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1 Plant Science 177 (2009) Contents lists available at ScienceDirect Plant Science journal homepage: Photosynthesis of cotton near-isogenic lines introgressed with QTLs for productivity and drought related traits Avishag Levi a, Lianne Ovnat a, Andrew H. Paterson b, Yehoshua Saranga a, * a The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel b Plant Genome Mapping Laboratory, University of Georgia, Athens, GA 30602, USA ARTICLE INFO ABSTRACT Article history: Received 3 February 2009 Received in revised form 5 April 2009 Accepted 7 April 2009 Available online 16 April 2009 Keywords: Gas exchange Gossypium Leaf water potential NIL MAS Quantitative trait loci (QTLs) for yield and drought related traits were exchanged via marker-assisted selection (MAS) between elite cultivars of two cotton species, Gossypium barbadense (GB) cv. F-177 and Gossypium hirsutum (GH) cv. Siv on. The resulting near-isogenic lines (NILs) manifested in many cases the expected drought-adaptive traits, but rarely exhibited an advantage in yield relative to the recipient parents. In the current study we characterized the photosynthetic activity of two selected NILs and their recipient parents under dryland and irrigated field conditions. The GB NIL exhibited a stable net rate of CO 2 assimilation (A) across a wide range of leaf water potentials with a notable advantage over its recipient parent, F-177, under severe drought, associated with lower stomatal limitation, greater maximum velocity for carboxylation of Rubisco and greater electron transport rate. The GH NIL exhibited greater mesophyll conductance under drought conditions than its recipient parent, Siv on, but these genotypes did not differ in A. Nevertheless, both NILs did not differ from their recipient parents in yield. This study provides further support to our previous conclusion that MAS is a useful means to enhance drought-adaptive traits of cotton but complimentary conventional breeding is required to achieve high yield potential. ß 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Cotton (Gossypium spp.; Malvaceae family) is the world s leading fiber crop ( and among the most important oilseed crops. Gossypium hirsutum L. and Gossypium barbadense L. (noted hereafter as GH and GB, respectively) are the two predominant elite cotton species, usually grown during the summer in arid and semiarid regions where water availability is often limited. Regardless of whether it is irrigated or not, cotton is often exposed to drought, which adversely affects both yield and lint quality [1]. Drought, induced by soil and/or atmospheric water deficit, poses the most severe environmental constraint to plant survival and crop productivity [2]. With increasing aridity and population growth, water is expected to become even scarcer in the near future [3]. Developing drought resistant crop plants is vital to meeting the increasing demands for agricultural products and mitigating the effects of the anticipated environmental shift towards greater aridity [4,5]. This approach, however, requires * Corresponding author. Tel.: ; fax: address: saranga@agri.huji.ac.il (Y. Saranga). comprehensive understanding of plant responses to water stress and their underlying physiological and genetic mechanisms. Photosynthetic down-regulation and/or inhibition under water stress conditions are major determinants of plant growth, survival and yield in drought prone-areas [6]. Photosynthesis in plants is composed of interconnected biophysical processes (CO 2 diffusion through stomata and the mesophyll) and biochemical processes (located in the chloroplast thylakoid membranes, stroma, mitochondria and the cytosol of the cell). Dissection of the biological factors underlying net rate of CO 2 assimilation (A) isanimportant step towards understanding the effects of genetics and environment on A and plant productivity [7]. Photosynthetic rate is progressively diminished during drought [8], yet, the nature of limitations imposed by water deficit on leaf carbon assimilation has been under debate [9 12]. It is generally accepted that, under field conditions, reduced stomatal conductance (g s ) is the primary cause of decreased photosynthesis in response to moderate drought stress [10,11,13]. In addition, mesophyll conductance (g m, the conductance for CO 2 diffusion from the intercellular space to the chloroplast stroma) was strongly correlated with A in a variety of plant species ([14] and references therein) and shown to impose an important limitation on A under drought [11,15]. Several other reports suggested that impaired biochemical processes such as Rubisco activity or capacity for RuBP regeneration are the main limitations to /$ see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi: /j.plantsci

