Photosynthetic responses to salinity stress of halophytic seashore paspalum ecotypes

Plant Science 166 (2004) 1417–1425

Photosynthetic responses to salinity stress of halophytic seashore paspalum ecotypes Geungjoo Lee a,1 , Robert N. Carrow b,∗ , Ronny R. Duncan b a

Department of Crop and Soil Sciences, Center for Applied Genetic Technologies, University of Georgia, Athens, GA 30602-6810, USA b Department of Crop and Soil Sciences, University of Georgia, Griffin, GA 30223-1797, USA Received 31 August 2003; received in revised form 19 December 2003; accepted 22 December 2003

Abstract Changes in photosynthetic capacity in variable salinity ranges were applied to explore mechanisms of salinity tolerance in seashore paspalum (Paspalum vaginatum Swartz) ecotypes. Nine ecotypes exhibiting a wide range of salinity tolerance were grown in a greenhouse using nutrient/sand culture, with six salinity levels of 1.1–49.7 dS m−1 (denoted as ECw 0 to ECw 50; electrical conductivity of water). With increasing salinity, chlorophyll concentrations decreased significantly only at ECw 50 in comparison with nonsaline control. As salinity increased, initial chlorophyll fluorescence (F0 ) increased, while maximum fluorescence (Fm ) and the variable to maximum fluorescence ratio (Fv /Fm ) tended to decrease. As salinity increased, reflectance at visible wavelengths (507–706 nm) was enhanced, whereas it decreased at wavelength ≥760 nm. Compared to Adalayd, more tolerant SI 93-2 and HI 101 exhibited significantly lower reflectance in the photosynthetically active range (PAR), and higher reflectance beyond the PAR. All seashore paspalums maintained active photosynthetic capacity, as indicated by minor reductions in pigments, high light-harvesting capacity, and high maximum photochemical efficiency (high Fv /Fm values of 0.75–0.81) at high salt levels. SI 93-2 and HI 101 were characterized by high Fm and high Fv /Fm at ECw 50 with minimal changes with increasing salinity. Also, SI 93-2 and HI 101 exhibited higher density and canopy cover. This resulted in low reflectance, as indicated by significantly higher IR/R (ratio of infrared to red wavelength) and NDVI (normalized difference vegetation index) indexes, and lower stress indexes (Stresses 1 and 2) compared to the least salt-tolerant Adalayd. IR/R and Stress 1 indexes were found to be useful tools for salinity tolerance evaluation, accounting for 85% of shoot and 51% of root growth variations, respectively. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Salinity tolerance; Paspalum vaginatum; Turfgrass; Chlorophyll concentration; Chlorophyll fluorescence; Multispectral reflectance

1. Introduction Plant biomass production depends on the accumulation of carbon products through photosynthesis, but elevated salinity can adversely affect photosynthesis [1,2]. Photosynthetic capacity of many plant species is reduced in the presence of salinity, which is associated with stomatal closure [3,4], inAbbreviations: ECw , electrical conductivity of water; F0 , initial fluorescence; Fm , maximum fluorescence; Fv , variable fluorescence; Fv /Fm , variable to maximum fluorescence ratio; PAR, photosynthetically active range: 400–706 nm; LAI, leaf area index; NDVI, normalized difference vegetation index = (RF935 − RF661)/(RF935 + RF661); IR/R, ratio of infrared to red wavelength = RF935/RF661; PS II, photosystem II; LHC, light harvesting complex ∗ Corresponding author. Tel.:+1-770-228-7277; fax:+1-770-229-3215. E-mail addresses: [email protected] (G. Lee), [email protected] (R.N. Carrow). 1 Tel.: +1-706-542-0915; fax: +1-706-583-8120.

creased mesophyll resistance for CO2 diffusion [4], reduced efficiency of Rubisco for carbon fixation [4], damage to photosynthetic systems by excessive energy [5], and structural disorganization [4,6,7]. In addition to reduced photosynthesis, reduced growth of glycophytes exposed to salinity may be the result of accelerated respiration and relatively higher photorespiration, which leads to faster consumption of photosynthates that are otherwise used to support growth [8–10]. Many attempts have been made to detect differences in salinity tolerance of crop species or cultivars by measuring photosynthetic parameters since plants may undergo adaptations in response to salinity. These include observation of the modification of composition and function of the photosynthetic apparatus [4,6,7], changes in chlorophyll concentration [11,12], quenching ability of excessive energy through chlorophyll fluorescence [13–16], and canopy reflectance of incident light [17–19]. Plants with salinity tolerance are

