Comparative study on morphological and physiological traits related to drought resistance between xeric and mesic Hordeum spontaneum lines in Israel

 

Chen Guoxiong, Tamar Krugman, Tzion Fahima, Abraham B. Korol,

and Eviatar Nevo

 Institute of Evolution, University of Haifa, Haifa 31905, Israel

 

Hard-copy edition pages 22 - 33.

Abstract

 

Three lines of Hordeum spontaneum, 10-30 from mesic, 20-05 and 23-38 from xeric habitat in Israel, and two cultivars of Hordeum vulgare, Noga from Israel and Mona from Sweden were compared in seed germination, 10-30 and 23-38 in seedling stage for the morphological and physiological differences that confer drought resistance or susceptibility. Seed germination was conducted in Petri dish with filter paper at 25 ^oC. The subsequent seedlings were transplanted to fine sand medium in pots (0.55 L). Drought treatment was applied by withholding water for 8 days starting at 2 leaves stage. Cultivars had larger seed size than wild types. Xeric lines had smaller seeds, lower germination percent, and thinner root, but faster subsequent seedling growth than mesic lines. Drought reduced total leaf length and shoot dry weight to a larger extent in 10-30 than in 23-38. Root dry weight of 23-38 was increased by drought treatment, while root dry weight of 10-30 was irrespective of drought treatment. Stomatal conductance was lower in 23-38 than in 10-30 with or without drought treatment, however, its reduction under drought was more pronounced in 23-38 than in 10-30. 23-38 had higher capacity of osmotic adjustment than 10-30. In conclusion, P23-38 is a drought resistant genotype in comparison with P10-30. Its drought resistance is characterized by a low stomatal conductance, high osmotic adjustment, extensive root growth, and small reduction of shoot growth in drought stressed conditions. 

 

Introduction

Drought is a major abiotic stress that severely affects agricultural systems and food production (Boyer 1982). Even intermittent water stress at critical stages of cereal crops may reduce yield (Ludlow & Muchow 1990). To improve drought resistance is probably one of the most difficult tasks for the cereal breeders, because of the complexity of drought conditions in the fields, and of the diversity of drought tolerance strategies developed by the plants. Wild cereals are important for drought resistance since they possess vast genetic diversity that may be missing in crop species (Nevo et al. 1984, 1986; Nevo 1992).

 

There are few traits, such as osmotic adjustment (OA) and stomatal conductance, considered to be important for drought resistance. OA is defined as a decrease of osmotic potential within the cells due to active accumulation of compatible osmolytes during a period of cell water deficit (Ludlow and Muchow 1990). It has been shown that OA contributes to yield stability under drought (Morgan 1984) through keeping leaf turgor and elongation (Turner 1986), maintaining root development and soil moisture extraction (Morgan &Condon 1986), delaying leaf senescence (Hsiao et al. 1984). Stomatal conductance is a plant property related to the ease with which water vapor escapes from plant leaves through small pores in the leaves know as stomata. Stomata closure hence decreasing stomatal conductance to prevent excess water loss is one of mechanisms of plant avoiding drought stress  (Levitt, 1980; McWilliam, 1989). Recently, mapping quantitative trait loci (QTLs) for osmotic adjustment in cultivated cereals has been conducted in wheat (Morgan & Tan 1996), rice (Lilley et al. 1996) and barley (Teulat et al. 1998, 2001), for stomatal conductance in rice (price et al., 1997), maize (Sanguineti et al., 1999) and cotton (Ulloa et al., 2000). However, up to now QTLs for osmotic adjustment and stomatal conductance in more pronounced drought-resistance of wild barley have not been mapped.

 

The primary purpose of this project are to map the candidate genes associated with drought resistant mechanisms, such as OA and stomatal conductance in Hordeum spontaneum by use of DNA-based molecular markers. As the first stage, we compared two lines of wild barley, 23-38 from xeric habitat, 10-30 from mesic habitat, in the difference of seed germination, shoot and root growth, stomatal conductance and leaf OA.