2 A. Levi et al. / Plant Science 177 (2009) photosynthesis under drought conditions (reviewed by ref. [12]). Leaf photochemistry has been shown to be extremely resistant to water stress [16]; for example isolated Xanthium strumarium mesophyll cells exhibited unchanged electron transport down to water potential of 4.0 MPa and only 20% inhibition at 6.4 MPa [17]. However, other studies showed reduction in the total electron transport and in the quantum efficiency of PSII photochemistry with increasing water deficit [12,13,18]. These apparent discrepancies may arise from the fact that different studies were performed under different environmental conditions, using different species and undergoing different drought intensities. Breeding efforts to improve crop adaptation to water-limited conditions through direct selection have been hindered by the complex genetic basis of plant productivity and drought responses, characterized by low heritability and large genotype environenvironment (G E) interaction [19 21]. The application of marker-assisted selection (MAS) to introgress specific quantitative trait loci (QTLs) from a donor line into a recipient line received considerable attention in the last years [22,23]. However, only a few examples of MAS for traits associated with drought resistance have been reported [24] and none has been reported in cotton. In a previous study, QTLs for yield and drought related physiological traits, turgid osmotic potential (OP), carbon isotope ratio (d 13 C, an indicator of water use efficiency), and leaf chlorophyll content (Chl) were exchanged via MAS between elite cultivars of the two cotton species GB cv. F-177 and GH cv. Siv on [25]. A considerable number of the resulting near-isogenic lines (NILs) exhibited the expected physiological traits that were targeted for introgression as well as remarkable modifications in non-targeted traits (leaf morphology, stomatal conductance and specific leaf weight). However, the NILs rarely exhibited an advantage in yield under water-limited conditions. In this study, cotton NILs, introgressed with selected genomic regions associated with productivity and physiological traits, and their recipient parents, GB cv. F-177 and GH cv. Siv on, were studied under contrasting water conditions to characterize the factors underlying their photosynthetic activity. 2. Material and methods 2.1. Development of NILs Seven genomic regions containing QTL conferring productivity and drought related traits, OP, d 13 C and Chl, were selected for the development of NILs [25]. A marker-assisted backcross program was conducted for the introgression of targeted regions using GH cv. Siv on and GB cv. F-177 as donor and recipient genotypes. The procedure used well-established protocols for genomic DNA extraction [26] and RFLP marker analysis [27]. Donor genotypes were drawn from the original F 3 mapping population, genotyped with 279 DNA markers. Plants containing the favorable allele at the targeted region, with a minimum of chromatin from the donor species (an average of about 40%) were backcrossed three times to the recipient parent to produce BC 3 F 1 progenies. In BC 2 F 1 as well as in BC 3 F 1 progenies, 25 plants per target region were genotyped with the appropriate markers, flanking and within the target regions, to identify plants heterozygous at the target markers. For each target region, about 10 BC 3 F 1 plants were selfed to produce 40 BC 3 F 2 progenies, which were genotyped to identify plants homozygous at the target markers. Subsequently, BC 3 F 3 progenies genotyped with a total of 105 microsatellite markers revealed an average 6.6% of the donor genome (including the target region). Two of the resultant NILs that exhibited significant modifications as compared with their recipient parent were