0168-9452/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2003.12.029

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thought to have mechanisms that allow them to maintain photosynthesis in the presence of high levels of salt. Incident light is reflected from plant surfaces, transmitted through the canopy, or absorbed by substances such as water, structural or metabolic compounds, and chlorophyll pigments [20]. The reflectance percentage of the incident light, therefore, indirectly indicates the changes in physiological, morphological, and biochemical status of the plant in a given environment. Under stress conditions, spectral reflectance is sensitive at visible (380–760 nm) or infrared wavelengths (760–2500 nm) [17,18,21], and is highly correlated with chlorophyll concentration (r > 0.97), leaf area index (LAI) (r > 0.97), biomass yield (r > 0.87), and water concentration [19,21–23]. This suggests that canopy reflectance would be a good parameter for measuring plant response to environmental stresses such as salinity. Plants under stress utilize less light energy for photosynthesis. Some of the excessive energy is quenched into chlorophyll fluorescence to minimize damage to photosynthetic systems, particularly in photosystem II (PSII) and subsequent electron carriers [13,24–26]. Earlier studies indicate that measurement of chlorophyll fluorescence can be used in assessing salinity tolerance of plants that vary in tolerance [14,27,28]. Water-related problems are enhanced in turfgrass sites using recycled water. Managers for perennial turfgrass must deal with reduced growth caused by salinity stress. One strategy to improve turfgrass survival and recovery from salt stress is to develop cultivars with better tolerance. Photosynthetic indexes such as chlorophyll concentration, kinetics of chlorophyll fluorescence, and multispectral reflectance could provide information about which photosynthetic parameters should be targeted in breeding efforts to improve seashore paspalums (Paspalum vaginatum Swartz) with greater salinity tolerance. Seashore paspalums has demonstrated superior salt tolerance compared to other turfgrasses [29–31]. Seashore paspalums were found to grow on coastal sites having seawater (34,486 mg l−1 total soluble salts or electrical conductivity of ECw of 54 dS m−1 ). The objectives of this study were: (a) to evaluate differences in photosynthetic responses of seashore paspalums that vary in salinity tolerance; (b) to define the characteristics of photosynthetic parameters associated with salinity tolerance; and (c) to assess the potential use of chlorophyll concentration, chlorophyll fluorescence, and multispectral reflectance as screening indexes for salinity tolerance in halophytic seashore paspalums.

2. Materials and methods 2.1. Growth conditions and treatments Nine seashore paspalum ecotypes which had previously exhibited difference in salt tolerance were used for this study [29]. The study was conducted using a solution/sand culture under greenhouse conditions at the Georgia Agricultural

Experiment Station in Griffin, GA. Prior to being transplanted into plastic pots (13.0 cm long × 10.0 cm wide × 12.5 cm high), these ecotypes were maintained with common management practices of irrigation (once a day) and cutting them back to 2.5 cm (once a week). A modified half-strength of Hoaglad and Arnon’s [32] nutrient solution (no. 2) was used. An Fe-EDTA chelate (Sprint 138, 6% Fe, Ames, IA) as an iron source was used to provide 5 mg l−1 of Fe. Nine pots were positioned in a wooden frame, which was placed into a nutrient container containing 28 l of nutrient solution (1.2 dS m−1 and pH = 6.3 ± 0.5). After a 4-week acclimation period, the nutrient solution was gradually salinized with sea-salt mixture to 10.3, 20.5, 30.7, 39.5, 49.7 dS m−1 [31]. To minimize salt shock, electrical conductivity of the solution (ECw ) was gradually increased by the addition of 6.9 g l−1 of the sea salt mixture every day until the final salinity levels were achieved. Solutions were replaced weekly, aerated constantly, and maintained at constant volume by adding deionized water every 2–3 days. The salinity level was monitored by measuring ECw twice a week at 25 ◦ C with an Orion 160 conductivity meter (Boston, MA). 2.2. Evaluation of photosynthetic parameters Chlorophyll concentration was analyzed from 0.05 g fresh weight (FW) of the leaf tissue, which was excised from the third young and fully expanded leaves. Samples were weighed immediately after harvest and placed into light-protecting vials. Ten milliliters of N,N-dimethylformamide (DMF) extraction buffer was added to each vial and the vials were stored at 4 ◦ C for 12 h. Vials were then transferred to room temperature and gently shaken on a horizontal shaker for 12 h. The extract was analyzed for absorbance at 647 and 664.5 nm on a Beckman DU-600 spectrophotometer (Beckman Instruments, Fullerton, CA ). Chlorophyll a, chlorophyll b, and total chlorophyll concentrations (Chl a, Chl b, TChl) were calculated by using the following equations: Chl a = 12.70A664.5 − 2.79A647 ; Chl b = 20.70A647 − 4.62A664.5 ; TChl = 17.90A647 + 8.08A664.5 , where A = absorbance in a 1 cm cuvette [33]. Chlorophyll fluorescence was measured on the third or fourth fully expanded young leaves by using an FIM 1500 fluorescence induction monitor (Analytical Development, Hoddesdon, England). Each leaf was excised, immediately clipped into a leafclip (32 mm wide × 80 mm long), and the shutter plate closed to induce dark-adaptation for 30 min at room temperature. An array of six high intensity light emitting diodes (LEDs) provided red light with a peak wavelength of 650 nm to illuminate the exposed leaf surface (4 mm diameter). Maximum intensity of the illuminating light was approximately 3000 ␮mol m−2 s−1 at leaf surface. Measured fluorescence parameters included F0 (initial fluorescence measured at the onset of illumination), Fm (maximum fluorescence), Fv (variable fluorescence = Fm − F0 ), and Fv /Fm (ratio of variable to maximum fluorescence

