least six replicate plants. Data for lines lacking the long arm or the whole of chromosome 4D are shown in bold type NaC1 (tool 9m - 3)
Chinese Spring (hexaploid)
Langdon (tetraploid)
Euploid
Nullisomic 4D tetrasomic 4A
Ditelosomic 4D long
Ditelsomic 4D short
Euploid
Substitution 4D (4A)
Substitution 4D (4B)
Height(cm)
0 75 125
106+ 5 108_+ 5 88+ 6
102+ 9 92_+ 5 75_+ 6
84-t-ll 82+ 3 77_+ 4
94+10 97_+ 2 58+ 7
114+ 5 109_+ 4 76-+ 5
110!15 87_+12 97+ 6
92_+8 82___5 57-+2
Plant weight (g)
0 75 125
38+ 7 35-+ 5 18_+ 4
29-+11 16_+ 4 6_+ 2
38-+13 26_+ 8 17_+ 3
9+ 3 14-+ 1 2_+1
47-+ 9 32_+ 5 8_+ 2
16_+ 4 8-+ 7_+ I
7_+2 5_+2 2_+1
Seedweight (g)
0 75 125
12_+ 2 8_+ 2 4_+ 1
5_+ 2 2_+ 1 1_+ 0
5_+ 2 3_+ 2_+ 0
1_+1 2_+0 0_+ 0
15_+ 3 6_+ 3 1_+ 0
6_+ 4 2_+ I 1_+ 0
1_+0 0_+0 0_+0
Numberof tillers
0 75 125
15_+ 2 15_+ 2 10_+ I
13_+ 3 8_+ 2 4_+ 1
21_+ 6 15_+ 3 12+ 1
7_+ 2 9_+ l 3_+ 1
14_+ 2 12-+ 2 6_+ 1
7_+ 3 4+_ I 5_+ 1
5_+1 4_+1 2_+0
Total number of seeds
0 75 125
430_+64 460-+71 318_+54
144_+65 98_+27 61_+23
170_+83 127_+50 98_+23
41_+20 111-+14 16_+10
Number of seeds pertiller
0 75 125
26_+ 2 28_+ 2 30_+ 3
9_+ 2 10_+ 2 11_+ 3
6_+ 2 8_+ 2 8_+ 2
7_+ 3 12_+ 2 3_+ 1
4 3 6 _ + 7 9 145-+85 2 6 0 _ + 3 5 70_+23 68_+18 67_+11 31_+ 1 22_+ 1 9_+ 2
19_+ 3 14_+ 3 15_+ 2
16-+8 11_+1 5+4 3_+1 3_+1 2_+1
594
J. Gorham et al. : K+-Na + discrimination in wheat
gest fully e x p a n d e d leaves o f C h i n e s e S p r i n g h e x a p l o i d w h e a t a n d a n u m b e r o f a n e u p l o i d lines g r o w n for 21 d in 125 m o l . m - 3 NaC1 plus 6.25 m o l - m - 3 CaCI2 are s h o w n in Table 3. T h e lines l a c k i n g t h e whole, o r j u s t the l o n g a r m , o f c h r o m o s o m e 4 D h a d h i g h l e a f N a § a n d low l e a f K + c o n c e n t r a t i o n s , resulting in c o n s i d e r a b l y l o w e r K + / N a + ratios. T h e C a 2+ a n d M a 2+ c o n c e n t r a t i o n s were slightly l o w e r t h a n in e u p l o i d C h i n e s e S p r i n g o r the o t h e r a n e u p l o i d lines, b u t o t h e r w i s e there were n o c o n s i s t e n t effects o f the 4 D c h r o m o s o m e o n the c o n c e n t r a t i o n s o f o t h e r ions. S i m i l a r results were o b t a i n e d in a s e c o n d h a r v e s t t a k e n 21 d l a t e r (results not shown).
Effect of salt on grain yield. T h e results o f a n e x p e r i m e n t in w h i c h the effects o f salt o n g r o w t h a n d g r a i n yield o f a n e u p l o i d lines o f C h i n e s e S p r i n g a n d L a n g d o n were e x a m i n e d a r e s h o w n in Table 4. P l a n t h e i g h t w a s the p a r a m e t e r least affected b y a n e u p l o i d y o r salt t r e a t m e n t , w h e r e a s p l a n t w e i g h t was affected b y b o t h . E x c e p t for the C h i n e s e S p r i n g line d i t e l o s o m i c for the l o n g a r m o f c h r o m o s o m e 4D, all o f the o t h e r a n e u p l o i d lines were s m a l l e r at all salt c o n c e n t r a t i o n s (0, 75 a n d 125 m o l t o - 3 NaC1) t h a n the e u p l o i d lines. G e n e r a l l y , the a n e u -
p l o i d lines were w o r s e t h a n the e u p l o i d w h e a t s in t e r m s o f t o t a l seed weight, n u m b e r s o f seed a n d tillers, a n d n u m b e r o f seeds p e r tiller. T h e Chinese S p r i n g line ditel o s m i c f o r the l o n g a r m o f c h r o m o s o m e 4 D a n d the L a n g d o n 4 D ( 4 A ) s u b s t i t u t i o n line were the best o f the a n e u p l o i d lines as j u d g e d b y the effect o f salt o n seed p r o d u c t i o n . D e s p i t e the p o o r a b s o l u t e p e r f o r m a n c e o f the a n e u p l o i d lines there is s o m e i n d i c a t i o n o f a beneficial effect o f c h r o m o s o m e 4D. W h e n the t o t a l n u m b e r o f seeds p e r p l a n t at 125 m o l . m - 3 NaC1 is expressed as a p e r c e n t a g e o f the c o n t r o l (no salt) values, then the lines w i t h c h r o m o s o m e 4 D ( n o r m a l t y p e in Table 4) h a d a m e a n v a l u e o f 5 2 % a n d the lines l a c k i n g c h r o m o s o m e 4 D ( b o l d t y p e in Table 4) h a d a m e a n o f 3 2 % . O n l y the 4D(4B) s u b s t i t u t i o n gave l o w e r relative yields t h a n the lines l a c k i n g c h r o m o s o m e 4D. T h e 4D(4B) s u b s t i t u tion, even w h e n m o n o s o m i c f o r 4B, is k n o w n to h a v e p o o r fertility ( J o p p a a n d W i l l i a m s 1988). W h e n the g r a i n f r o m this e x p e r i m e n t was a n a l y s e d for i n o r g a n i c - i o n c o n t e n t s it was f o u n d (Table 5) t h a t the lines l a c k i n g the 4 D c h r o m o s o m e (or its l o n g a r m ) h a d c o n s i d e r a b l y h i g h e r N a + c o n t e n t s t h a n the o t h e r lines, especially w h e n g r o w n a t 1 2 5 m o l - m 3 NaC1. C o n v e r s e l y K § c o n t e n t s were g e n e r a l l y lower, except
Table 5. Inorganic-ion contents (mmol. (kg DW) 1) of the grain of aneuploid lines of Chinese Spring and Langdon wheats grown in hydroponic culture with nutrient solution plus 0, 75 or 125 mol-m-3 NaC1 (+0, 3.75 or 6.25 tool.m-3 CaC12). Values are the means of four replicates _+SEs. Data for lines lacking the long arm or the whole of chromosome 4D are shown in bold type NaC1 (tool- m - 3)
Chinese Spring (hexaploid) Euploid
Sodium
Potassium
Calcium
Magnesium
Chloride
Nullisomic 4D tetrasomic 4A
0 75 125
4 _+ 1 51 _+23 76 -+23
0 75 125
92 _+ 6 156 _+24 161 _+21
0 75 125 0 75 125
3 9 4 23 38 26
_+ -+ _+ _+ _+ •
0 4 1 3 4 3
13 -+ 2 80 -+27 120 _+17 84
_+ 1
151 _+25 140 _+13 3 3 4 24 24 31
• • • • _+ _+
1 1 1 4 4 5
Langdon (tetraploid) Ditelosomic 4D long 12 -+ 2 22 • 5 34 _+ 8 88 _+ 6 104 _+ 4 114 _+20 4 5 3 25 24 21
_+ -+ _+ _+ _+ _+
I 2 1 3 3 2
Ditelosomic 4D short 10 -+ 3 117 441
•
84
_+
55-+14
_+ 51
257_+82
12 _+4 54 _+12 128 _+45
112_+16
177
_+ 17
164_+43
82 + 9 141 _+14 182 _+24
3 4 8 28 30
_+ 1 _+ 1 • 4 • 4 _+ 2 _+ 19
2-+ 5_+ 8_+ 21• 30_+ 26_+
3 _+I 5_+0 6_+1 23 _+3 28 _+3 37 _+5
47
81_+ 7
0 1 2 2 2 6
17 _+ 1 _+ 22 583 _ + 1 4 9
17_+ 2 107_+35 239_+51
17 • 4 158 _+37 142 _+13
33 _+ 1 --4- 16
16_+ 3 52-+13
_+
0
45_+10
21 _+ 4 20 _+4 29 + 5
1 _+ 0 5 _+ 1 5 -+ 0
5_+ 1 11_+ 1 9_+ 2
6+1 5-+1 4___2
3_+ 0 1_+ 0 3_+ 0
2_+0 2+1 i _+ 0
10 _+ 2 91 _+29 213 _+28
12 _+ 4 27 _+ 3 71 _+23
0 75 125
25 _+10 31 _+ 4 38 _+ 1
29 + 3 69 _+11
27 _+ 1 21 _+ 4
37
28
Orthophosphate
0 75 125
3 _+ 1 2 _+ 0 2 • 0
2 _+ 0 4 _+ l 5 -+ 1
6 • 1 2 • 0 2 • 0
Sulphate
0 75 125
2 _+ 0 2 _+ 0 2 _+ 0
2 + 0 3 + 0 4 -+ 0
3 _+ 0 2 _+ 0 1 _+ 0
2 _+ 0 2 _ 0 3 _+ 0
K+/Na +
0 75 125
31.7• 8.9 5.2_+ 1.3 2.7-+ 0.5
6.9_+ 0.7 3.1_+ 1.1 1.2• 0.1
7.6_+ 1.0 6.2_+ J.3 3.6_+ 0.5
+ 3
Substitution 4D (41)
194 _+ 25
5
11 _+ 2 62 _+11 122 _+30
_+ 3
9_+ 2
25
0 75 125
Nitrate
Euploid
155
100 43
14.3_+ 1.3+ 0.4-+
6.4 0.1 0.0
12.2_+ 4.1 2.4_+ 0.4 0.9-+ 0.3
13.7-+ 5.0 3.4-+ 0.9 1.8_+ 0.3
J. Gorham et al. : K+-Na + discrimination in wheat
595
that the Chinese Spring line ditelosomic for the long arm o f chromosome 4D had the lowest K + content when subjected to salinity. Thus the lines lacking the 4D chromosome had the lowest grain K + / N a + ratios. In contrast to leaf C1- concentrations, those in the grain were greatly increased in the absence of c h r o m o s o m e 4D, especially in the Chinese Spring line ditelosomic for the short arm o f c h r o m o s o m e 4D. There were no consistent differences between the other inorganic ions in the grains of the various aneuploid lines tested. Discrimination between K + and Na + at low external sodium concentrations. In a number of experiments with the tetraploid wheat L a n g d o n and synthetic hexaploid w h e a t s or Chinese Spring it was observed that the enhanced ability of the hexaploid wheats to discriminate in the uptake of Na + and K + was expressed over a wide range of external N a + concentrations upto 250 m o l - m -3 NaCI (data not shown). The results of a typical experiment at the lower end of the concentration range are shown in Fig. 3, where the leaf Na + and K + concentrations o f L a n g d o n and Chinese Spring are plotted against external salinity in the range 5-50 tool. m -3 NaC1. The greater accumulation of N a + in the leaves of L a n g d o n is evident even at 5 tool. m - 3 NaC1, whereas the K + concentrations are not significantly different until 30 m o l - m - 3 NaC1. Uptake o f 22Na into low-salt-grown seedlings. Uptake from solutions containing lower concentrations o f Na + were investigated by following the incorporation of 22Na into excised or intact roots o f seedlings which had been grown on a medium containing only Ca and Mg salts. Uptake into excised roots was determined after short (/2rain) or long (60rain) incubations in 2 t o o l .