selected for the current study: (1) NIL 1-4, GB cv. F-177 as a recipient parent introgressed with a genomic region (LGA02 flanked by par792, pgh232a markers) from GH as a donor line, conferring high seed-cotton yield, low OP and high Chl. This line exhibited significantly lower OP, greater Chl and lower leaf size as compared with its recipient parent [25]. (2) NIL 3-2, GH cv. Siv on as a recipient parent introgressed with a genomic region (Chr. 25 flanked by par969, par839 markers) from GB as a donor line, conferring high seed-cotton yield and low OP. This line exhibited lower OP under severe water stress, lower specific leaf weight (SLW, leaf thickness) and Chl, higher stomatal density and smaller epidermal cells as compared with the recipient parent [25] Experimental design and growth conditions Two NILs and their parental genotypes were field grown under two water treatments: well-watered control and dryland. The trial was sown on 1/4/08 at Bnei-Darom farm located in the coastal plain of Israel ( N E). The soil at this location is sandy clay composed of 51.3% sand, 6.9% silt and 41.8% clay. The temperature during the field experiment ranged between a minimum of C and a maximum of C, in April and July, respectively. A split-plot factorial (line water regime) block design with four replicates was employed, with irrigation treatment in main plots and genotypes in sub-plots. Each plot consisted of a single row of 4 m 0.96 m (length row spacing) with 9 plants/m of row. The amounts of water applied to the well-watered treatment were calculated according to Class A pan evaporation multiplied by crop coefficients, consistently with commercial cotton practices. Fine adjustments of water application were made to maintain the recommended daily growth rates of the main stem (reflecting plant water status). Starting from 1/6/08, the wellwatered treatment was irrigated via a drip system twice a week (as practiced in commercial cultivation) with total amounts of 475 mm. The dryland treatment received only 30 mm of water for seed germination, and relied on stored soil moisture for the remaining season. Notably, no precipitation is expected during the summer (April October) in Israel, as also occurred in the experimental season Photosynthesis measurements Gas exchange of the youngest fully expanded leaf was measured using a portable photosynthesis system (Li-6400, Li- COR Inc., Lincoln, Nebraska, USA). Leaf cuvette was set at photosynthetic photon flux density (PPFD) of 1500 mmol m 2 s 1 1 and temperature of 30 8C, and the actual leaf temperature during the measurements was C. For measurements of A vs. C i (A/C i curves), the [CO 2 ] in the leaf cuvette was set at 11 levels (370, 270, 170, 100, 50, 370, 600, 800, 1000, 1200 and 1500 ppm). For daily gas exchange curves the [CO 2 ] was set at 370 ppm, equivalent to the ambient [CO 2 ]. Gas exchange measurements were conducted during flowering time between 89 and 105 days after sowing, days after commencing the application of contrasting water regimes. Six and nine A/C i measurements were conducted in GB and GH genotypes, respectively. Measurements for GB genotypes were carried out on 29/6/08 and 30/6/08 and for GH genotypes on 3/7/ 08, 6/7/08 and 10/7/08. Each day, two genotypes (NIL and its recipient parent) under the two water treatments were measured three times between morning (about 07:00 h) and noon time (about 13:00 h). Daily gas exchange curves were established on 14/7/08. Four genotypes (two NILs and their recipient parents) under the two water treatments were

3 90 A. Levi et al. / Plant Science 177 (2009) Table 1 Seed-cotton yield (SC), osmotic potential (OP), leaf chlorophyll content (Chl) and specific leaf weight (SLW) of G. barbadense and G. hirsutum genotypes under well-watered and dryland treatments. SC (g m 2 ) OP (MPa) Chl (SPAD values) SLW (mg cm 2 ) Well-watered Dryland Well-watered Dryland Well-watered Dryland Well-watered Dryland G. barbadense genotypes F b (0.08) x y y x y x NIL (0.08) a x y x y y x y x G. hirsutum genotypes Siv on a 8.3a 10.9a x y x y y x y x NIL b 7.1b 9.8b x y x y y x y x Different letters indicate significant differences (p 0.05) between genotypes within each species and water treatments (a, b) or between water treatments within each genotype (x, y). Probability levels