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included in the model, and partial R2 and coefficient values were assessed for the relation of variables.

indicating the quantum yield). Measurements of three leaves from the same pot were averaged to obtain a mean. Just prior to final harvest, spectral reflectance was measured on the 130 cm−2 turfgrass canopy in each pot at the wavelengths of 507, 559, 661, 706, 760, 813, and 935 nm using a hand-held MSR 16 multispectral radiometer (Crop Scan, Fargo, ND) [34]. Additionally, the following growth and stress indexes were determined:

3. Results 3.1. Chlorophyll concentration Because there were no significant difference in total chlorophyll (Tchl) concentration between ECw 10 versus ECw 20, and ECw 30 and ECw 40, data for Tchl were provided at only four salinity levels (Table 1). Tchl concentration among nine seashore paspalums was statistically similar across salt levels. As salinity increased, Tchl concentration decreased significantly with the minimum at ECw 50 for seven out of nine ecotypes. Due to nonsignificant changes in Chl b across salinity levels and among grass entries, significant differences in Tchl concentration (Chl a + b) at ECw 50 could mainly be ascribed to changes in Chl a concentration (data not shown). No significant difference in Tchl concentration between more and less salt tolerant ecotypes was evident at any salinity level.

1. Normalized difference vegetation index (NDVI), computed as (RF935 − RF661)/(RF935 + RF661). 2. IR/R, computed as RF935/RF661. 3. Stress 1 index, computed as RF706/RF760. 4. Stress 2 index, computed as RF706/RF813, where RF = % reflectance at a given wavelength. Data were collected around solar noon to minimize the disturbance of spectral reflectance from solar angle, and measurements were made under minimal cloud cover. The photodiodes on the multispectral radiometer were perpendicular to the grass surface and were positioned 15 cm above the grass canopy. 2.3. Experimental design and statistical analysis

3.2. Chlorophyll fluorescence The experimental design was a split-plot design with six replications. Salinity level and ecotype were the main plot and the subplot, respectively. Six salinity levels (one salt level/container) were arranged randomly in each of the six replications. All data were statistically analyzed using least significant difference (LSD) to separate entry means within each salinity level [35]. Photosynthetic components were compared across salinity levels by entry to determine significant differences among salinity levels. Multiple regression analysis was used to determine the most significant photosynthesis parameter contributing to shoot and root performance of all entries across salinity levels. All variables exhibiting a P ≤ 0.05 significance level were

Initial fluorescence (F0 ) differences were not significantly different among the nine entries (Table 2). Within an entry, seven seashore paspalums demonstrated no significant effect of salinity on F0 . F0 levels of Sea Isle 2000 and TCR 6 were influenced by salinity, with the highest values at ECw 50. Salinity stress generally resulted in lower maximum chlorophyll fluorescence (Fm ) (Table 3). Six ecotypes exhibited a quadratic response in Fm , with the highest values at ECw 20, and decrease with further increase in salinity. The more salt-tolerant SI 93-2 and HI 101 ecotypes exhibited significantly higher Fm values than Adalayd at highest salin-

Table 1 Total chlorophyll concentration (Tchl) and Chl a/b ratio in the parenthesis of seashore paspalums under different salinity levelsa Entry

Tchl (mg g−1 FW) at ECw 0

SI 93-2 HI 101 Sea Isle 2000 TCR 1 TCR 6 Sea Isle 1 HI 34 SI 90 Adalayd

0.59 0.69 0.61 0.66 0.61 0.62 0.58 0.65 0.62

F-testb LSD (0.05)b

0.11 0.08

(4.4) (4.4) (4.5) (4.3) (4.3) (4.4) (4.3) (4.5) (4.4)