 

Materials and methods

Plant materials The plant materials used in this study included three wild-type barley genotypes, P10-30, P23-38, P20-05, and two cultivars, Mona and Noga. P10-30 originally collected from Maalot of drought index of 0.37, P23-38 from Wadi Qilt of 0.04, P20-05 from Sede Boker of 0.03, in Israel. Mona is from Sweden, and Noga is a Israeli cultivar. Seeds of these genotypes are produced in the Institute of Evolution, University of Haifa, in 1995, with the exception of Noga bought from local company. Germination observation was conducted twice, the first time with seeds of P10-30, P20-05, P23-38 and Noga, the second time with the addition of Mona. Seeds were wet on filter paper in Petri dishes with distilled water followed by cold treatment at 4 ^oC for 2 days. Cold treatment of P20-05 and Noga were prolonged to 4 days in the second batch. Cold treated seeds were incubated at 25 ^oC in an incubator for 2 days, meanwhile the measurement of water uptake of seeds (in first batch), germination percent and subsequent seedlings growth (in both batch) were performed. The seedlings of P10-30 and P23-38 of similar size in the first batch germination were transplanted in pots (0.55 L) with fine sand medium. The seedlings was irrigated once two days with 100 ml per pot of nutrient solution: Ca(NO₃)₂ 1 mM, KNO₃ 1 mM, KH₂PO₄ 0.5 mM, MgSO₄ 0.5 mM, Fe-EDTA 25 μM, MnCl₂ 3 μM, ZnSO₄ 4 μM, H₃BO₃ 2 μM, CuSO₄ 1 μM. After the second true leaf reached up to the first true leaf length, the drought treatment via water withholding was started, and it was maintained 8 days without watering when the sand water content was about 50% field capacity (FC).

 

Growth measurement Seedlings 2-day old after germination were used to measure shoot and root length with ruler, root thickness with WScanArray2/3 Image Analyzer (Galai production LTD, Israel). After starting drought treatment, total leaf blade lengths of seedlings with or without irrigation were measured with ruler every two days. Fresh and dry weight (drying at 70 ^oC, 48 hours in oven) of  shoot and root of seedlings were measured after finishing the experiment.

 

Water relations Stomatal conductance was measured on the lower surface of a youngest fully expanded leaf (second or third from the apex) with LI-1600 Steady State Porometer (LI-COR Inc., USA) between 1400 to 1500 h on the fifth day without watering when the media water content was about 71% FC. A youngest fully expanded leaf (second or third from the apex) was cut with sharp blade and immediately put into a 15 ml plastic vial into which distilled water was filled subsequently. After saturated at 4 ^oC for 12 hours, the leaf was dotted dry with tissue paper and put in a centrifuge tube following freeze in –20^oC refrigerator for 12 hours. The leaf was thawed and centrifuged to get cell sap that was used to measure osmotic potential with an osmometer (5520 Vapour Pressure Osmometer, Wescor, USA).

 

Results

There were significant difference in seed weight among genotype studied (Table 1). Seed weight of Noga, the cultivar, is much higher than that of the three wild genotypes, indicating the significant selection force on barley seed mass. Among wild type genomes, the seed of P10-30 has highest weight, P23-38 the second, P20-05 the third, this is coincident with the rainfall gradient from north to south Israel, from 800, to 150 and 100 mm annual rainfall. One may refer that the seed mass decreased with rainfall.

 

 

       Table 1. Seed mass of different genotypes

Genotype	P10-30	P20-05	P23-38	Noga
Seed weight (mg/grain)	35	19	29	66
Standard Deviations	5	1	2	3
Significance (p<0.05; n=4)	b	d	c	a

 

Seeds of P10-30, P20-05, P23-38 and Noga were from the Gene bank of

the Institute of Evolution. 24 seeds of each genotype were divided into 4

groups that were weighed.

 

Imbibition rate of P20-05 seed was highest, Noga lowest, P10-30 and P23-38 in middle after 48 hours wet at 4^oC (Figure 1). P10-30 seed increased water uptake faster than the others after 12 hours at 25^oC following 48 hours at 4^oC, because of germination earlier and more than others. At least, we can conclude that wild genotype seed imbibed quicker than cultivar, and that seed of wild genotype from severe drought habitat imbibed faster than drought and mesic habitat.