between 0.05 p 0.1 are indicated in parenthesis. measured eight times in rotation between 07:00 and 16:00 h using the same four plant replicates. A/C i curves were fitted using a non-linear model [28]: A ¼ að1 e bx Þþc where x is C i and a, b and c are parameters. This model revealed a significant fit (r 2 > 0.98) with all CO 2 response data sets. Stomatal limitation (L s ) was calculated as the depression between A under C i = 370 ppm ða Ci Þ and A under ambient [CO 2 ] C a = 370 ppm ða Ca Þ [29] as: L s ¼ 100 ða C i A Ca Þ A Ci Mesophyll conductance (g m ), the maximum velocity for carboxylation of Rubisco (V cmax ) and electron transport rate (J) were determined based on A vs. C c (A/C c ) curves, using leaf temperature of 30 8C and adjusting it to 25 8C, as suggested by Sharkey et al. [7] Drought related traits and yield measurements In parallel to each A/C i measurement, three youngest fully expanded leaves of neighboring plants were harvested and leaf water potential (C w ) was assessed immediately using a pressure chamber (ARI-2, Arimad, Kfar Haruv, Israel). Samples for measurements of OP, Chl and SLW were collected at early morning hours on , 107 days after sowing. For OP, a whole leaf was sampled from each plot, placed in a test tube with its petiole immersed in distilled water and rehydrated for 5 h at 4 8C (to obtain full turgor). Subsequently, leaves were frozen in liquid N and kept at 18 8C until measured. Samples were defrosted, and OP of the leaf sap was assessed using a vapor-pressure osmometer (Model 5520; Wescor Inc., Logan, UT, USA). Chl was measured in-situ on three leaves per plot using a chlorophyll meter (SPAD-502, Minolta Camera Co., Osaka, Japan). For SLW, three leaf discs (11 mm diameter) were sampled from the youngest fully expanded leaf and from each of the two leaves below it, using two plants per plot. The 18 discs collected per plot were oven-dried (80 8C) and weighed. Seedcotton yield (SC) was harvested manually at full boll opening from 1 m or 2 m of plants row in well-watered and dryland treatments, respectively Statistical analysis The JMP statistical package (SAS Institute 2007) was used for statistical analysis. A factorial model was employed separately for each genotype group (NIL and its recipient parent). For the photosynthetic variables, analysis of variance was applied with genotype (G), irrigation (I) and G I as fixed effects, measurements date as random effect, and measurements time (T) and T I as covariate effects. For SC, Chl, OP and SLW, analysis of variance was applied with genotype (G) and irrigation (I) as fixed effects and block (B) and B I as random effects (detailed analysis is not presented). Student s t-test was used to compare between each NIL and its recipient parent separately under each irrigation treatment as well as between irrigation treatments for each of the genotypes. 3. Results 3.1. Whole plant responses to drought The level of water stress imposed by the dryland treatment affected significantly plant productivity and drought related traits as compared with the well-watered control (Table 1). Under the dryland treatment, the average SC was 31% of the yield under irrigation, OP was reduced by MPa, while Chl and SLW were significantly increased. GB genotypes did not differ from one another under the well-watered treatment, whereas under the dryland treatment NIL 1-4 exhibited lower OP (p = 0.08) and greater Chl, but non-significant changes in SC and SLW than its recipient parent, F-177 (Table 1). GH genotypes exhibited similar values of SC and OP under both treatments as well as Chl under irrigation, whereas Chl under dryland treatment and SLW under both treatments were lower in NIL 3-2 than its recipient parent, Siv on Diurnal patterns of gas exchange The values of A and g s measured under ambient [CO 2 ](A 370 and g s370, respectively) throughout the daytime were usually lower in the dryland treatment than the well-watered treatment (Figs. 1 and 2). GB genotypes exhibited similar patterns of diurnal A 370, g s370 under well-watered treatment (Fig. 1a, b), with high values in the morning and gradual decrease during the day. Under the dryland treatment at morning time (7:30 10:00), F-177 exhibited higher A 370 and g s370 than NIL 1-4. During mid-day (12:00), A 370 and g s370 of F-177 decreased dramatically, while in NIL 1-4 these variables remained stable (Fig. 1c, d). Similar to GB, under the well-watered treatment GH genotypes exhibited high A 370 and g s370 in the morning and gradual decrease during the day (Fig. 2a, b). However, in contrast to GB, significant differences between genotypes were manifested under wellwatered treatment, with Siv on showing higher A 370 and g s370 throughout most of the day. Under dryland treatment, GH genotypes exhibited the highest values of A 370 and g s370 at early morning followed by reduction during late morning, peaking at