240
Table 6. Rates of influx of 22Na (mmol-(kg FW)-I-h -1) from 2 mol.m-3 NaC1 into excised, low-salt-grown roots of Langdon and Chinese Spring wheats. Each value is the mean of three replicates_+ SE Influx time/wash time (rain)
Langdon
Chinese Spring
2 tool-m- 3 NaC1 alone 12/ 5 45/30
1.49+0.02 2.52 _ 0.06
1.29_+0.05 1.864- 0.19
2 mol. m- 3 NaC1 plus 0.5 mol-m -3 KC1 15/ 5 60/30
1.04+0.08 0.93 4-0.20
0.94_+0.26 0.66 -+0.09
24 22 20 .-..18
///o ' /
o L-12 7
9
10
E 8 ..i." O
6
E t-" 4 2 ~Z
0 -2
I
I
I
I
,5
10
15
20
25
TIME (h) Fig. 4. Uptake of 22Na into the roots (e, 9 and the shoots (T, v) of low-salt-grown seedlings of Chinese Spring (hexaploid, o, v) and Langdon (tetraploid, e, v) wheats. The uptake solution contained 1 mol.m 3 NaC1 and 0.5 mol-m - 3 C a S O 4. The SEs did not exceed three times the size of the symbols
0 220 0
200
v".~.%
180
K ~v--
Q. 160 x 0 140
v
"?E 120 .2." 100 0
E
+ "./ +
121 Z
"6
(D ._1
/
8o
No
6O
4o -O
20
o 0'
10 '
20 '
~0
go
EXTERNAL NaCl ( m o l m -3 )
5'o
Fig. 3. Concentrations (mol-mol 3 expressed sap) of Na + (e, v) and K + (9 ,7) in leaves of Chinese Spring hexaploid wheat (+D genome, o, o) and Langdon tetraploid wheat (--D genome, ~v, v) grown for 15 d in basic nutrient solution containing low NaC1 concentrations. The SEs were not more than twice the size of the symbols used
m -3 NaC1, followed by 5- or 30-rain washes in cold (4 ~ C), unlabelled medium (Table 6). In all cases the uptake rates were higher in L a n g d o n than in Chinese Spring, and were decreased in the presence of 0.5 molt o - 3 KC1. The long-term (vacuolar) influx was reduced to a greater extent by KC1 than the short-term (cytoplasmic) influx. When uptake into whole seedlings from a solution containing 1 tool. m - 3 NaC1 was followed (Fig. 4) it was observed that, although the initial uptake into the roots of L a n g d o n was slightly higher than into those of Chinese Spring, after about 20 h Chinese Spring had a slightly higher 22Na content than Langdon. Transport of 22Na to the shoots was measurable after a few hours and was considerably greater in L a n g d o n than in Chinese Spring. Uptake of 22Na into the shoots of aneuploid lines of L a n g d o n and Chinese Spring was also investigated (Table 7). The lines lacking chromosome 4D accumulated more ZZNa than those lines lacking this chromosome, but the Chinese Spring lines ditelosomic for the short
596
J. Gorham et al. : K+-Na + discrimination in wheat
Table 7. Uptake of 2ZNa from 1 mol.m -3 NaC1 (plus 0.1 mol.
m-3 KC1) over 48 (expt. 2) and 72 h (expt. 1) into shoots of intact, low-salt-grown seedlings of aneuploid lines of Chinese Spring and Langdon, expressed as mean percentage uptake into Langdon • SEs. Data for lines lacking the long arm (or the whole) of chromosome 4D are shown in bold type 72 h Langdon Langdon 4D (4A) substitution Langdon 4D (4B) substitution Langdon 4D disomic addition Chinese Spring Chinese Spring ditelosomic 4D short Chinese Spring ditelosomic 4D long Chinese Spring tetra 4D Chinese Spring nulli 4D tetra 4A
48 h
100 • 10
6+ 6+ 7_+ 10+ 23 • 11 • 5_+ 30 +
1
100 -+10 8 _+ 2
I
8+ 1
3 2 4 3 1
8-+ 10-+ 21 -+ 5 _+ 7_+
2 2 3 2 1
4
21 -+ 3
Table 8. Fluxes of 22Na (gmol-kg-l.h -1) from intact, low-salt-
grown wheat roots loaded with 1 mol.m 3 22NaC1 for 24 h Flux
T. turgidurn cv. Langdon
T. aestivum cv. Chinese Spring
Vacuolar effiux ~co~c) Cytoplasmic efflux ~oo {cyt) Transport to shoot ~cx K-stimulated efflux ~oo(K-aep)
173_+ 8 673 _+188 513 • 59 80_+ 11
121___13" 473 _-+_93 34_ 1"** 162_+22"
Means significantly different at the 5% (*) or 0.1% (***) probability levels a r m of 4D and nullisomic for 4D b o t h accumulated less 22Na than Langdon.
Efflux of 22Na from intact seedlings, Low-salt-grown seedlings of Chinese Spring and L a n g d o n wheats were incubated in 1 tool. m - 3 22NaC1 for 24 h before the start of an efflux experiment in which the unlabelled washing solution was changed every 10 rain. The flux rates were calculated according to Jeschke and J a m b o r (1981) and are shown in Table 8. The cytoplasmic efflux was greater than the vacuolar efflux for both species, but the differences between L a n g d o n and Chinese Spring were small for b o t h effluxes. In contrast the xylem flux (transport to the shoots) was very much greater in L a n g d o n than in Chinese Spring. When KC1 (1 m o l . m - 3 ) was added to the washing solution after the establishment of steadystate vacuolar 22Na efflux, an additional K-stimulated ZZNa efflux was observed which occurred over the next 60 min. This K-stimulated efflux was significantly higher in Chinese Spring than in Langdon. Discussion
The data presented here show that the long a r m of chrom o s o m e 4D contains a trait (controlled by one or more genes ?) which affects the accumulation of both N a + and K + in the leaves o f wheat. This trait is operative over a wide range of external salt concentrations and appears to be constitutive. In the grain the contents of N a +,
K + and C1- are altered by the presence of c h r o m o s o m e 4D. The difference in the effect of c h r o m o s o m e 4D on C1- transport to the shoots and to the grain m a y reflect different influences on transport via the xylem (to the shoots) and the phloem (to the grain). Net transport rates of N a + and K + to the shoots are affected by the presence of the enhanced trait for K + - N a + discrimination, but leaf anion and root anion and cation concentrations are not. Studies of 22Na fluxes have confirmed these findings, with influx into and efflux out of the cytoplasm and vacuoles only slightly higher in the roots of the tetraploid wheat L a n g d o n than in the more salt-tolerant hexaploid cultivar Chinese Spring, but transport to the shoots m u c h less in Chinese Spring. Similar results have been obtained with excised roots (D.W. Rains, University of California, Davis, personal communication). It should be noted that, at the start of the efflux period, the Chinese Spring roots would have contained slightly more 221'qa than the roots of the tetraploid wheat. Davis (1984) has reported that, of two hexaploid wheats, the more salt-tolerant cultivar transported less Z2Na to the shoots than the less tolerant cultivar. Other fluxes were similar in the two cultivars. The comparison between Chinese Spring and Langdon is not ideal since the difference between the two is both the presence or absence of the whole of the D genome and any differences in their A and B genomes. These 2ZNa uptake and efflux experiments are being repeated using the aneuploid lines described above. A feature of both L a n g d o n and Chinese Spring was the K-stimulated N a + effiux (Table 8). High rates of transient N a + efflux on addition o f K + have been observed in several cereal grasses (Jeschke 1983; Jeschke and Nassery 1981). The results presented here, and other observations of ours involving X-ray microanalysis of roots and observation of dye m o v e m e n t indicate that neither uptake into the root nor transpirational effects can explain the K + - N a + discrimination controlled by c h r o m o s o m e 4D. Thus it seems likely that the discrimination between N a + and K + occurs mainly at the point of xylem loading, or possibly at the endodermis. Jeschke (1983) suggests that selectivity for K + over N a + at the symplasmxylem b o u n d a r y occurs in barley, but it is difficult to obtain reliable estimates of ion concentrations in the cytoplasm of the xylem p a r e n c h y m a cells. Sodium accumulation in the shoots of the hexaploid wheats was less than in the shoots of Ae. squarrosa, indicating some interaction between the D genome and the A and B genomes. It is not k n o w n whether this represents an interaction with a specific mechanism controlling ion m o v e m e n t in tetraploid wheat, or whether it is a m o r e general consequence of the difference in ploidy level and growth rate (see B a m a k h r a m a h et al. 1984 for a general discussion of the effects of ploidy on growth and yield of wheat). Furthermore, the Chinese Spring lines lacking the whole, or just the long arm, of chromosome 4D had 22Na-uptake rates (Table 7) or grain N a + contents (Table 5) different f r o m those of Langdon. In this case also it is not possible to distinguish between a specific interaction between the tetraploid wheat gen o m e and other transport mechanism(s) on D-genome