ECw 10 bc a bc ab a–c a–c c ab a–c

0.63 0.59 0.61 0.64 0.65 0.55 0.64 0.62 0.66 0.26 0.08

(4.2) (4.3) (4.8) (4.5) (4.3) (4.2) (4.3) (4.3) (4.3)

ECw 30 ab ab ab a a b a ab a

0.58 0.61 0.59 0.61 0.58 0.61 0.59 0.62 0.65 0.77 0.09

(4.4) (4.4) (4.3) (4.3) (4.3) (3.7) (4.3) (4.3) (4.3)

F-testb

LSD (0.05)b

0.32

0.12 0.09 0.08 0.05 0.09 0.09 0.11 0.09 0.09

ECw 50 a a a a a a a a a

0.50 0.50 0.47 0.51 0.54 0.45 0.47 0.50 0.54

(4.3) (4.3) (4.3) (4.5) (4.3) (4.1) (4.3) (4.3) (4.2)

a–c a–c bc ab a c bc a–c a

∗∗

∗∗∗ ∗∗∗

0.09 ∗∗ ∗ ∗∗ ∗

0.06 0.06

Means within a column followed by the same letter are not significantly different at the P = 0.05 level for the given salinity. F-test and LSD test (0.05) are to compare mean performances among entries or salinity levels where the denoted symbols indicate significant difference at the 0.001 (***), 0.01 (**), and 0.05 (*) levels. a

b

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Table 2 Initial chlorophyll fluorescence index (F0 ) among seashore paspalum ecotypes under different salinity levelsa Entry

F0 at ECw 0

Ecw 10

ECw 20

ECw 30

ECw 40

ECw 50

SI 93-2 HI 101 Sea Isle 2000 TCR 1 TCR 6 Sea Isle 1 HI 34 SI 90 Adalayd

580 539 542 577 580 554 566 547 525

600 555 527 570 554 594 554 565 528

597 599 547 561 580 582 559 569 543

591 532 560 561 541 531 565 548 546

580 544 545 572 599 561 570 582 550

589 546 618 577 661 547 571 561 607

F-testb LSD (0.05)b

0.16 44

a,b

a ab ab a a ab ab ab b

a ab b ab ab a ab ab b

0.10 54

a a b ab ab ab ab ab b

0.09 40

a b ab ab b b ab b b

0.11 38

ab b b ab a ab ab ab b

0.24 45

bc c ab bc a c bc bc a–c

F-testb

LSD (0.05)b

0.98 0.16

66 67 52 59 68 64 57 52 68

∗

0.99 ∗

0.38 0.99 0.73 0.20

0.07 67

See explanations in Table 1.

Table 3 Maximum chlorophyll fluorescence index (Fm ) among seashore paspalum ecotypes under different salinity levelsa Entry

F-testb

Fm at ECw 0

ECw 10

ECw 20

ECw 30

ECw 40

ECw 50

SI 93-2 HI 101 Sea Isle 2000 TCR 1 TCR 6 Sea Isle 1 HI 34 SI 90 Adalayd

3354 3150 3199 3333 3299 3230 3201 3149 3078

3329 3228 3171 3220 3091 3361 3111 3204 3110

3290 3288 3220 3205 3288 3377 3229 3267 3126

3196 3093 3156 2920 2982 3087 3176 3037 2750

3240 3140 3132 3145 3336 3249 3067 2899 2823

2888 2886 2835 2711 2851 2742 2757 2771 2438

F-testb LSD (0.05)b

0.14 205

a,b

a a–c a–c ab ab a–c a–c bc c

0.16 216

a ab ab ab b a b ab b

ab ab ab ab ab a ab ab b

0.52 211

0.07 287

a a a ab ab a a ab b

∗∗

206

ab a–c a–c a–c a ab b–d d d

a a a ab a ab ab a b

∗∗

0.14 ∗

0.08 ∗

∗∗∗

0.12 ∗∗

∗∗∗

LSD (0.05)b

238 340 233 448 304 256 362 265 235

0.21 328

See explanations in Table 1.

ity of ECw 40 and ECw 50. Compared to the levels at ECw 0, Fm decreased by 14 and 8% at ECw 50 in SI 93-2 and HI 101, respectively, but 21% for Adalayd. Similarly, variable chlorophyll fluorescence (Fv = Fm −F0 ) also tended to have a quadratic response, with maximum values at ECw 20, and then a decrease with increasing salinity (data not shown). SI 93-2 and HI 101 exhibited a higher Fv than the less tolerant Adalayd at ECw 40, the only salinity at which differences in Fv were significant. The decrease in Fv from ECw 0 to ECw 50 was more pronounced in Adalayd than in SI 93-2 and HI 101. With increasing salinity, Fv /Fm ratio decreased significantly in Sea Isle 2000, TCR 6, HI 34, SI 90, and Adalayd only at ECw 50 compared to the control (Table 4). The variation in the Fv /Fm among entries was evident at ECw 50. At this salinity, the more tolerant ecotypes had an Fv /Fm of 0.80–0.81, while the least tolerant Adalayd had 0.75. Up to ECw 40, the halophytic seashore paspalums exhibited Fv /Fm ratio ≥0.80, which is the value normally observed for many plants in growing season in non stress conditions.