4 A. Levi et al. / Plant Science 177 (2009) Fig. 1. Diurnal curves of gas exchange in F-177 and NIL 1-4 under ambient [CO 2 ], net CO 2 assimilation (A 370 ; a, c) and stomatal conductance (g s370 ; b, d). Measurements were conducted under well-watered (a, b) and dryland (c, d) treatments. Each data point is the average of four measurements SE. Note the different scales for g s370 under wellwatered and dryland treatments. mid-day and decreasing again in the afternoon, with only small differences between genotypes (Fig. 2c, d) A/C i response curves Selected A/C i curves of each genotype under well-watered and dryland treatments at morning and noon are presented (Figs. 3 and 4). The GB genotypes, NIL 1-4 and its recipient parent F-177, usually exhibited similar A/C i curves under the well-watered treatment at both morning (Fig. 3a) and noon (Fig. 3b) measurements, as well as at morning time under the dryland treatment (Fig. 3c). However, at noon time A/C i curves of the dryland treatments revealed a dramatic difference between these genotypes (Fig. 3d). While NIL 1-4 was not affected neither by water availability nor by time of the day, F-177 exhibited a remarkable reduction in both C i and A. Fig. 2. Diurnal curves of gas exchange in Siv on and NIL 3-2 under ambient [CO 2 ], net CO 2 assimilation (A 370 ; a, c) and stomatal conductance (g s370 ; b, d). Measurements were conducted under well-watered (a, b) and dryland (c, d) treatments. Each data point is the average of four measurements SE. Note the different scales for g s370 under wellwatered and dryland treatments.

5 92 A. Levi et al. / Plant Science 177 (2009) Fig. 3. Typical curves of net CO 2 assimilation (A) as a function of intercellular CO 2 concentration (C i ) in F-177 and NIL 1-4 under well-watered (a, b) and dryland (c, d) treatments. Measurements were conducted at morning time (a, c) and at noon time (b, d). Fig. 4. Typical curves of net CO 2 assimilation (A) as a function of intercellular CO 2 concentration (C i ) in Siv on and NIL 3-2 under well-watered (a, b) and dryland (c, d) treatments. Measurements were conducted at morning time (a, c) and at noon time (b, d).

6 A. Levi et al. / Plant Science 177 (2009) Table 2 Analysis of variance for net CO 2 assimilation rate under ambient [CO 2 ](A 370 ), stomatal conductance under ambient [CO 2 ] (g s370 ), stomatal limitation (L s ), mesophyll conductance (g m ), the maximum velocity for carboxylation of Rubisco (V cmax ) and electron transport rate (J); separate ANOVA was conduced for G. barbadense and G. hirsutum genotypes. Source of variation d.f. F ratio A 370 g s370 L s g m V cmax J G. barbadense genotypes Genotype (G) Irrigation (I) ** 33.0 *** *** 7.8 ** 7.9 ** 11.9 ** G I * ** * 5.8 * Date * * 0.8 Time (T) T I Error 15 G. hirsutum genotypes Genotype (G) * 14.5 *** 18.7 *** Irrigation (I) *** 99.7 *** *** 45.0 *** 59.1 *** 30.9 *** G I ** Date * Time (T) ** 5.8 * 16.0 *** 11.0 * T I * 2 Error 20 (22) a * Indicates significance at p ** Indicates significance at p *** Indicates significance at p a d.f. for L s and g s370 is indicated in parentheses. Different trends were observed in the GH genotypes. Under the well-watered treatment the recipient parent, Siv on, exhibited higher A than NIL 3-2 throughout most of the C i range (Fig. 4a, b). However, under dryland treatment, both genotypes exhibited similar A/C i curves (Fig. 4c, d). It appears that while NIL 3-2 exhibited relative stability across the irrigation treatment, Siv on showed reduced levels of A under the dryland relative to the wellwatered conditions (Fig. 4a, b vs. Fig. 4c, d) Gas exchange under ambient [CO 2 ] as related to plant water status Gas exchange values under ambient CO 2 (370 ppm) were