 

In the first batch of germination test, Noga seed had the lowest germination rate, (Table 2). After prolong cold treat time, 4 days at 4 ^oC, Noga seed germination rate reach up to 85%. However, prolonging cold treat did not raise P20-05 germination rate. P20-05 seed germination in nature requires exposure of caryopses in their dispersal units to a long period of high temperatures, which may prevent germination from occurring after an unexpected summer rain following the time of maturation (Gutterman and Nevo, 1994). Mona seed germination rate was absolutely 100%. There was no significant different germination rate among P10-30, P23-38 and Noga.

 

     Table 2. Seed germination rate of different barley genotypes

Genotype	P10-30	P20-05	P23-38	Noga	Mona
Batch 1	96 ± 8 a	42 ± 10 c	75 ± 9 b	29 ± 8 c	nd
Batch 2	88 ± 14 ab	34 ± 5 c	88 ± 10 ab	85 ± 11 b	100 ± 0 a

 

Seeds were wetted and cold treated at 4 ^oC for 2 days. Germination rates were counted  after 2 days in an incubator at 25 ^oC. Batch 1 was carried out in December of 2000, batch 2 in January of 2001. Specially in Batch 2, seeds of P20-05 and Noga  were cold treated for extra 2 days. Values are mean ± standard deviation, following letters within each row indicate statistical significant difference (p<0.05, Post Hoc LSD test , n=4).

 

Primary root and shoot growth depends on seed size. In order to compare the root and shoot length growth of seedlings of different genotypes after germination in Petri dishes, the length of root or shoot was expressed as relative length in mm per mg seed (Table 3). Relative length of root and shoot were higher in wild barley than in cultivars, indicating wild barley growth after germination was potentially faster than cultivar. There was no significant difference between wild and cultivar barley in root number and root thickness. However, within the wild barley group, P23-38 and P20-05 had thinner root than P10-30, which maybe imply that desert barley has thinner root, hence more ability rooting deeper than mesic barley. P23-38 had much higher relative root length than P10-30 and P20-05, whereas P20-05 had much higher relative shoot length than P10-30 and P23-38. Two different strategies to adapt drought habitat were suggested here, P23-38 with big seed spent relative more energy to grow root, and P20-05 with small seed invest relative more energy to build up shoot to conduct photosynthesis to support rooting.

 

Seedlings of similar size were transplanted to pot (0.55 L) with sand, and irrigated with nutrient solution. The total leaf length of seedlings showed similar before drought treatment, it became longer in P23-38 than P10-30 after 2days. Drought effect appeared after 4 days water withhold. The difference between drought and control was bigger in P10-30 than P23-38, which indicated that drought effect on total leaf length was stronger in P10-30 than in P23-38.   

 

 

   Table 3. Relative root length, root number, thickness and relative shoot length of seedlings 2 days old.

	P10-30	P20-05	P23-38	Noga	Mona
Relative root length (mm/mg seed)	7.2 ±2.8    b	6.6 ± 1.9    b	15.5 ± 1.6  a	4.2 ± 0.6   c	4.8 ± 0.8     c
Root number	4 ± 0.5 c	3 ± 0.5 d	7 ± 0.5 a	6 ± 0.4 b	4 ± 0.3  c
Root thickness (mm)	0.72 ± 0.1 a	0.63 ± 0.06 bc	0.62 ± 0.07 c	0.68 ± 0.08 ab	0.67 ± 0.09 b
Relative shoot length (mm/mg seed)	1.2 ± 0.2 c	2.3 ± 0.4    a	1.4 ± 0.1    b	0.7 ± 0.0   d	0.6 ± 0.0     d

 

The seedlings’ data were collected after 2 days growing in an incubator at 25^oC after germination. Values are mean ± standard deviation, following letters within each row indicate statistical significant difference (p<0.05, Post Hoc LSD test , n=12).