J. Gorham et al. : K+-Na + discrimination in wheat c h r o m o s o m e s , and a non-specific effect o f the difference in ploidy level. A large n u m b e r o f i m p o r t a n t physiological a n d m o r p h o l o g i c a l characters (e.g. the R h t 2 dwarfing gene f r o m N o r i n 10) are k n o w n to be located on D g e n o m e c h r o m o s o m e s ( M c I n t o s h 1973). We have a t t e m p t e d to answer the question o f w h a t effect the e n h a n c e d trait for K + - - N a + discrimination on c h r o m o s o m e 4 D has on g r o w t h a n d grain yield in saline conditions. There is some evidence which indicates that N a + is m o r e toxic to w h e a t t h a n C1- ( K i n g s b u r y and Epstein 1986) and that leaf K + / N a + ratio is a factor in determining salt tolerance in the b r o a d e s t sense ( R a n a 1986; R a s h i d 1986). The results presented in Table 4 show quite clearly that, in absolute terms, the detrimental effects o f a n e u p l o i d y are larger than a n y effect o f the 4 D K + - N a + discrimination character. All o f the aneuploid lines p r o d u c e d less grain, and in m o s t cases less straw, than the euploid wheats. This agrees with the d a t a o f J o p p a a n d Williams (1988) for L a n g d o n disomic substitution lines. A l t h o u g h there were indications o f a beneficial effect o f c h r o m o s o m e 4D, it will only be possible realistically to assess the i m p a c t o f the enh a n c e d K + - N a + discrimination character on wholeplant p e r f o r m a n c e a n d yield c o m p o n e n t s w h e n it can be transferred, w i t h o u t the rest o f the long a r m o f chrom o s o m e 4D, to a tetraploid wheat. Such r e c o m b i n a t i o n m a y be possible t h r o u g h hybrids involving tetraploid wheats deficient for the h o m o e o l o g o u s pairing suppressor genes. A n o t h e r a p p r o a c h to assessing the i m p a c t o f c h r o m o s o m e 4 D w o u l d be to obtain i o n - t r a n s p o r t m u t a n t s (like the scabrous diminutive m u t a n t o f Capsicum annuum, Tal a n d Benzioni 1977), preferably in a diploid species. It is i m p o r t a n t to emphasize that the e n h a n c e d K + -N a + discrimination character described here is n o t seen as the only m e c h a n i s m controlling salt tolerance in the Triticeae. O t h e r mechanisms, which p r o b a b l y operate in totally different ways, are f o u n d in other m e m b e r s o f this tribe ( G o r h a m a n d W y n Jones 1989). F u r t h e r m o r e barley, which is generally m o r e salt tolerant than tetraploid or hexaploid w h e a t (Maas 1986), has leaf N a + c o n c e n t r a t i o n s similar to those f o u n d in tetraploid w h e a t when g r o w n in the same conditions. Thus the c h r o m o s o m e - 4 D - c o n t r o l l e d e n h a n c e d K + - N a + discrimination trait is only one m e c h a n i s m which m u s t be integrated with other characters in the p y r a m i d i n g a p p r o a c h to designing a n d breeding crop plants with greater salt tolerance (Yeo and Flowers 1986; G o r h a m a n d W y n Jones 1989). The authors gratefully acknowledge the financial support of the Overseas Development Administration of the United Kingdom, and would like to thank Dr. L.R. Joppa and Dr. C.N. Law for supplying the seeds of aneuploid lines of Langdon and Chinese Spring wheats.
References Bamakhramah, H.S., Halloran, G.M., Wilson, J.H. (J 984) Components of yield in diploid, tetraploid and hexaploid wheats (Triticure spp.). Ann Bot. 54, 51-60 Davis, R.F. (1984) Sodium fluxes in intact roots of wheat varieties
597 differing in salt tolerance. In: Membrane transport in plants, pp. 489-490, Cram, W.J., Janacek, K., Rybova, R., Sigler, K. eds. Academia, Praha Francois, L.E., Maas, E.V., Donovan T.J., Youngs, V.L. (1986) Effect of salinity on grain yield and quality, vegetative growth, and germination of semi-dwarf and durum wheat. Agron. J. 78, 1053 1058 Gorham, J., Wyn Jones, R.G. (1989) A physiologists approach to improving the salt tolerance of wheat. Rachis (in press) Gorham, J., Hardy, C., Wyn Jones, R.G., Joppa, L.R., Law, C.N. (1987) Chromosomal location of a K/Na discrimination character in the D genome of wheat. Theor. Appl. Genet. 74, 584-588 Jeschke, W.D. (1983) Cation fluxes in excised and intact roots in relation to specific and varietal differences. In: Genetics aspects of plant nutrition, pp. 71 86, Saric, M.R., Loughman, B.C., eds. Nijhoff, The Hague, Netherlands Jeschke, W.D., Jambor, W. (1981) Determination of unidirectional sodium fluxes in roots of intact sunflower seedlings. J. Exp. Bot. 32, 1257-1272 Jeschke, W.D., Nassery, H. (1981) K + - N a + selectivity in roots of Triticum, Helianthus and Allium. Physiol. Plant. 52, 217-224 Joppa, L.R. (1987) Aneuploid analysis in tetraploid wheat. In: Wheat and wheat improvement, pp. 151 166, Heyne, E.G., ed. Monograph 13, Am. Soc. Agron., Madison, Wis., USA Joppa, L.R., Williams, N.D. (1988) Langdon durum disomic substitution lines and aneuploid analysis in tetraploid wheat. Gehome 30, 22~228 Joshi, Y.C., Dwivedi, R.S., Quadar, A., Bal, A.R. (1982) Salt tolerance in diploid, tetraploid and hexaploid wheat. Ind. J. Plant Physiol. 25, 421-422 Kingsbury, R.W., Epstein, E. (1986) Salt sensitivity in wheat. A case for specific ion toxicity. Plant Physiol. 80, 651 654 Maas, E.V. (1986) Crop tolerance to saline soil and water. In: Prospects for biosaline research (Proc. U.S.-Pakistan Biosaline Workshop) pp. 205 219, Ahmad, R., San Pietro, A., eds. Karachi University, Pakistan McIntosh, R.A. (1973) A catalogue of gene symbols for wheat. In: Proc. 4th Int. Wheat Genet. Syrup., pp. 893-938. University of Columbia, Mo. USA Rana, R.S. (1986) Genetic diversity for salt-stress resistance of wheat in India. Rachis 5, 32-37 Rana, R.S., Singh, K.N., Ahuja, P.S. (1980) Chromosomal variation and plant tolerance to sodic and saline soils. In: Symposium papers (Int. Syrup. Salt-affected Soil), pp. 487-493, Central Soil Salinity Research Inst., Karnal, India Rashid, A. (1986) Mechanisms of salt tolerance in wheat. PhD thesis, University of Agriculture, Faisalabad, Pakistan Sayed, H.I. (1985) Diversity of salt tolerance in a germplasm collection of wheat (Tritieum spp.). Theor. Appl. Genet. 69, 651 657 Shah, S.H., Gorham, J., Forster, B.P., Wyn Jones, R.G. (1987) Salt tolerance in the Triticeae: The contribution of the D genome to cation selectivity in wheat. J. Exp. Bot. 36, 254-269 Tal, M., Benzioni, A. (1977) Ion imbalance in Capsicum annuum, scabrous diminutive, a wiry mutant of pepper. I. Sodium fluxes. J. Exp. Bot. 28, 1337-1341 Weimberg, R. (1987) Solute adjustment in leaves of two species of wheat at two different stages of growth in response to salinity. Physiol. Plant. 70, 381 388 Weimberg, R. (1988) Modification of foliar solute concentrations by calcium in two species of wheat stressed with sodium chloride and/or potassium chloride. Physiol. Plant. 73, 418-425 Wyn Jones, R.G., Gorham, J. (1989) Physiological effects of salinity. Scope for genetic improvement. In: Proc. Int. Syrup. on Improving Winter Cereals under Temperature and Salinity Stresses, Cordoba, 1987. ICARDA, Syria (in press) Yeo, A.R., Flowers, T.J. (1986) Salinity resistance in rice (Oryza sativa L.) and a pyramiding approach to breeding varieties for saline soils. Aust. J. Plant Physiol. 13, 161-173 Received 30 June; accepted 10 November 1989
P l a n t a 9 Springer-Verlag1990
Partial characterization of the trait for enhanced K +-Na + discrimination in the D genome of wheat J. Gorham, R.G. Wyn Jones, and A. Bristol School of Biological Sciences, University College of North Wales, Bangor, Gwynedd, LL57 2UW, UK
Abstract. The long arm of chromosome 4D of wheat (Tritieum aestivum L.) contains a gene (or genes) which influences the ability of wheat plants to discriminate between N a + and K +. This discrimination most obviously affects transport from the roots to the shoots, in which less N a + and m o r e K + accumulate in those plants which contain the long arm of chromosome 4D. Concentrations o f N a + and K + in the roots, and C1- concentrations in the roots and shoots, are not significantly affected by this trait, but N a +, K + and C1- contents of the grain are reduced. The trait operates over a wide range o f salinities and appears to be constitutive. At the m o m e n t it is not possible to determine accurately the effect of this trait on growth or grain yield because the aneuploid lines which are available are much less vigorous and less fertile than their euploid parents.