of a special interest. Analysis of variance for A 370 and g s370 (based on CO 2 responses curves) revealed highly significant effects of irrigation and no effect of genotype in all cases (Table 2), as well as significant G I interactions for A 370 in GB. Values of A 370 and g s370 were significantly lower under dryland than the well-watered conditions, with the exception of A in NIL 1-4, which exhibited a small, non-significant reduction (Table 3). The GB genotypes did not differ from one another under the well-watered treatment neither in A 370 nor in g s370. However, under the dryland treatment, NIL 1-4 exhibited significantly higher A 370 as well as higher g s370 (not significant) than its recipient parent, F-177. In contrast, among the GH genotypes, NIL 3-2 exhibited lower A 370 values under the well-watered treatment than its recipient parent, Siv on, whereas in all other cases no differences were found between these genotypes (Table 3). Data of A 370 and g s370 were also used to analyze the association between leaf water status and gas exchange. Values of C w measured between 7:00 and 13:00 ranged under the well-watered control between 1.2 and 2.0 MPa and under the dryland conditions between 2.0 and 2.9 MPA. Among the GB genotypes, F-177 exhibited a significant decline in A 370 with reduced C w, whereas NIL 1-4 exhibited stable A 370 values across the entire range of C w (Fig. 5a). As a consequence of these relationships, NIL 1-4 exhibited greater A 370 than F-177 under low water potentials (< 2.0). The respective g s370 vs. C w curves revealed a moderate slope in NIL 1-4 than F-177 (Fig. 5b). In GH, however, the two genotypes exhibited similar relationships between gas exchange and C w (Fig. 5c, d) Biophysical and biochemical photosynthetic variables Analysis of variance for L s, g m, V cmax, and J revealed highly significant effects of genotypes and irrigation in most cases (Table 2), as well as significant G I interactions, particularly in GB. Both GB and GH genotypes exhibited 2 3 fold higher L s under the dryland treatment than the well-watered control (Table 3). Significantly different values of L s were found between the GB genotypes under the dryland treatments, with F-177 being 10% higher than NIL 1-4, whereas the GH genotypes did not differ from one another in L s values. Dryland treatment reduced g m of both GB and GH genotypes; however, NIL 1-4 exhibited relatively stable g m across treatments. (Table 3). The GB genotypes did not differ from one another in g m values. However, in GH genotypes under the dryland treatment, g m of NIL 3-2 nearly doubled as compared with Siv on (p = 0.06). The biochemical variables, V cmax and J, of Siv on, NIL 1-4 and NIL 3-2 were significantly higher under dryland than the well-watered conditions, while F-177 exhibited stability of these variables across treatments (Table 3). Among the GB genotypes, under the dryland treatment NIL 1-4 exhibited a significantly higher V cmax and J values than its recipient parent, F-177. In contrast, among the GH genotypes, NIL 3-2 exhibited lower V cmax and J values under the well-watered treatment, than its recipient parent, Siv on. A third biochemical variables, the rate of triose phosphate utilization Table 3 Net CO 2 assimilation rate under ambient [CO 2 ](A 370 ), stomatal conductance under ambient [CO 2 ](g s370 ), stomatal limitation (L s ), mesophyll conductance (g m ), maximum velocity for carboxylation of Rubisco (V cmax ) and electron transport rate (J) ofg. barbadense and G. hirsutum genotypes under well-watered and dryland treatments. All variables were derived from CO 2 response curves. A 370 (mmol m 2 s 1 ) g s370 (mol m 2 s 1 ) L s (%) g m (mmol m 2 s 1 Pa 1 ) V cmax (mmol m 2 s 1 ) J (mmol m 2 s 1 ) Well-watered Dryland Well-watered Dryland Well-watered Dryland Well-watered Dryland Well-watered Dryland Well-watered Dryland G. barbadense genotypes F b a b b x y x y y x x y NIL a b a a x y y x y x y x G. hirsutum genotypes Siv on 34.0a a a x y x y y x x y y x y x NIL b (0.06) 128.3b b x y x y y x x y y x y x Different letters indicate significant differences (p 0.05) between genotypes within each species and water treatments (a, b) or between water treatments within each genotype (x, y). Probability levels between 0.05 p 0.1 are indicated in parenthesis.