3.3. Spectral reflectance Within the PAR ranges (400–706 nm), the highest salinity (ECw 50) resulted in enhanced reflectance (i.e., less PAR absorption) relative to ECw 0 or ECw 10 for most ecotypes (Table 5). The more salt-tolerant SI 93-2 and HI 101 consistently exhibited significantly lower reflectance than the less tolerant SI 90 and Adalayd at ECw 10, ECw 40, and ECw 50. Adalayd was the entry most vulnerable to salt stress based on increasing reflectance at salinity levels ≥30 dS m−1 compared to ECw 0 in the PAR spectra. Reflectance percentage above PAR ranges (i.e., IR at 760–935 nm) was similar to halophytic shoot responses (Table 6). In all seashore paspalums except Adalayd, reflectance increased slightly between ECw 10 and ECw 10 or ECw 20 and then decreased. Reflectance in Adalayd decreased continuously as salinity increased. Adalayd was also the most vulnerable to salt stress based on a significant decrease in reflectance percentage at 935 nm at the salinity levels ≥30 dS m−1 compared to ECw 0.

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Table 4 Ratio between variable and maximum chlorophyll fluorescence (Fv /Fm ) among seashore paspalum ecotypes under different salinity levelsa Entry

Fv /Fm at ECw 0

ECw 10

ECw 20

ECw 30

ECw 40

ECw 50

SI 93-2 HI 101 Sea Isle 2000 TCR 1 TCR 6 Sea Isle 1 HI 34 SI 90 Adalayd

0.83 0.83 0.83 0.83 0.82 0.83 0.82 0.83 0.83

0.82 0.83 0.83 0.82 0.82 0.82 0.82 0.82 0.83

0.82 0.82 0.83 0.83 0.82 0.83 0.83 0.83 0.83

0.81 0.83 0.82 0.80 0.82 0.83 0.83 0.82 0.80

0.82 0.83 0.82 0.82 0.82 0.83 0.81 0.80 0.81

0.80 0.81 0.78 0.77 0.76 0.80 0.79 0.80 0.75

F-testb

0.72 0.01

LSD (0.05)b a,b

a a a a a a a a a

b ab a b b ab ab ab ab

c bc a ab a–c ab a–c a–c ab

∗

0.27 0.01

ab a ab b ab a ab ab b

0.19 0.02

0.01

a a a a a a a b a

∗

∗

0.02

0.04

a a ab ab ab a a a b

F-testb

LSD (0.05)b

0.24 0.16

0.03 0.04 0.03 0.05 0.04 0.03 0.02 0.02 0.03

∗

0.20 ∗

0.22 ∗ ∗ ∗∗∗

See explanations in Table 1.

Table 5 Spectral reflectance at the wavelength of 706 nm (RF706) among seashore paspalum ecotypes under different salinity levelsa Entry

F-testb

RF706 (%) at ECw 0

ECw 10

ECw 20

ECw 30

ECw 40

ECw 50

SI 93-2 HI 101 Sea Isle 2000 TCR 1 TCR 6 Sea Isle 1 HI 34 SI 90 Adalayd

10.07 a–c 9.53 b–d 9.41 cd 10.01 a–c 10.28 ab 9.63 b–d 9.87 a–d 10.55 a 9.16 d

9.53 9.49 9.22 10.46 9.59 9.82 10.47 10.35 9.67

9.32 9.75 9.50 10.09 9.21 9.25 9.36 9.90 10.06

10.56 11.00 10.25 10.59 11.09 10.41 10.70 10.52 11.27

9.90 10.08 9.74 10.44 9.66 10.48 10.08 11.29 11.78

11.48 11.03 11.44 11.76 11.10 11.79 11.84 11.99 14.32

F-testb

∗

LSD (0.05)b

0.76

a,b

c c c a c a–c a ab bc

∗∗

a a a a a a a a a

0.30 0.90

0.76

a a a a a a a a a

0.67 1.18

c c c bc c bc c ab a

∗∗∗

∗∗∗

0.99

0.88

bc c bc bc c bc bc b a

∗∗ ∗∗ ∗∗

0.07 ∗∗

∗∗∗ ∗∗∗

0.06 ∗∗∗

LSD (0.05)b

1.12 1.06 1.04 1.20 1.14 0.93 0.83 1.39 1.36

See explanations in Table 1.