Key words: Ion transport - Salinity (ion uptake) - Salt tolerance (genetic trait) - Triticum (salt tolerance)
chromosomes (from the hexaploid wheat Chinese Spring) had been substituted for their homoeologues in the A and B genomes of the tetraploid wheat cultivar L a n g d o n (Joppa 1987; Joppa and Williams 1988). The use of ditelosomic lines of the hexaploid wheat Chinese Spring enabled us to confirm that the enhanced trait for K + - N a + discrimination was located on chromosome 4D, and specifically on the long arm of this chromosome. This paper presents a more detailed examination of the effects of D-genome chromosomes on the salt tolerance and ion-uptake patterns in wheat, and brings together new ion-uptake and other data with some results which have been presented as parts o f contributions to a number of recent conferences (e.g. Wyn Jones and G o r h a m 1989). We suggest a possible site for the discrimination between N a + and K + and discuss the practical exploitation of this trait in improving the overall salt tolerance of tetraploid wheat.
Material and methods Introduction A number of studies have demonstrated that hexaploid bread wheat (Triticum aestivum L., genome B B A A D D ) is more salt-tolerant (in the broadest sense) than tetraploid (T. turgidum L., genome BBAA), or diploid wheats (T. monococcum L., genome AA) (Rana et al. 1980; Joshi et al. 1982; Francois etal. 1986; Weimberg 1987, 1988; but see also Sayed 1985). Our own studies have shown that the D genome (from Aegilops squarrosa L., genome D D ) contains a trait for enhanced K + - N a + discrimination, and that this trait is expressed in synthetic hexaploid wheats derived from hybrids between tetraploid wheat and Ae. squarrosa (Shah et al. 1987). We have been able to demonstrate that this trait is located on chromosome 4D ( G o r h a m et al. 1987) by examining Na + and K + concentrations in leaves of salt-treated plants of disomic substitution lines in which D-genome
Growth of plants. Triticum turgidum cv. Langdon, Triticum aestivum cv. Chinese Spring and synthetic wheats and their various aneuploid lines were obtained from Dr. L.R. Joppa (USDA-ARS, Fargo, N.D., USA) and Dr. C. Law (FPSR, Cambridge, UK) and grown in saline hydroponic culture as described by Gotham et al. (/987). The concentrations of the major nutrients were as follows: K +, 6.8mol-m-3; NO~-, 8.4mol-m-3; PO2, 1.7mol-m-3; C a 2+, 0.73mol-m-3; Mg2+, 0.92molm-3; Na +, 0.3molm -3. Salt (NaCI+CaC12 to give an Na+/Ca 2+ ratio of 20:1) was added to the culture solution at a rate of 25 mol.m 3.d-1 to give the final amounts stated in the legends. Production of the Langdon (tetraploid wheat) disomic substitution lines, which incorporate Dgenome chromosomes from Chinese Spring hexaploid wheat, has been described by Joppa (1987). The presence of monosomic or telosomic representatives of the substituted A- or B-genome chromosomes is indicated in Table 1. For experiments with low-salt-grown seedlings, grain was germinated in 0.5 mol-m 3 C a S O 4 alone in a growth cabinet maintained at 22~ C with 16 h of light daily supplied by a mixture of warm-white fluorescent tubes and tungsten light bulbs at 300 gmol photons, m- 2. s- 1 of photosynthetically active radiation (PAR).
J. Gorham et al. : K+-Na § discrimination in wheat Two days after germination the solution was replaced with a mixture containing 2 m o l . m -3 Ca(NO3)2 and 1 mol.m 3 MgSO4, and the seedlings grown for a further 5 d prior to use in 22Nauptake experiments.
Sap extraction and analysis. Leaf saps were extracted by a freezethaw technique followed by mechanical disruption and centrifngation (Gorham et al., 1987). Inorganic-ion concentrations in the expressed saps were determined by ion chromatography, also as previously described. Roots were first washed for 10 min in 20 mol. m - 3 Ca-acetate solutions which were adjusted to the same osmotic pressures as the culture solutions by the addition of sorbitol, and then blotted with tissue paper to remove surface moisture. Measurement of photosynthesis and transpiration. Measurements of carbon-dioxide exchange rates (CER) and transpiration rates (E) were made in situ on plants growing in hydroponic culture in the greenhouse using an LCA-2 infra-red gas analyser, a DL1 data logger, an ASU/MF air supply unit (taking air from 3 m above the greenhouse) and a Parkinson narrow leaf chamber, all supplied by the Analytical Development Co., Hoddesdon, Hefts., UK. Measurements were made in bright sunlight ( > 1 mol photons.m 2. s- 1 PAR) and were taken within 30 s of applying the leaf chamber (i.e. before the start of stomatal closure). Studies of 22Naflux. Measurements of 22Na fluxes into intact, lowsalt-grown wheat seedlings were made essentially as described by Jeschke and Jambor (1981). The experimental vessels were 20-cm 2 plastic syringe barrels fitted with plastic taps. 22Na was measured in a well-type gamma counter. To concentrate the 22Na in efflux solutions an excess of Dowex 50 (H + form) ion-exchange resin was shaken with the solution in a test tube. Most of the activity was then found to be in the bottom 1-2 cm 3 of the tube and could be positioned inside the well of the counter. Statistical analysis. All results are expressed as means + standard errors. Means were compared using the t-test facility of the STATPACK (University of York, UK) statistical package.
Results Photosynthesis, transpiration and inorganic-ion accumulation in Langdon disomic substitution lines. C o n c e n t r a t i o n s
600 - -
591 o f i n o r g a n i c c a t i o n s in the s a p f r o m the y o u n g e s t fullye x p a n d e d leaves o f L a n g d o n , Aegilops squarrosa R L 5003, the s y n t h e t i c h e x a p l o i d ( L a n g d o n / R L 5003) a n d the full set o f D - g e n o m e d i s o m i c s u b s t i t u t i o n lines g r o w n for 21 d in the p r e s e n c e o f 1 5 0 m o l . m - 3 NaC1 a n d 7.5 m o l . m - 3 CaC12 a r e s h o w n in Fig. 1. T h e e q u i v a l e n t d a t a for i n o r g a n i c a n i o n s are p r e s e n t e d in Fig. 2. C o n c e n t r a t i o n s o f N a § were l o w e s t in the full s y n t h e t i c hexap l o i d a n d in the s u b s t i t u t i o n lines i n v o l v i n g c h r o m o s o m e 4 D (i.e. the 4 D ( 4 A ) a n d 4D(4B) s u b s t i t u t i o n lines). In a g r e e m e n t w i t h p r e v i o u s o b s e r v a t i o n s on s y n t h e t i c hexap l o i d s w h e a t s ( S h a h et al. 1987) all three lines h a d l o w e r N a + c o n c e n t r a t i o n s t h a n Ae. squarrosa. C o n c e n t r a t i o n s o f K + were h i g h e r in the synthetic, Ae. squarrosa a n d the t w o 4 D s u b s t i t u t i o n lines t h a n in L a n g d o n o r a n y o f the o t h e r s u b s t i t u t i o n lines. T h e 2D(2B) s u b s t i t u t i o n line h a d significantly ( 0 . 1 % ) h i g h e r l e a f N § c o n c e n t r a tions t h a n L a n g d o n . C o n c e n t r a t i o n s o f C a 2 § a n d M g 2§ in l e a f s a p were n o t s i g n i f i c a n t l y d i f f e r e n t a m o n g the v a r i o u s lines (Fig. 1), b e i n g r e d u c e d b y salinity in all cases ( d a t a n o t shown). C h l o r i d e c o n c e n t r a t i o n s in the leaves were n o t g r e a t ly affected b y the s u b s t i t u t i o n o f c h r o m o s o m e 4 D for 4 A o r 4B (Fig. 2), b u t were slightly l o w e r in these two lines, a n d in Ae. squarrosa a n d the s y n t h e t i c h e x a p l o i d , t h a n in L a n g d o n . T h e 2D(2B), 3D(3B) a n d 7D(7B) L a n g d o n s u b s t i t u t i o n lines h a d l e a f C1- c o n c e n t r a t i o n s considerably higher than those of Langdon. Leaf NO~ c o n c e n t r a t i o n s were h i g h e r in Ae. squarrosa, the s y n t h e t ic h e x a p l o i d , a n d the 4 D ( 4 B ) a n d I D ( 1 A ) s u b s t i t u t i o n lines t h a n in L a n g d o n , b u t l o w e r in m o s t o f the o t h e r s u b s t i t u t i o n lines. A clear effect o f c h r o m o s o m e 5 D c a n be seen in the l e a f S O ] - c o n c e n t r a t i o n s . T h e s e were h i g h e r in the 5 D ( 5 A ) a n d 5D(5B) s u b s t i t u t i o n lines t h a n in L a n g d o n o r the o t h e r s u b s t i t u t i o n lines, w h e t h e r the p l a n t s were g r o w n in the presence (Fig. 2) o r a b s e n c e ( d a t a n o t s h o w n ) o f salt. E l e v a t e d S O l - c o n c e n t r a t i o n s were n o t f o u n d in Ae. squarrosa o r the s y n t h e t i c hexa-
iiiiiLiiiiiiii N a +
500
400
300 Fig. 1. Inorganic cations (mol-m-3 expressed sap) in leaves of wheats grown for 21 d in nutrient solution plus 150 mol. m 3 NaC1 (+7.5 mol-m -3 CaCI2). LANG = Langdon tetraploid wheat; AE.SQ. = Aegilops squarrosa; SYN= synthetic hexaploid wheat; 1D (IA) etc. = Langdon disomic substitution lines
200 100
ID 2D 3D 4D 5D 6D 7D 1D 2D 3D 4D 5D .6D 7D LANG AE. SYN (IA) (2A) (3A) (4A) (5A) (6A) (7A) (1B) (2B) (3B) (4B) (5B) (6B) (7B) SQ.