7 94 A. Levi et al. / Plant Science 177 (2009) Fig. 5. Gas exchange under ambient [CO 2 ] vs. leaf water potential (C w )ing. barbadense genotypes and G. hirsutum genotypes; net CO 2 assimilation (A 370 ; a, c) and stomatal conductance (g s370 ; b, d). Measurements were conducted under well-watered (closed symbols) and dryland (open symbols) treatments. (TPU), exhibited similar trends to those found for V cmax and J, however, since TPU was not detectable in all cases data are not presented. 4. Discussion 4.1. Photosynthetic responses to drought Photosynthesis is the basis of plant production and a major determinant of crop yield [30]. In the current study, two cotton NILs introgressed with QTLs associated with improved yield and drought related traits [31,32] and their recipient parents were subjected to a detailed field study of their photosynthetic responses to drought conditions. The effect of water stress on the dryland treated plants was manifested by several variables. While under the well-watered treatment SC was at the acceptable level for commercially grown cotton, it was 70% lower under the dryland treatment. This corresponded to low mid-day C w, 2.7 and 1.8 MPa under the dryland and well-watered treatments, respectively, as well as to mid-day g s370 of 0.2 and 0.6 mol m 2 s 1 under the same respective treatments. These responses indicate that a severe level of stress was imposed by the dryland treatment. Stomatal closure and reduced g m are among the major physiological events occurring in response to decreased water availability, affecting net CO 2 assimilation [11,15]. Indeed, lower C w imposed by dryland treatment was associated with reduced g s370, greater L s, lower g m and reduced A 370 (Fig. 5; Table 3) in most genotypes, with acceptation of NIL 1-4 (discussed below). Despite the severe water stress applied in dryland treatment, the biochemical variables, V cmax and J, exhibited similar (F-177) or even higher (Siv on and both NILs) values under dryland treatment relative to the well-watered, control (Table 3). The concurrent increase of J and V cmax in the current study under drought was in agreement with the positive correlation between these biochemical variables [33 35]. Enhanced electron transport under moderate drought stress was previously reported in field-grown cotton [36]. Pot-grown cotton exhibited reduced J and carboxylation efficiency only under extreme water stress, where relative soil water content was <15% and g s <0.1 mol m 2 s 1 [37]. In fieldgrown grapevines, J remained relatively stable across a range of g s ( mol m 2 s 1 ) [38]. A compiled data of many different species showed that Rubisco activity, RuBP content, total soluble protein and Nitrate Reductase activity remains unaffected from maximum g s down to 0.1 mol m 2 s 1, suggesting that within this range assimilation of CO 2 was not limited by the biochemical capacity [11]. In agreement with the literature, in the current study, where g s values were rarely below 0.1 mol m 2 s 1, it seems that reduced diffusional conductances (stomatal and mesophyll) and not biochemical components, were the predominant factors limiting A under drought GB genotypes responses to drought conditions The photosynthetic capacity of NIL 1-4 was not affected significantly by the experimental treatments (Table 3) and across a wide range of C w (Fig. 5a). This unique phenomenon was unmatched in any of the other tested genotypes. Under drought conditions NIL 1-4 exhibited reduced g s370 (and increased L s ), similarly to all other genotypes, as well as improved biochemical capacity (greater J and V cmax ; Table 3), similarly to the GH genotypes. Among the variables measured in the current study, a capacity to maintain a stable g m across treatments, observed only in NIL 1-4 (Table 3), could have contributed to its outstanding stable A. NIL 1-4 exhibited greater A than F-177 under dryland conditions, as reflected by the noon measurement of both diurnal curves (Fig. 1a), and A/C i curves (Fig. 3d). NIL 1-4 was reported to have smaller leaf size [25], which could be associated with higher C w [39] and consequently with greater photosynthetic capacity. However, our current results showed clearly that NIL 1-4 had greater A than F-177 under a range of C w below 2.0 MPa (Fig. 5a). Under the dryland treatment NIL 1-4 exhibited higher g s370 (not significant) and lower L s (Table 3) relative to F-177. The differences in stomatal behavior between these genotypes could result from the lower OP measured in NIL 1-4 under drought conditions