 

P23-38 had longer total leaf length than P10-30 but the leaf number is the same (data not show). Fresh weight is affected by water content. Therefore, to compare drought effect on seedling growth, the ratio in dry weight of drought to control, the relative weight, is a good trait. Root dry weight showed no significant difference between P23-38 and P10-30 under drought or control (Table 4). However, P10-30 shoot dry weight under drought was lower than control, whereas P23-38 shoot dry weight exhibited no significant difference between drought and control. Higher relative weight in root than in shoot indicates that root was less affected by drought. P23-38 had higher relative weight than P10-30, indicating that P23-38 had stronger drought resistance than P10-30. 

 

   The relatively higher drought resistant of P23-38 was related to its ability to contain relatively higher water content than P10-30 (Table 5). Shoot water content of P23-38 was higher than P10-30 under either drought or control. Root water content of P23-38 was slightly higher than P10-30 under drought and control. The reason may be that stomatal conductance was higher in P10-30 than P23-38 under either drought or control (Figure 3). P23-38 decreased water loss through lowered stomatal conductance compared with P10-30.

 

 Table 4. Shoot and root dry weight and the ratio of shoot to root of  two barley genotype seedlings as affected by drought.

Genotype	P10-30		P23-38		P10-30 P23-38
Treatment	D	C	D	C	D/C
DWs (mg)	61 ± 10 b	86 ± 15 a	64 ± 13 b	75 ± 6 ab	0.71	0.86
DWr (mg)	38 ± 6 ab	39 ± 6 ab	42 ± 12 a	29 ± 4 b	0.97	1.45
S/R	1.6 ± 0.3 c	2.2 ± 0.1 b	1.5 ± 0.1 c	2.6 ± 0.2 a	0.73	0.59

 

Shoot and root of drought (D) and control (C) treated seedlings were oven dried at 65^oC for 48 hours. Shoot dry weight (DWs), root dry weight (DWr) and ratio of shoot to root (S/R) are expressed as mean ± standard deviation, following letters within each row indicate statistical significant difference (p<0.05, Post Hoc LSD test , n=3).

 

 

 

 

Table 5. Shoot and root water status of two wild barley genotypes under soil gradually drought 

Genotype	Shoot water	Root water
and	content (%)	content (%)
treatment
P1D	85.4 ± 0.5 d	86.7 ± 0.8 b
P1W	89.9 ± 0.3 b	93.6 ± 0.7 a
P2D	86.5 ± 0.2 c	87.1 ± 1.0 b
P2W	90.6 ± 0.4 a	94.0 ± 0.1 a

 

Shoot and root water content of 20-day-old seedlings of P10-30 (P1) and P23-38 (P2) under drought (D) and control (W) (8 days withholding water, water content was about 50% field capacity) were measured. Values are mean ± standard deviation, following letters within columns indicate statistical significant difference (p<0.05, Post Hoc LSD test , n=3).

 

 

Soil water lost as percent of saturated soil weight during 8 days drought treatment was higher in the pot growing P10-30 than P23-38 (Table 6). Assuming that the water lost from soil was comprised of soil evaporation and plant transpiration, and that soil evaporation was the same among pots, we could refer that P10-30 transpiration was higher than P23-38 transpiration.

 

 

Table 6. Soil water loss rate as affected by plant genotypes

 

Genotype	P10-30	P23-38
Soil water loss	13.1 ± 0.4 a	12.5 ± 0.1 b

 

The soil in the pots was excessly watered with nutrient solution, weighted after no more drainage. The water lost during 8 days drought treatment were determined by the soil weight lost at the percent of saturated soil weight. Values are mean ± standard deviation, following letters indicate statistical significant difference (p<0.05, Post Hoc LSD test , n=3

 

Saturated detached leaf blade lost water at different rate (Figure 4). Drought treated leaf blade lose water much slower than control leaf blade in both genotypes. The reason may be that drought increased leaf blade ABA content, hence, increased stomatal control to reduce water loss. Drought treated leaf blade of P10-30 lose water slightly faster than that of P23-38, which may be explained by that drought treated leaf of P10-30 had lower osmotic potential than of P23-38 (Figure 5). Control leaf blade of P10-30 lost water slightly slower than of P23-38, which may be have the reason that control leaf of P10-30 had higher osmotic potential than of P23-38.  Stomatal conductance separated the four lines in Figure 5 in two groups, then leaf osmotic potential explained the difference within the group. Therefore, stomatal conductance is a more important trait than leaf osmotic potential to explain the ability of plant to resist water loss.