Reflectance at infrared range was significant among the nine grasses at the different salinity levels, which demonstrated the higher reflectance in salt-tolerant seashore paspalums (Table 6).

3.4. Growth and stress indexes Two growth-related indexes (IR/R and NDVI) showed similar results so that only IR/R data were presented

Table 6 Spectral reflectance at the wavelength of 935 nm (RF935) among seashore paspalum ecotypes under different salinity levelsa Entry

RF935 (%) at ECw 0

ECw 20

ECw 30

ECw 40

ECw 50

46.88 46.80 47.55 46.23 48.37 45.91 43.08 46.47 44.19

46.00 47.67 49.10 48.85 45.16 48.81 43.43 44.97 43.69

46.39 46.32 46.04 46.67 44.13 47.79 44.76 42.70 40.12

46.16 44.83 45.49 43.57 43.90 46.42 42.39 41.51 39.76

46.13 44.00 43.12 43.93 43.00 44.51 39.89 39.53 35.53

SI 93-2 HI 101 Sea Isle 2000 TCR 1 TCR 6 Sea Isle 1 HI 34 SI 90 Adalayd

45.35 41.86 46.62 46.82 45.04 42.36 39.82 43.55 44.62

F-testb

∗∗∗

∗∗

∗∗∗

∗∗∗

∗∗∗

∗∗∗

LSD (0.05)b

3.15

2.59

2.91

2.71

2.40

2.99

a,b

a–c de ab a a–c c–e e b–d a–d

ECw 10

See explanations in Table 1.

a a a ab a ab c ab bc

b–d a–c a ab cd ab d cd d

ab ab ab ab bc a bc cd d

ab a–c a–c cd bd a d de e

a ab b ab b ab c c d

F-testb

LSD (0.05)b

0.97

3.43 2.82 3.41 4.49 4.08 3.11 3.86 3.35 3.4

∗∗ ∗

0.19 0.16 ∗∗

0.08 ∗∗

∗∗∗

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Table 7 Spectral reflectance ratio between wavelengths of infrared and red (IR/R) among seashore paspalum ecotypes under different salinity levelsa Entry

IR/R (RF935/RF661) at

SI 93-2 HI 101 Sea Isle 2000 TCR 1 TCR 6 Sea Isle 1 HI 34 SI 90 Adalayd

19.84 18.80 23.62 20.63 18.75 19.21 15.97 18.46 20.79

ECw 0

F-testb LSD (0.05)b a,b

b b a b bc b c bc b

F-testb

ECw 10

ECw 20

ECw 30

ECw 40

ECw 50

23.34 21.59 22.90 20.49 22.20 20.05 16.58 17.46 18.43

18.85 19.30 18.51 19.19 18.43 20.05 16.48 18.06 14.65

14.05 12.80 14.23 14.70 12.29 14.35 12.70 13.53 9.32

14.21 12.52 13.61 12.10 14.08 12.09 11.49 9.16 8.20

9.29 9.04 8.20 8.19 9.46 8.32 7.29 7.05 4.37

a ab ab a–c ab bc d cd cd

∗∗∗

∗∗∗

∗∗∗

2.80

3.04

1.89

ab ab ab ab ab a cd bc d

a a a a ab a a a b

0.07 3.33

a ab ab ab a ab b c c

∗∗∗

∗∗∗

2.19

1.36

a a ab ab a ab b b c

∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

LSD (0.05)b

4.52 4.13 4.35 4.25 4.60 4.54 3.28 3.28 4.17

See explanations in Table 1.

(Table 7). Significant differences among entries were found at all salinity levels, with higher values for SI 93-2 and HI 101 than for Adalayd. For the intermediate and more salt-tolerant ecotypes, the maximum growth indexes occurred at ECw 10 or ECw 20, whereas Adalayd showed the maximum values at ECw 0. Compared to growth indexes at ECw 0, Adalayd exhibited significant reduction at ECw 20 for IR/R or NDVI index. The more tolerant SI 93-2 exhibited significant reduction in growth indexes only at ECw 50 (Table 7). Stress indexes (Stresses 1 and 2) were also similar so that only Stress 1 data were presented (Table 8). Stress 1 index for intermediate and tolerant ecotypes showed quadratic responses, with the minimum values at ECw 10 or ECw 20. In contrast, a linear increase was noted for Adalayd, with a minimum value at ECw 0. Differences among entries were significant at all salinity levels. From salinity ≥ECw 20, SI 93-2 and HI 101 exhibited significantly lower values of Stresses I and 2 indexes than the less tolerant Adalayd.