592
J. Gorham et al. : K+-Na + discrimination in wheat Ol-
700 600 500
400 300 Fig. 2. Inorganic anions (mol-m 3 expressed sap) in leaves of wheats grown for 21 d in nutrient solution plus 150 molto -3 NaC1 (+7.5 mol-m -3 CaClz). L A N G = Langdon tetraploid wheat; AE.SQ. = Aegilops squarrosa; S Y N = synthetic hexaploid wheat; 1D (1A) etc. = Longdon disomic substitution lines
200 100
1D 2D 3D 4D 5D 6D 7D 1D 2D 3D 4D 5D 6D 7D LANGAE. SYN (IA) (2A) (3A) (4A) (5A) (6A) (7A) (1B) (2B) (3B) (4B) (5B) (6B) (7B) SQ.
root cation concentrations (mol-m-3 in expressed sap) in the tetraploid wheat Langdon, Aegilops squarrosa, and a synthetic hexaploid wheat, and in disomic substitution lines of D-genome chromosomes in Langdon. Values are means of four (Langdon, Aegilops squarrosa and the synthetic hexaploid) or six replicates -+ SEs
Table 2. Effect of 150 mol.m 3 (+7.5 mol.m 3 CaClJ NaC1 on photosynthesis (COz exchange rate, CER) and transpiration (E) in the tetraploid wheat Langdon, Aegilops squarrosa, and a synthetic hexaploid wheat, and in disomic substitution lines of D-genome chromosomes in Langdom Values are the means of six replicates _ SEs
NaC1 added (mol. m - 3)
NaC1 added (mol- m - 3)
Table 1. Effect of 150 mol.m -3 NaC1 (+7.5 mol-m-3CaC12) on
Potassium 0
Langdon (BBAA) 81.0_+ 6.7 Aegilops 64.0_+ 8.0 squarrosa (DD) Synthetic 102.0-+ 8.1 (BBAADD)
Sodium 150
0
150
30.3-+3.7 63.3-+3.1
1.8-+0.6 1.4-+0.3
37.5-+3.2 36.8-+1.2
47.5-+0.9
2.0-+0.8
37.5-+4.8
Langdon disomic substitution lines 1D(1A) 77.8-+ 7.5 35.7_+7.5 2D (2A) 75,8_+ 7.0 29.2_+4.0 3D(3A) 79.5_+ 9.2 37.5-+7.3 4D (4A) 73.2-+ 8.8 29.8_+4.1 5D(5A) a 68.7_+11.9 34.7-+3.5 6D (6A) 97.0-+11.2 19.7-+3.6 7D (7A) 51.7__. 9.2 12.8-+3.1
2.6-+0.9 1.2_+0.5 2.1-+0.3 3.5-+0.9 5.9_+3.3 2.4-+0.6 2.8-+0.4
28.0-+6.5 36.0-+5.2 15.5-+3,1 47.5-+8.8 31.0-+4.0 23.3+4,6 21.7___6.3
1D (1B) 2D (2B) 3D (3B) a 4D (4B) a 5D (5B) ~ 6D (6B) ~ 7D(7B)
3.8-+1.7 2.0-+0.4 3.3+1.6 6.5-+4.3 1.8-+0.4 2.4-+0,8 0.3_+0.3
30.3-+6.2 35.8-+5.1 25.0-+4.6 27.7__6.8 23.3-+5.3 22.0+3.1 19.2+5.3
77.7-+ 7.4 68.7-+ 6.1 81.3-+ 7.5 60.0-+ 6.9 62.3_+ 6.6 66.7-+ 7.7 21.2--+11.3
32.7-+7.5 26.3-+4.8 23.7-+4,9 24,5-+8.3 22.5-+4.5 21,7_+ 1,5 8,8_+1,7
Langdon (BBAA) Aegilops squarrosa (DD) Synthetic (BBAADD)
Photosynthesis CER (~tmol.m-2.s -1)
Transpiration E (mmol.m-2.s -t)
0
150
0
150
10.4-+1.1 11.2-+1.2
5.3 -+0.5 7.9-+1.8
8.7-+1.4 10.5-+0.8
3.8-+0.5 5.4-+0.9
13.6 -+ 1.4
8.9 • 2.0
9.7-+0.6
6.1-+1.4
7.6_+1.2 6.3_+1.0 8.2_+0.7 6.5__.1.0 6.1_+1.2 8.6_+0.9 7.2_+1.4
3.3_+1.2 3.1_+0.5 2.3_+0.8 4.1___0.6 4.2_+1.5 4.3_+0.6 3.6_+0.9
Langdon disomic substutition lines 1D (1A) 8.7_+1.4 3.9_+1.4 2D (2A) 7.9_+1.1 4.1_+0.7 3D (3A) 11.6_+1.5 2.6_+1.0 4D (4A) 10.6_+1.7 4.5_+1.9 5D (5A) 8.3_+1.2 4.6_+0.7 6D (6A) 12.4-+1.0 5.4-+0.4 7D (7A) 10.1_+2.0 3.8_+0.7
" Mono- or telosomic for the replaced A or B chromosome
1D 2D 3D 4D 5D 6D 7D
(1B) (2B) (3B) (4B) (5B) (6B) (7B)
11.9_+2.2 12.7_+0.8 11.1 _+1,4 9.8-+1.7 9.6_+1.4 10.0_+0.9 9.3_+0.9
5.6_+1.0 10.4_+1.6 3.8_+0.4 2.6_+0.8 10.4_+1.1 3.0+_1.0 3,5 _+0.9 9.5 _+1.3 3.1 +0,6 6.2_+1.6 7.9-+1.3 4,8--+0.7 4.9_+1.6 8.0-+1.2 3.1 _+IA 5.2_+0.6 8.5_+0.8 3.9_+0.3 2.3-+1.0 8.0+_1.0 2.4-+ 0.9
ploid. I n o r g a n i c P O 2 c o n c e n t r a t i o n s in the s u b s t i t u t i o n lines were n o t significantly different f r o m those in L a n g don. C o n c e n t r a t i o n s o f N a § a n d K + in the r o o t s o f these p l a n t s (Table 1) w e r e n o t affected by the p r e s e n c e o f
the 4 D c h r o m o s o m e to the s a m e e x t e n t as were l e a f c o n c e n t r a t i o n s . A t 150 m o l - m a NaC1 the r o o t N a + conc e n t r a t i o n s were a l m o s t identical in L a n g d o n , A e . s q u a r r o s a a n d the synthetic h e x a p l o i d . S o d i u m c o n c e n t r a t i o n s in the L a n g d o n s u b s t i t u t i o n lines were generally lower,
J. Gorham et al. : K+-Na + discrimination in wheat
593
with the exception o f the 4 D ( 4 A ) s u b s t i t u t i o n . I n the p l a n t s g r o w n in the presence of salt, r o o t K + c o n c e n t r a tions were higher in Ae. squarrosa a n d the h e x a p l o i d t h a n in L a n g d o n , b u t lower i n the 7 D ( 7 A ) a n d 7D(7B) s u b s t i t u t i o n lines. T h e synthetic h e x a p l o i d h a d a slightly higher p h o t o synthetic rate per u n i t leaf area in the absence o f salt t h a n the diploid or t e t r a p l o i d wheats (Table 2). This difference was m a i n t a i n e d a n d e n h a n c e d w h e n the p l a n t s were g r o w n in 150 m o l . m - 3 NaC1. T h e relative reductions in the rates o f p h o t o s y n t h e s i s a n d t r a n s p i r a t i o n caused by salt were greatest in L a n g d o n a n d least in the synthetic hexaploid. I n the absence o f salt the rates of p h o t o s y n t h e s i s a n d t r a n s p i r a t i o n exhibited b y the
L a n g d o n s u b s t i t u t i o n lines were n o t greatly different f r o m those o f L a n g d o n itself. I n the presence o f salt the rates for the 4 D ( 4 A ) a n d 4D(4B) s u b s t i t u t i o n lines were a m o n g the highest for the s u b s t i t u t i o n lines, b u t were n o t significantly different f r o m the rates exhibited by L a n g d o n . These two lines exhibited a m o n g the smallest decreases in p h o t o s y n t h e s i s a n d t r a n s p i r a t i o n in the presence o f salt, especially a m o n g the B - g e n o m e substit u t i o n lines. Values for some o f the other s u b s t i t u t i o n lines (e.g. 2D(2B)) were significantly lower t h a n those for L a n g d o n .