 

Discussion

Seed size differs with accessions of different origins, it was related to drought resistance in many cases. Large 100-seed mass was considered as a possible characteristic that may improve the drought resistance of short-duration pigeonpea (Lopez et al., 1996). The experiment of 21 species from three woody genera (Eucalyptus, Hakea and Banksia) suggest that large seed could provide the seedling with the mineral nutrients, rather than carbon-based metabolites, needed for maximizing initial root growth. Reaching reliable moisture before summer (drought avoidance) is an alternative strategy to physiological tolerance of drought (Milberg et al., 1998). Experiments have shown that seedlings of larger-seeded species are better able to survive drought (Westoby et al., 1996). Large seed of spring-sown chickpea in the rainfed farming systems of West Asia and North Africa were associated with drought escape (early flowering) (Silim and Saxena, 1993). Faster early growth from larger seeds may be advantageous in establishing plants under dry soil conditions (Mian and Nafziger, 1994). Seed size was showed not related to drought resistance in some cases. For example, within 19 inbred faba bean lines of different origin, those from the drought-prone regions characterized by smaller plant size (r = 0.93), and more pods and seeds per plant (r gtoreq 0.90) regardless of seed size (Amede et al., 1999). However, barley accessions studied in this paper showed that cultivar has much bigger seed than wild type, and that seed size increased with rainfall of its habitat. One may inferred that small seed-size barley was of drought resistance. This is in contradiction with the results of above literatures, with the reason that different species response to drought in different ways. P20-05 with the smallest seed took longer time to imbibe and absorbed relatively more water than the others before germination (Figure 1), meaning that P20-05 requires enough soil moisture to germinate seed in the nature. This may be one of the strategies of P20-05 adapting to drought habitat. This germination strategy could make sure there is enough moisture in the soil to be used by subsequent seedling growth which characterized by low relative root growth and high relative shoot growth (Table 3). The small seed mass of P20-05 was allocated relatively more to shoot growth than to root growth, in order to let the shoot conduct photosynthesis as soon as possible in the early seedling growth stage, then establish extension root system to adapt drought in the late seedling growth stage. P23-38 seed size was similar to P10-30, their imbibition rates and germination percentage were not significant different. However, contrasting sharply with P20-05, P23-38 seedling showed highest relative root growth and the thinnest root (Table 3). Therefore, P23-38 adapts drought habitat with the strategy of fast relative root growth and deeper rooting in the early seedling growth stage.

 

For wheat seeds, large one gave significantly higher seminal root number than small one regardless of water potential (Al et al., 1998). There was no relationship between seed size and seminal root number in barley (Table 3). However, the seminal root number seems related to drought resistance. Excluded P20-05 because of its special strategy to adapt drought habitat, we could see that drought resistant wild-type P23-38 had more seminal root than P10-30 and that drought resistant cultivar Noga had more seminal root than Mona. Further study was required to conform this phenomenon. If it is true, seminal root number is a very good trait for drought resistant screen.

 

More adverse effects of low osmotic pressure were on shoot than on root growth of Boerhaavia diffusa seedlings (Bajpai, 1997). Germination of seeds in PEG solutions during a period of 5 d caused a growth reduction of shoots of barley seedling, its root dry matter, however, increased with increasing concentration of the stressor (Leinhos et al., 1996). Root and shoot weights of all wheat cultivars were reduced when osmotic potential was decreased, but the extent of reduction in root growth was less than that for shoots (Baalbaki et al., 1999). Results of this paper agreed with these literatures. There were decreases in total leaf blade length (figure 2) and shoot dry weight (Table 4) but no significant decreases in root dry weight of both P10-30 and P23-38 stressed by soil drying. The effect of soil drying on shoot dry weight of P23-38 was less than P10-30. Soil drying decreased slightly P10-30 root dry weight but increased P23-38 root dry weight. P23-38 root growth was enhanced by drought. Extensive root growth under drought conditions is a major drought avoidance mechanism (Takele, 2000). Therefore, P23-38 is of drought resistance in comparison with P10-30.