3.5. Multiple regression analysis Based on multiple regression analysis using chlorophyll concentration, chlorophyll fluorescence, and spectral reflectance parameters, the parameters that were closely associated with variations of shoot and root growth were determined (Table 9). The equations associated with photosynthetic parameters used in this study were the following: • Shoot growth = −8.6 + 0.1(IR/R) − 0.1(RF935) + 0.1(RF760) + 0.3(RF706) + 9.2(NDVI), R2 = 0.96. • Root growth = 0.2 − 0.6(Stress 1) + 0.0001(Fm ), R2 = 0.54. Looking at partial R2 values, shoot and root growth was best explained by IR/R (85%) and Stress 1 index (51%), respectively. 4. Discussion Photosynthesis involves a process of light harvesting to synthesize organic compounds in which chemical energy is

Table 8 Difference in Stress 1 index (RF706/RF760) among seashore paspalum ecotypes under different salinity levelsa Entry

Stress 1 index (RF706/RF760) at

SI 93-2 HI 101 Sea Isle 2000 TCR 1 TCR 6 Sea Isle 1 HI 34 SI 90 Adalayd F-testb LSD (0.05)b a,b

ECw 0

ECw 10

ECw 20

ECw 30

ECw 40

ECw 50

0.26 0.26 0.28 0.26 0.26 0.27 0.32 0.29 0.23

0.24 0.22 0.25 0.27 0.22 0.28 0.32 0.26 0.26

0.22 0.23 0.22 0.23 0.22 0.20 0.24 0.24 0.26

0.28 0.30 0.27 0.28 0.30 0.26 0.30 0.32 0.37

0.28 0.27 0.26 0.29 0.27 0.28 0.28 0.35 0.37

0.33 0.32 0.35 0.35 0.32 0.33 0.40 0.41 0.52

bc bc a–c bc bc a–c a ab c

0.10 0.05

See explanations in Table 1.

b–d cd b–d a–c d ab a b–d b–d

bc bc bc bc bc c ab ab a

b b b b b b b ab a

b b b b b b b a a

∗∗

∗∗

∗

∗∗∗

∗∗∗

0.05

0.03

0.05

0.05

0.05

d d cd cd d d bc b a

F-testb

LSD (0.05)b

0.15

0.08 0.07 0.07 0.09 0.07 0.08 0.08 0.09 0.09

∗ ∗

0.20 ∗

0.06 ∗

∗∗ ∗∗∗

G. Lee et al. / Plant Science 166 (2004) 1417–1425 Table 9 Multiple regression analysis of photosynthetic variables contributing to shoot and root growth under salinity stressa Variable

Coefficient

Partial R2 (%)

P-value

Shoot growth Intercept IRR RF935 RF760 RF706 NDVI

−8.63 0.08 −0.13 0.06 0.32 9.15

– 85 3 3 4 1

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

96∗∗∗

Overallb Root growth Intercept Stress1 Fm Overallb

0.24 −0.58 0.0001

– 51 3

0.0494 0.0052 0.0477

54∗∗∗

a Included all variables to meet 0.05 significance level for entry into the model. b Significant at the 0.001 probability level.

stored and used later to activate cellular processes in plants. Since the energy in a photon is high for cellular molecules, the absorbed energy beyond the photosynthesis requirement must be dissipated, which is achieved by excitation transfer to neighboring pigments, thermal de-excitation, and chlorophyll fluorescence emission [24]. Thus, all plants under salinity stress must have an active system for efficient allocation of excess energy without damage in photosynthetic apparatus. At higher salinity levels of ECw 40 or ECw 50, where significant growth differences were found between SI 93-2 and HI 101 (more tolerant) versus Adalayd (less tolerant) [37], correlation between shoot dry weight and Chl a, Chl b, and Tchl concentrations were nonsignificant (p = 0.05) with r = 0.21, 0.59, and 0.20, respectively (data not shown). Thus, chlorophyll concentration of halophytic seashore paspalums seems to be insensitive to salinity up to ECw 50, and it is unlikely to result in reduced growth under salinity ranges in this study. This is consistent with the reports for other monocots including rice, wheat, and maize [12,27,36]. However, since chlorophyll concentration was measured on a fresh weight basis, higher water concentration might dilute actual concentration for salt-tolerant SI 93-2 and HI 101 at ECw 50 [37]. Our results exhibited increased F0 with increasing salinity and the highest values of F0 occurred at the salinity levels ≥40 dS m−1 for intermediate tolerant ecotypes and Adalayd (Table 2). This is in agreement with findings reported from other salt-sensitive genotypes of rice, barley, and wheat [12,14,27]. The sources of F0 include Chl a molecules in PS II and attached light harvesting complex [13,38,39]. Salinity tolerance of seashore paspalum, therefore, seemed to be associated with low levels of F0 at ECw 50 (589 and 546 for SI 93-2 and HI 101 versus 607 for Adalayd), and a relatively low increase in F0 values from ECw 0 to higher