Ion accumulation in Chinese Spring aneuploids. Sap osm o t i c pressures a n d i n o r g a n i c - i o n c o n t e n t s o f the y o u n -
Table 3. Sap osmotic pressure (mOsmol.kg 1) and inorganic solute concentrations (mol-m 3 in expressed sap) in aneuploid lines of Chinese Spring hexaploid wheat grown for three weeks in nutrient solution plus 125 mol.m-3 NaC1 plus 6.25 mol.m -a CaC12. Values are the means of at least five replicates_+ SEs. Data for lines lacking the long arm or the whole of chromosome 4D are shown in bold type
Sap osmotic pressure Sodium Potassium Calcium Magnesium Chloride Nitrate Orphosphosphate Sulphate K++Na + K+/Na +
Euploid chinese
Ditelosomic 4D short
Ditelosomic 4D long
Tetra 4D
Nullisomic 4B tetrasomic 4A
Nullisomic 4B tetrasomic 4D
Nullisomic 4D tetrasomic 4A
541 _+37 85 _+16 223 _+ 6 27 _+ 3 28 _+ 3 180 _+15 30 _+ 4 28 _+ 3 8 -+ 1 329 _+25 3.6_+ 0.6
594 _+61 261 _+26 71 _+ 8 13 -+ 3 17 _+ 5 230 -+41 17 _+ 4 32 _+ 3 10 _+ 8 332 +29 0.6_+ 0.4
550 _+58 103 +25 232 +19 24 _+ 5 26 _+ 5 210 _+34 29 • 8 24 _+ 2 8 -+ 2 335 _+34 4.0_+ 1.4
673 _+101 78 -+ 12 232 _+ 16 24 _+ 3 37 _+ 5 195 _+ 33 35 _+ 6 37 -+ 5 13 -+ 2 301 _+ 13 3.5_+ 0.7
552 _+36 95 _+17 206 _+12 25 _+ 3 29 _+ 5 184 +19 29 • 6 26 _+ 4 10 _+ 1 333 _+36 3.4_+ 0.6
495 61 231 20 27 165
592 _+77 274 _+31 124 _+38 16 _+ 1 19 + 4 161 _+12 30 _+ 6 44 -+ 3 12 _+ 1 408 _+54 0.5_+ 0.2
_+23 -+11 -+12 _+ 2 _+ 3 _+15 55 _+12 20 -+ 2 7 _+ I 292 -+10 5.5_+ 1.4
Table 4. Growth and yield data for Chinese Spring and Langdon aneuploid lines of wheat grown in hydroponic culture with nutrient solution plus 0, 75 or 125 mol-m -3 NaC1 (+0, 3.75 or 6.25 mol.m -a CaC12). Values are the means_+SEs of data from at least six replicate plants. Data for lines lacking the long arm or the whole of chromosome 4D are shown in bold type NaC1 (tool 9m - 3)
Chinese Spring (hexaploid)
Langdon (tetraploid)
Euploid
Nullisomic 4D tetrasomic 4A
Ditelosomic 4D long
Ditelsomic 4D short
Euploid
Substitution 4D (4A)
Substitution 4D (4B)
Height(cm)
0 75 125
106+ 5 108_+ 5 88+ 6
102+ 9 92_+ 5 75_+ 6
84-t-ll 82+ 3 77_+ 4
94+10 97_+ 2 58+ 7
114+ 5 109_+ 4 76-+ 5
110!15 87_+12 97+ 6
92_+8 82___5 57-+2
Plant weight (g)
0 75 125
38+ 7 35-+ 5 18_+ 4
29-+11 16_+ 4 6_+ 2
38-+13 26_+ 8 17_+ 3
9+ 3 14-+ 1 2_+1
47-+ 9 32_+ 5 8_+ 2
16_+ 4 8-+ 7_+ I
7_+2 5_+2 2_+1
Seedweight (g)
0 75 125
12_+ 2 8_+ 2 4_+ 1
5_+ 2 2_+ 1 1_+ 0
5_+ 2 3_+ 2_+ 0
1_+1 2_+0 0_+ 0
15_+ 3 6_+ 3 1_+ 0
6_+ 4 2_+ I 1_+ 0
1_+0 0_+0 0_+0
Numberof tillers
0 75 125
15_+ 2 15_+ 2 10_+ I
13_+ 3 8_+ 2 4_+ 1
21_+ 6 15_+ 3 12+ 1
7_+ 2 9_+ l 3_+ 1
14_+ 2 12-+ 2 6_+ 1
7_+ 3 4+_ I 5_+ 1
5_+1 4_+1 2_+0
Total number of seeds
0 75 125
430_+64 460-+71 318_+54
144_+65 98_+27 61_+23
170_+83 127_+50 98_+23
41_+20 111-+14 16_+10
Number of seeds pertiller
0 75 125
26_+ 2 28_+ 2 30_+ 3
9_+ 2 10_+ 2 11_+ 3
6_+ 2 8_+ 2 8_+ 2
7_+ 3 12_+ 2 3_+ 1
4 3 6 _ + 7 9 145-+85 2 6 0 _ + 3 5 70_+23 68_+18 67_+11 31_+ 1 22_+ 1 9_+ 2
19_+ 3 14_+ 3 15_+ 2
16-+8 11_+1 5+4 3_+1 3_+1 2_+1
594
J. Gorham et al. : K+-Na + discrimination in wheat
gest fully e x p a n d e d leaves o f C h i n e s e S p r i n g h e x a p l o i d w h e a t a n d a n u m b e r o f a n e u p l o i d lines g r o w n for 21 d in 125 m o l . m - 3 NaC1 plus 6.25 m o l - m - 3 CaCI2 are s h o w n in Table 3. T h e lines l a c k i n g t h e whole, o r j u s t the l o n g a r m , o f c h r o m o s o m e 4 D h a d h i g h l e a f N a § a n d low l e a f K + c o n c e n t r a t i o n s , resulting in c o n s i d e r a b l y l o w e r K + / N a + ratios. T h e C a 2+ a n d M a 2+ c o n c e n t r a t i o n s were slightly l o w e r t h a n in e u p l o i d C h i n e s e S p r i n g o r the o t h e r a n e u p l o i d lines, b u t o t h e r w i s e there were n o c o n s i s t e n t effects o f the 4 D c h r o m o s o m e o n the c o n c e n t r a t i o n s o f o t h e r ions. S i m i l a r results were o b t a i n e d in a s e c o n d h a r v e s t t a k e n 21 d l a t e r (results not shown).
Effect of salt on grain yield. T h e results o f a n e x p e r i m e n t in w h i c h the effects o f salt o n g r o w t h a n d g r a i n yield o f a n e u p l o i d lines o f C h i n e s e S p r i n g a n d L a n g d o n were e x a m i n e d a r e s h o w n in Table 4. P l a n t h e i g h t w a s the p a r a m e t e r least affected b y a n e u p l o i d y o r salt t r e a t m e n t , w h e r e a s p l a n t w e i g h t was affected b y b o t h . E x c e p t for the C h i n e s e S p r i n g line d i t e l o s o m i c for the l o n g a r m o f c h r o m o s o m e 4D, all o f the o t h e r a n e u p l o i d lines were s m a l l e r at all salt c o n c e n t r a t i o n s (0, 75 a n d 125 m o l t o - 3 NaC1) t h a n the e u p l o i d lines. G e n e r a l l y , the a n e u -
p l o i d lines were w o r s e t h a n the e u p l o i d w h e a t s in t e r m s o f t o t a l seed weight, n u m b e r s o f seed a n d tillers, a n d n u m b e r o f seeds p e r tiller. T h e Chinese S p r i n g line ditel o s m i c f o r the l o n g a r m o f c h r o m o s o m e 4 D a n d the L a n g d o n 4 D ( 4 A ) s u b s t i t u t i o n line were the best o f the a n e u p l o i d lines as j u d g e d b y the effect o f salt o n seed p r o d u c t i o n . D e s p i t e the p o o r a b s o l u t e p e r f o r m a n c e o f the a n e u p l o i d lines there is s o m e i n d i c a t i o n o f a beneficial effect o f c h r o m o s o m e 4D. W h e n the t o t a l n u m b e r o f seeds p e r p l a n t at 125 m o l . m - 3 NaC1 is expressed as a p e r c e n t a g e o f the c o n t r o l (no salt) values, then the lines w i t h c h r o m o s o m e 4 D ( n o r m a l t y p e in Table 4) h a d a m e a n v a l u e o f 5 2 % a n d the lines l a c k i n g c h r o m o s o m e 4 D ( b o l d t y p e in Table 4) h a d a m e a n o f 3 2 % . O n l y the 4D(4B) s u b s t i t u t i o n gave l o w e r relative yields t h a n the lines l a c k i n g c h r o m o s o m e 4D. T h e 4D(4B) s u b s t i t u tion, even w h e n m o n o s o m i c f o r 4B, is k n o w n to h a v e p o o r fertility ( J o p p a a n d W i l l i a m s 1988). W h e n the g r a i n f r o m this e x p e r i m e n t was a n a l y s e d for i n o r g a n i c - i o n c o n t e n t s it was f o u n d (Table 5) t h a t the lines l a c k i n g the 4 D c h r o m o s o m e (or its l o n g a r m ) h a d c o n s i d e r a b l y h i g h e r N a + c o n t e n t s t h a n the o t h e r lines, especially w h e n g r o w n a t 1 2 5 m o l - m 3 NaC1. C o n v e r s e l y K § c o n t e n t s were g e n e r a l l y lower, except
Table 5. Inorganic-ion contents (mmol. (kg DW) 1) of the grain of aneuploid lines of Chinese Spring and Langdon wheats grown in hydroponic culture with nutrient solution plus 0, 75 or 125 mol-m-3 NaC1 (+0, 3.75 or 6.25 tool.m-3 CaC12). Values are the means of four replicates _+SEs. Data for lines lacking the long arm or the whole of chromosome 4D are shown in bold type NaC1 (tool- m - 3)
Chinese Spring (hexaploid) Euploid
Sodium
Potassium
Calcium
Magnesium
Chloride