 

Exposure of plants to drought stress led to noticeable decrease in photosynthesis rate and stomatal conductance (Siddique et al., 1999). Both P10-30 and P23-38 decreased stomatal conductance under soil drying, P23-38 reduced to lower level than P10-30 (Figure 3). However, the experiment conducted by Borel et al (1997) showed that stomatal control had a low genetic variability in barley lines, in spite of the large genetic differences between lines of contrasting origins (Syrian or French). Stomatal conductance is sensitive to environmental conditions, such as light, moisture in the air or soil, even wind. It also differs in leaf ages. Keeping the conditions similar, stomatal conductance might show genetic variability in contrasting origins. If stomatal conductance were measured in many lines, the measurement would take long time. If the time were more than 2 hours, the environmental variation would be bigger than genetic variation. Therefore, using stomatal conductance as a trait to conduct QTL mapping of a big population is unpractical.

 

Higher osmotic adjustment capacities were noted in drought resistant varieties across contrasting environments (Amau et al., 1997; Teulat et al., 1997 ). Drought tolerant genotypes maintained turgor by decreasing osmotic potential at lower leaf water potential and they showed higher osmotic adjustment (Khan et al., 1999). P23-38 showed higher osmotic adjustment, agreeing with these results. However, there were some exceptions. In Tichedrett, a landrace genotype with a very extensive root development, OA was not observed, but it exhibited a capacity to maintain its photosynthetic activity under water stress (Amau et al., 1997). Differences in drought resistance between 19 inbred faba bean lines were manifested through plant size-induced water demand but were not associated with osmotic adjustment (Amede et al., 1999). Hafid and his co-workers Suggested that the maintenance of higher root growth and osmotic adjustment in water stress conditions could confer an improved resistance of this species to drought (Bajji et al. 2000).

 

In conclusion, P23-38 is a drought resistant genotype in comparison with P10-30. Its drought resistance is characterized by low stomatal conductance, high osmotic adjustment, extensive root growth, and small reduction of shoot growth in drought stressed conditions.

 

 

Figure 1. Water uptake of seeds of different barley genotypes.Seeds of P10-30, P20-05, P23-38 and Noga were put on filter papers in Petri dishes. Filter Papers were wet with distilled water. Petri dishes with seeds were wrapped with aluminum foil and kept at 4^oC for 2 days. Following that, the Patri dishes were kept in an incubator at 25^oC for another one days. The seeds were weighted after they were dotted to dry with tissue paper, then they were put back to Petri dishes with filter papers which was always wet by adding distilled water.

 

Figure 2. Total leaf length of different genotypes as affected by drought.                                                                                  

Seedlings of P10-30 (P1) and P23-38 (P2) were grown in pots (0.55 L) with sand irrigated with nutrient solution. The treatment of with (W) or without (D) irrigation of nutrient solution was conducted after the second true leaf reached to the same height of the first true leaf in most of the seedlings. Leaf length was measured with ruler at the beginning of the treatment and afterward every two days. Error bar represented ¼ of standard error.

 

Figure 3. Leaf stomatal conductance of two wild barley genotypes as affected by drought.

Leaf lower surface stomatal conductance of P10-30 (P1) and P23-38 (P2) under drought (D) and control (W) were measured from 2 to 3 PM on the fifth day without watering when the media water content was about 71% field capacity.

 

 

 

Figure 4. Leaf blade drying as affected by drought. The first fully expanded leaf blades were cut from P10-30 (P1) and P23-38 (P2) seedlings under drought(D) and control (W), and weighted and put into distilled water in vials immediately. Following 12 hours saturated at 4^oC, leaf blades were dotted todry with tissue paper and weighted every hours, they were drying on tissue paper in the condition of laboratory from 12 to 18 o’clock.

 

 

Figure 5. Leaf osmotic potential of two wild barley genotypes as affected by drought. Leaf osmotic potential of P10-30 (P1) and P23-38 (P2) seedlings under drought (D) and control (W) were measured on the first fully expanded leaves after the leaves were saturated in distilled water for 12 hours in dark at 4^oC.

 

 

 

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