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salinity level (1.6 and 1.3% for SI 93-2 and HI 101 versus 15.6% increase for Adalayd), with similar observations reported with rice [12]. In contrast to F0 , Fm is achieved when QA or the plastoquinone pool (PQ pool) is fully reduced by electrons [38]. Based on significantly higher Fm values at ECw 40 or ECw 50 and relatively lower decreasing rate, SI 93-2 and HI 101 are better able to transfer electrons from the PS II reaction center to other acceptors (i.e., pheophytin a, QA , QB , and PQ pool) than the less tolerant SI 90 or Adalayd (Table 3). Decrease in Fm indicates irreversible or slowly reversible damage to the photosynthesis system, which is termed “photoinhibition”, and the target is mostly localized in PSII [24,25]. With increasing salinity, quantum efficiency (enhanced carbon assimilation and oxygen evolution per quantum use) decreased as can be seen in reduction in the Fv /Fm ratio (Table 4). It has been reported that increasing F0 and decreasing Fm in response to stresses can be attributed to separation of LHC II from the PS II complex, inactivation of PS II reaction center, and perturbation of electron flow within the PQ pool [12,13,38,39]. Thus, the greater values of Fv /Fm for the tolerant SI 93-2 and HI 101 (0.80 and 0.81, respectively) seem to allow maximum photochemical efficiency of PSII and higher biomass yield at ECw 50 [37]. The range of Fv /Fm for various monocot and dicot families with different life styles has been reported to be from 0.80 (early in the growing season) to 0.60 or less (latter part of growing season) [40]. The high levels of Fv /Fm (0.75–0.83 across salinity treatments) indicate that seashore paspalums were able to maintain maximum photochemical efficiency of PS II at salinity levels approaching to that of sea water (54 dS m−1 ), and demonstrate a very high degree of salinity tolerance relative to other turfgrass species [30]. Some plants show decreases in Fv /Fm that involve protective mechanism (i.e., changes in the xanthophyll cycle pool), which could be reversed upon transfer of plants to a stress-free environment or with changes in the environmental conditions [41]. Therefore, the recovery under stress-free conditions of seashore paspalums needs to be verified further to explain decreases in Fv /Fm value either by photoinhibiton or photoprotection. Also, factors associated with increase in F0 (reduction of the plastoquinone pool, chlorophyll respiration, or others) might need to be elucidated further (i.e., by using FR pre-illumination) [42]. The high spectral reflectance could be resulted by low grass density and canopy cover, which was proven by decreased growth indexes (IR/R and NDVI) and increased stress indexes (Stresses 1 and 2). Based on multiple regression analysis, variations in shoot and root growth under salinity stress are mostly accounted by positive IR/R and negative Stress 1 indexes (85 and 51%, respectively), indicating these indexes could be used as indirect screening tools for evaluation of salinity tolerance (Table 9). In conclusion, salinity tolerance of the seashore paspalums was associated with active photochemical quenching (high photosynthetic capacity) rather than shrinking

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of photosynthetic activity. This hypothesis is supported by the small reduction in chlorophyll pigments by salinity treatment and high maximum photochemical efficiency. For reflectance both in the PAR and IR ranges, significant differences occurred only at ECw 50 compared to ECw 0, indicating that most entries except Adalayd are able to maintain photosynthetic activity up to ECw 40. SI 93-2 and HI 101 also demonstrated high Fm and Fv /Fm at the highest ECw 50 as well as minimal changes in those values with increasing salinity. In addition, reflectance percentage in the PAR spectra was low for the more tolerant SI 93-2 and HI 101, which might be ascribed to greater grass density and canopy cover. This is confirmed by significantly higher values of growth indexes and lower stress indexes in the tolerant ecotypes, which might result in photosynthetic differences and yield differences. These results suggest potential use of photosynthetic parameters for rapid selection of salinity tolerant grasses at the intraspecific level.

Acknowledgements Funding from the US Golf Association and Georgia Turfgrass Foundation Trust is gratefully acknowledged.

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