Nullisomic 4D tetrasomic 4A
0 75 125
4 _+ 1 51 _+23 76 -+23
0 75 125
92 _+ 6 156 _+24 161 _+21
0 75 125 0 75 125
3 9 4 23 38 26
_+ -+ _+ _+ _+ •
0 4 1 3 4 3
13 -+ 2 80 -+27 120 _+17 84
_+ 1
151 _+25 140 _+13 3 3 4 24 24 31
• • • • _+ _+
1 1 1 4 4 5
Langdon (tetraploid) Ditelosomic 4D long 12 -+ 2 22 • 5 34 _+ 8 88 _+ 6 104 _+ 4 114 _+20 4 5 3 25 24 21
_+ -+ _+ _+ _+ _+
I 2 1 3 3 2
Ditelosomic 4D short 10 -+ 3 117 441
•
84
_+
55-+14
_+ 51
257_+82
12 _+4 54 _+12 128 _+45
112_+16
177
_+ 17
164_+43
82 + 9 141 _+14 182 _+24
3 4 8 28 30
_+ 1 _+ 1 • 4 • 4 _+ 2 _+ 19
2-+ 5_+ 8_+ 21• 30_+ 26_+
3 _+I 5_+0 6_+1 23 _+3 28 _+3 37 _+5
47
81_+ 7
0 1 2 2 2 6
17 _+ 1 _+ 22 583 _ + 1 4 9
17_+ 2 107_+35 239_+51
17 • 4 158 _+37 142 _+13
33 _+ 1 --4- 16
16_+ 3 52-+13
_+
0
45_+10
21 _+ 4 20 _+4 29 + 5
1 _+ 0 5 _+ 1 5 -+ 0
5_+ 1 11_+ 1 9_+ 2
6+1 5-+1 4___2
3_+ 0 1_+ 0 3_+ 0
2_+0 2+1 i _+ 0
10 _+ 2 91 _+29 213 _+28
12 _+ 4 27 _+ 3 71 _+23
0 75 125
25 _+10 31 _+ 4 38 _+ 1
29 + 3 69 _+11
27 _+ 1 21 _+ 4
37
28
Orthophosphate
0 75 125
3 _+ 1 2 _+ 0 2 • 0
2 _+ 0 4 _+ l 5 -+ 1
6 • 1 2 • 0 2 • 0
Sulphate
0 75 125
2 _+ 0 2 _+ 0 2 _+ 0
2 + 0 3 + 0 4 -+ 0
3 _+ 0 2 _+ 0 1 _+ 0
2 _+ 0 2 _ 0 3 _+ 0
K+/Na +
0 75 125
31.7• 8.9 5.2_+ 1.3 2.7-+ 0.5
6.9_+ 0.7 3.1_+ 1.1 1.2• 0.1
7.6_+ 1.0 6.2_+ J.3 3.6_+ 0.5
+ 3
Substitution 4D (41)
194 _+ 25
5
11 _+ 2 62 _+11 122 _+30
_+ 3
9_+ 2
25
0 75 125
Nitrate
Euploid
155
100 43
14.3_+ 1.3+ 0.4-+
6.4 0.1 0.0
12.2_+ 4.1 2.4_+ 0.4 0.9-+ 0.3
13.7-+ 5.0 3.4-+ 0.9 1.8_+ 0.3
J. Gorham et al. : K+-Na + discrimination in wheat
595
that the Chinese Spring line ditelosomic for the long arm o f chromosome 4D had the lowest K + content when subjected to salinity. Thus the lines lacking the 4D chromosome had the lowest grain K + / N a + ratios. In contrast to leaf C1- concentrations, those in the grain were greatly increased in the absence of c h r o m o s o m e 4D, especially in the Chinese Spring line ditelosomic for the short arm o f c h r o m o s o m e 4D. There were no consistent differences between the other inorganic ions in the grains of the various aneuploid lines tested. Discrimination between K + and Na + at low external sodium concentrations. In a number of experiments with the tetraploid wheat L a n g d o n and synthetic hexaploid w h e a t s or Chinese Spring it was observed that the enhanced ability of the hexaploid wheats to discriminate in the uptake of Na + and K + was expressed over a wide range of external N a + concentrations upto 250 m o l - m -3 NaCI (data not shown). The results of a typical experiment at the lower end of the concentration range are shown in Fig. 3, where the leaf Na + and K + concentrations o f L a n g d o n and Chinese Spring are plotted against external salinity in the range 5-50 tool. m -3 NaC1. The greater accumulation of N a + in the leaves of L a n g d o n is evident even at 5 tool. m - 3 NaC1, whereas the K + concentrations are not significantly different until 30 m o l - m - 3 NaC1. Uptake o f 22Na into low-salt-grown seedlings. Uptake from solutions containing lower concentrations o f Na + were investigated by following the incorporation of 22Na into excised or intact roots o f seedlings which had been grown on a medium containing only Ca and Mg salts. Uptake into excised roots was determined after short (/2rain) or long (60rain) incubations in 2 t o o l .
240
Table 6. Rates of influx of 22Na (mmol-(kg FW)-I-h -1) from 2 mol.m-3 NaC1 into excised, low-salt-grown roots of Langdon and Chinese Spring wheats. Each value is the mean of three replicates_+ SE Influx time/wash time (rain)
Langdon
Chinese Spring
2 tool-m- 3 NaC1 alone 12/ 5 45/30
1.49+0.02 2.52 _ 0.06
1.29_+0.05 1.864- 0.19
2 mol. m- 3 NaC1 plus 0.5 mol-m -3 KC1 15/ 5 60/30
1.04+0.08 0.93 4-0.20
0.94_+0.26 0.66 -+0.09
24 22 20 .-..18
///o ' /
o L-12 7
9
10
E 8 ..i." O
6
E t-" 4 2 ~Z
0 -2
I
I
I
I
,5
10
15
20
25
TIME (h) Fig. 4. Uptake of 22Na into the roots (e, 9 and the shoots (T, v) of low-salt-grown seedlings of Chinese Spring (hexaploid, o, v) and Langdon (tetraploid, e, v) wheats. The uptake solution contained 1 mol.m 3 NaC1 and 0.5 mol-m - 3 C a S O 4. The SEs did not exceed three times the size of the symbols
0 220 0
200
v".~.%
180
K ~v--
Q. 160 x 0 140
v
"?E 120 .2." 100 0
E
+ "./ +
121 Z
"6
(D ._1
/
8o
No
6O
4o -O
20
o 0'
10 '
20 '
~0
go
EXTERNAL NaCl ( m o l m -3 )
5'o
Fig. 3. Concentrations (mol-mol 3 expressed sap) of Na + (e, v) and K + (9 ,7) in leaves of Chinese Spring hexaploid wheat (+D genome, o, o) and Langdon tetraploid wheat (--D genome, ~v, v) grown for 15 d in basic nutrient solution containing low NaC1 concentrations. The SEs were not more than twice the size of the symbols used
m -3 NaC1, followed by 5- or 30-rain washes in cold (4 ~ C), unlabelled medium (Table 6). In all cases the uptake rates were higher in L a n g d o n than in Chinese Spring, and were decreased in the presence of 0.5 molt o - 3 KC1. The long-term (vacuolar) influx was reduced to a greater extent by KC1 than the short-term (cytoplasmic) influx. When uptake into whole seedlings from a solution containing 1 tool. m - 3 NaC1 was followed (Fig. 4) it was observed that, although the initial uptake into the roots of L a n g d o n was slightly higher than into those of Chinese Spring, after about 20 h Chinese Spring had a slightly higher 22Na content than Langdon. Transport of 22Na to the shoots was measurable after a few hours and was considerably greater in L a n g d o n than in Chinese Spring. Uptake of 22Na into the shoots of aneuploid lines of L a n g d o n and Chinese Spring was also investigated (Table 7). The lines lacking chromosome 4D accumulated more ZZNa than those lines lacking this chromosome, but the Chinese Spring lines ditelosomic for the short
596
J. Gorham et al. : K+-Na + discrimination in wheat
Table 7. Uptake of 2ZNa from 1 mol.m -3 NaC1 (plus 0.1 mol.
m-3 KC1) over 48 (expt. 2) and 72 h (expt. 1) into shoots of intact, low-salt-grown seedlings of aneuploid lines of Chinese Spring and Langdon, expressed as mean percentage uptake into Langdon • SEs. Data for lines lacking the long arm (or the whole) of chromosome 4D are shown in bold type 72 h Langdon Langdon 4D (4A) substitution Langdon 4D (4B) substitution Langdon 4D disomic addition Chinese Spring Chinese Spring ditelosomic 4D short Chinese Spring ditelosomic 4D long Chinese Spring tetra 4D Chinese Spring nulli 4D tetra 4A
48 h
100 • 10
6+ 6+ 7_+ 10+ 23 • 11 • 5_+ 30 +
1
100 -+10 8 _+ 2
I
8+ 1
3 2 4 3 1
8-+ 10-+ 21 -+ 5 _+ 7_+
2 2 3 2 1
4
21 -+ 3
Table 8. Fluxes of 22Na (gmol-kg-l.h -1) from intact, low-salt-
grown wheat roots loaded with 1 mol.m 3 22NaC1 for 24 h Flux
T. turgidurn cv. Langdon
T. aestivum cv. Chinese Spring
Vacuolar effiux ~co~c) Cytoplasmic efflux ~oo {cyt) Transport to shoot ~cx K-stimulated efflux ~oo(K-aep)
173_+ 8 673 _+188 513 • 59 80_+ 11
121___13" 473 _-+_93 34_ 1"** 162_+22"
Means significantly different at the 5% (*) or 0.1% (***) probability levels a r m of 4D and nullisomic for 4D b o t h accumulated less 22Na than Langdon.