8 A. Levi et al. / Plant Science 177 (2009) (Table 1; [25]). Low OP, as a result of solute accumulation, is known to maintain leaf turgor pressure and stomatal conductance under water deficit [40]. The differences between the GB genotypes in photosynthetic capacity under drought may have also influenced by different Chl, reflecting the strength of photosynthetic tissue [41]. Indeed, Chl and CO 2 exchange rate have been shown to be associated in cotton plants [42,43]. Hence, the higher Chl observed in NIL 1-4 under drought conditions, in the current study (Table 1) and previously [25], reflects the strength of the photosynthetic tissue in NIL 1-4. High Chl could increase light absorption, leading to higher J under drought in NIL 1-4 than F-177 (Table 3), which increases the availability of energy (NADPH and ATP) to the Calvin cycle GH genotypes responses to drought conditions Under well-watered treatment, Siv on exhibited higher rates of gas exchange than NIL 3-2 throughout most of the day (Fig. 2a, b) as well as across most CO 2 concentrations (Fig. 4a, b; Table 3). Under these conditions, Siv on exhibited greater values of V cmax and J than NIL 3-2 (Table 3), suggesting high efficiency of the photosynthesis system in Siv on. Under dryland conditions, however, the GH genotypes did not differ from one another in A throughout the day (Fig. 2c, d) or across a range of [CO 2 ](Fig. 4c, d). The only difference between the GH genotypes under dryland conditions was the lower values of g m observed in Siv on (Table 3). It is, therefore, apparent that a non-stomatal factor, g m, imposed a major limitation on A in Siv on under drought, thus eliminating the advantage shown by this genotype under well-watered conditions. In agreement with this, several field and greenhouse studies with GH cotton suggested that non-stomatal factors were primarily responsible for photosynthetic decline under drought [44 47] as well as under biotic stress [48]. NIL 3-2 exhibited significantly lower SLW (Table 1; [25]), smaller epidermal cells and higher stomatal density [25] than its recipient parent. Variation in leaf thickness (SLW) was related previously to differences in mesophyll anatomy in several species [49,50] including GH cotton [51], and can be negatively associated with g m [49,52]. Similarly to NIL 3-2, transgenic rice overexpressing barley aquaporin (HvPIP2;1) exhibited higher g m as well as several anatomical (mesophyll porosity, stomatal density, stomatal size) and physiological changes [14]. Thus, the lower g m in Siv on than NIL 3-2 may be derived from differences in tissue structure, which could limit CO 2 influx to carboxylation sites and reduce A in under drought [53]. 5. Conclusions Reduced CO 2 diffusion due to decreased stomatal and mesophyll conductances has been identified in the current study as the predominant factor limiting A under drought conditions. The sum of the diffusion resistances sets the limit to photosynthesis rates in stressed leaves [11]. Similar rates of A were revealed in the two GH genotypes under drought conditions; hence, differences in yield were not expected. In GB genotypes, however, NIL 1-4 introgressed with QTLs for higher yield and drought related traits exhibited improved photosynthetic capacity under drought conditions relative to F Moreover, NIL 1-4 also surpassed its recipient parent in several drought related traits (smaller leaf size, lower OP and greater Chl) [25]. Nonetheless, under water stress NIL 1-4 did not reveal yield advantage relative to its recipient parent. In a larger set of GB NILs, lower OP (greater OA) was associated with higher lint yield [25]. Nevertheless, we cannot preclude the possibility that metabolic cost of OA [40] has limited the yield potential in NIL 1-4. In addition, NIL 1-4 exhibited smaller leaf size [25] accompanied with compact canopy and low height [unpublished data], suggesting that lower leaf area may have limited the productivity of NIL 1-4. Low heritability of complex trait influenced by multiple gene networks is considered the major reason for the poor success of MAS for improved yield [24]. Thus, the current photosynthetic study provides further support to our previous conclusion [25] that MAS should be combined with conventional breeding to identify lines taking advantage of drought related QTL(s) while retaining high yield potential. 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