Efflux of 22Na from intact seedlings, Low-salt-grown seedlings of Chinese Spring and L a n g d o n wheats were incubated in 1 tool. m - 3 22NaC1 for 24 h before the start of an efflux experiment in which the unlabelled washing solution was changed every 10 rain. The flux rates were calculated according to Jeschke and J a m b o r (1981) and are shown in Table 8. The cytoplasmic efflux was greater than the vacuolar efflux for both species, but the differences between L a n g d o n and Chinese Spring were small for b o t h effluxes. In contrast the xylem flux (transport to the shoots) was very much greater in L a n g d o n than in Chinese Spring. When KC1 (1 m o l . m - 3 ) was added to the washing solution after the establishment of steadystate vacuolar 22Na efflux, an additional K-stimulated ZZNa efflux was observed which occurred over the next 60 min. This K-stimulated efflux was significantly higher in Chinese Spring than in Langdon. Discussion
The data presented here show that the long a r m of chrom o s o m e 4D contains a trait (controlled by one or more genes ?) which affects the accumulation of both N a + and K + in the leaves o f wheat. This trait is operative over a wide range of external salt concentrations and appears to be constitutive. In the grain the contents of N a +,
K + and C1- are altered by the presence of c h r o m o s o m e 4D. The difference in the effect of c h r o m o s o m e 4D on C1- transport to the shoots and to the grain m a y reflect different influences on transport via the xylem (to the shoots) and the phloem (to the grain). Net transport rates of N a + and K + to the shoots are affected by the presence of the enhanced trait for K + - N a + discrimination, but leaf anion and root anion and cation concentrations are not. Studies of 22Na fluxes have confirmed these findings, with influx into and efflux out of the cytoplasm and vacuoles only slightly higher in the roots of the tetraploid wheat L a n g d o n than in the more salt-tolerant hexaploid cultivar Chinese Spring, but transport to the shoots m u c h less in Chinese Spring. Similar results have been obtained with excised roots (D.W. Rains, University of California, Davis, personal communication). It should be noted that, at the start of the efflux period, the Chinese Spring roots would have contained slightly more 221'qa than the roots of the tetraploid wheat. Davis (1984) has reported that, of two hexaploid wheats, the more salt-tolerant cultivar transported less Z2Na to the shoots than the less tolerant cultivar. Other fluxes were similar in the two cultivars. The comparison between Chinese Spring and Langdon is not ideal since the difference between the two is both the presence or absence of the whole of the D genome and any differences in their A and B genomes. These 2ZNa uptake and efflux experiments are being repeated using the aneuploid lines described above. A feature of both L a n g d o n and Chinese Spring was the K-stimulated N a + effiux (Table 8). High rates of transient N a + efflux on addition o f K + have been observed in several cereal grasses (Jeschke 1983; Jeschke and Nassery 1981). The results presented here, and other observations of ours involving X-ray microanalysis of roots and observation of dye m o v e m e n t indicate that neither uptake into the root nor transpirational effects can explain the K + - N a + discrimination controlled by c h r o m o s o m e 4D. Thus it seems likely that the discrimination between N a + and K + occurs mainly at the point of xylem loading, or possibly at the endodermis. Jeschke (1983) suggests that selectivity for K + over N a + at the symplasmxylem b o u n d a r y occurs in barley, but it is difficult to obtain reliable estimates of ion concentrations in the cytoplasm of the xylem p a r e n c h y m a cells. Sodium accumulation in the shoots of the hexaploid wheats was less than in the shoots of Ae. squarrosa, indicating some interaction between the D genome and the A and B genomes. It is not k n o w n whether this represents an interaction with a specific mechanism controlling ion m o v e m e n t in tetraploid wheat, or whether it is a m o r e general consequence of the difference in ploidy level and growth rate (see B a m a k h r a m a h et al. 1984 for a general discussion of the effects of ploidy on growth and yield of wheat). Furthermore, the Chinese Spring lines lacking the whole, or just the long arm, of chromosome 4D had 22Na-uptake rates (Table 7) or grain N a + contents (Table 5) different f r o m those of Langdon. In this case also it is not possible to distinguish between a specific interaction between the tetraploid wheat gen o m e and other transport mechanism(s) on D-genome
J. Gorham et al. : K+-Na + discrimination in wheat c h r o m o s o m e s , and a non-specific effect o f the difference in ploidy level. A large n u m b e r o f i m p o r t a n t physiological a n d m o r p h o l o g i c a l characters (e.g. the R h t 2 dwarfing gene f r o m N o r i n 10) are k n o w n to be located on D g e n o m e c h r o m o s o m e s ( M c I n t o s h 1973). We have a t t e m p t e d to answer the question o f w h a t effect the e n h a n c e d trait for K + - - N a + discrimination on c h r o m o s o m e 4 D has on g r o w t h a n d grain yield in saline conditions. There is some evidence which indicates that N a + is m o r e toxic to w h e a t t h a n C1- ( K i n g s b u r y and Epstein 1986) and that leaf K + / N a + ratio is a factor in determining salt tolerance in the b r o a d e s t sense ( R a n a 1986; R a s h i d 1986). The results presented in Table 4 show quite clearly that, in absolute terms, the detrimental effects o f a n e u p l o i d y are larger than a n y effect o f the 4 D K + - N a + discrimination character. All o f the aneuploid lines p r o d u c e d less grain, and in m o s t cases less straw, than the euploid wheats. This agrees with the d a t a o f J o p p a a n d Williams (1988) for L a n g d o n disomic substitution lines. A l t h o u g h there were indications o f a beneficial effect o f c h r o m o s o m e 4D, it will only be possible realistically to assess the i m p a c t o f the enh a n c e d K + - N a + discrimination character on wholeplant p e r f o r m a n c e a n d yield c o m p o n e n t s w h e n it can be transferred, w i t h o u t the rest o f the long a r m o f chrom o s o m e 4D, to a tetraploid wheat. Such r e c o m b i n a t i o n m a y be possible t h r o u g h hybrids involving tetraploid wheats deficient for the h o m o e o l o g o u s pairing suppressor genes. A n o t h e r a p p r o a c h to assessing the i m p a c t o f c h r o m o s o m e 4 D w o u l d be to obtain i o n - t r a n s p o r t m u t a n t s (like the scabrous diminutive m u t a n t o f Capsicum annuum, Tal a n d Benzioni 1977), preferably in a diploid species. It is i m p o r t a n t to emphasize that the e n h a n c e d K + -N a + discrimination character described here is n o t seen as the only m e c h a n i s m controlling salt tolerance in the Triticeae. O t h e r mechanisms, which p r o b a b l y operate in totally different ways, are f o u n d in other m e m b e r s o f this tribe ( G o r h a m a n d W y n Jones 1989). F u r t h e r m o r e barley, which is generally m o r e salt tolerant than tetraploid or hexaploid w h e a t (Maas 1986), has leaf N a + c o n c e n t r a t i o n s similar to those f o u n d in tetraploid w h e a t when g r o w n in the same conditions. Thus the c h r o m o s o m e - 4 D - c o n t r o l l e d e n h a n c e d K + - N a + discrimination trait is only one m e c h a n i s m which m u s t be integrated with other characters in the p y r a m i d i n g a p p r o a c h to designing a n d breeding crop plants with greater salt tolerance (Yeo and Flowers 1986; G o r h a m a n d W y n Jones 1989). The authors gratefully acknowledge the financial support of the Overseas Development Administration of the United Kingdom, and would like to thank Dr. L.R. Joppa and Dr. C.N. Law for supplying the seeds of aneuploid lines of Langdon and Chinese Spring wheats.
References Bamakhramah, H.S., Halloran, G.M., Wilson, J.H. (J 984) Components of yield in diploid, tetraploid and hexaploid wheats (Triticure spp.). Ann Bot. 54, 51-60 Davis, R.F. (1984) Sodium fluxes in intact roots of wheat varieties
597 differing in salt tolerance. In: Membrane transport in plants, pp. 489-490, Cram, W.J., Janacek, K., Rybova, R., Sigler, K. eds. Academia, Praha Francois, L.E., Maas, E.V., Donovan T.J., Youngs, V.L. (1986) Effect of salinity on grain yield and quality, vegetative growth, and germination of semi-dwarf and durum wheat. Agron. J. 78, 1053 1058 Gorham, J., Wyn Jones, R.G. (1989) A physiologists approach to improving the salt tolerance of wheat. Rachis (in press) Gorham, J., Hardy, C., Wyn Jones, R.G., Joppa, L.R., Law, C.N. (1987) Chromosomal location of a K/Na discrimination character in the D genome of wheat. Theor. Appl. Genet. 74, 584-588 Jeschke, W.D. (1983) Cation fluxes in excised and intact roots in relation to specific and varietal differences. In: Genetics aspects of plant nutrition, pp. 71 86, Saric, M.R., Loughman, B.C., eds. Nijhoff, The Hague, Netherlands Jeschke, W.D., Jambor, W. (1981) Determination of unidirectional sodium fluxes in roots of intact sunflower seedlings. J. Exp. Bot. 32, 1257-1272 Jeschke, W.D., Nassery, H. (1981) K + - N a + selectivity in roots of Triticum, Helianthus and Allium. Physiol. Plant. 52, 217-224 Joppa, L.R. (1987) Aneuploid analysis in tetraploid wheat. In: Wheat and wheat improvement, pp. 151 166, Heyne, E.G., ed. Monograph 13, Am. Soc. Agron., Madison, Wis., USA Joppa, L.R., Williams, N.D. (1988) Langdon durum disomic substitution lines and aneuploid analysis in tetraploid wheat. Gehome 30, 22~228 Joshi, Y.C., Dwivedi, R.S., Quadar, A., Bal, A.R. (1982) Salt tolerance in diploid, tetraploid and hexaploid wheat. Ind. J. Plant Physiol. 25, 421-422 Kingsbury, R.W., Epstein, E. (1986) Salt sensitivity in wheat. A case for specific ion toxicity. Plant Physiol. 80, 651 654 Maas, E.V. (1986) Crop tolerance to saline soil and water. In: Prospects for biosaline research (Proc. U.S.-Pakistan Biosaline Workshop) pp. 205 219, Ahmad, R., San Pietro, A., eds. Karachi University, Pakistan McIntosh, R.A. (1973) A catalogue of gene symbols for wheat. In: Proc. 4th Int. Wheat Genet. Syrup., pp. 893-938. University of Columbia, Mo. USA Rana, R.S. (1986) Genetic diversity for salt-stress resistance of wheat in India. Rachis 5, 32-37 Rana, R.S., Singh, K.N., Ahuja, P.S. (1980) Chromosomal variation and plant tolerance to sodic and saline soils. In: Symposium papers (Int. Syrup. Salt-affected Soil), pp. 487-493, Central Soil Salinity Research Inst., Karnal, India Rashid, A. (1986) Mechanisms of salt tolerance in wheat. PhD thesis, University of Agriculture, Faisalabad, Pakistan Sayed, H.I. (1985) Diversity of salt tolerance in a germplasm collection of wheat (Tritieum spp.). Theor. Appl. Genet. 69, 651 657 Shah, S.H., Gorham, J., Forster, B.P., Wyn Jones, R.G. (1987) Salt tolerance in the Triticeae: The contribution of the D genome to cation selectivity in wheat. J. Exp. Bot. 36, 254-269 Tal, M., Benzioni, A. (1977) Ion imbalance in Capsicum annuum, scabrous diminutive, a wiry mutant of pepper. I. Sodium fluxes. J. Exp. Bot. 28, 1337-1341 Weimberg, R. (1987) Solute adjustment in leaves of two species of wheat at two different stages of growth in response to salinity. Physiol. Plant. 70, 381 388 Weimberg, R. (1988) Modification of foliar solute concentrations by calcium in two species of wheat stressed with sodium chloride and/or potassium chloride. Physiol. Plant. 73, 418-425 Wyn Jones, R.G., Gorham, J. (1989) Physiological effects of salinity. Scope for genetic improvement. In: Proc. Int. Syrup. on Improving Winter Cereals under Temperature and Salinity Stresses, Cordoba, 1987. ICARDA, Syria (in press) Yeo, A.R., Flowers, T.J. (1986) Salinity resistance in rice (Oryza sativa L.) and a pyramiding approach to breeding varieties for saline soils. Aust. J. Plant Physiol. 13, 161-173 Received 30 June; accepted 10 November 1989
