Potassium application affects carbohydrate metabolism in the leaf subtending the cotton (Gossypium hirsutum L.) boll and its relationship with boll biomass

Field Crops Research 179 (2015) 120–131

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Potassium application affects carbohydrate metabolism in the leaf subtending the cotton (Gossypium hirsutum L.) boll and its relationship with boll biomass Wei Hu a , Jiashuo Yang a , Yali Meng a , Youhua Wang a , Binglin Chen a , Wenqing Zhao a , Derrick M. Oosterhuis b , Zhiguo Zhou a,∗ a b

Key Laboratory of Crop Growth Regulation, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, PR China Department of Crop, Soil, and Environmental Sciences, University of Arkansas, 1366 West Altheimer Drive, Fayetteville, AR 72704, USA

a r t i c l e

i n f o

Article history: Received 15 March 2015 Received in revised form 23 April 2015 Accepted 24 April 2015 Keywords: Cotton (Gossypium hirsutum L.) Potassium fertilization Leaf subtending the cotton boll Carbohydrate metabolism Boll biomass

a b s t r a c t Field experiments were conducted in 2012 and 2013 with two cotton (Gossypium hirsutum L.) cultivars (Simian 3, low-K tolerant; Siza 3, low-K sensitive) under three levels of potassium (K) fertilization (0, 150 and 300 kg K2 O ha−1 ). Results showed that K application increased leaf K concentration, net photosynthetic rate (Pn), stomatal conductance (Gs), plant biomass and stimulated boll biomass (capsule wall, seed and lint biomass). K application increased the proportion of lint biomass, decreased the proportion of seed biomass, and did not affect the proportion of capsule wall biomass. Specific leaf weight (SLW), maximum/minimum sucrose contents and nonstructural carbohydrate (hexose, sucrose, starch) decreased, but sucrose transformation rate in LSCB increased in both cultivars after K application, the leaf critical K levels for hexose content, sucrose content and starch content were 1.1%, 1.3%, 1.2–1.4% in Simian 3 and were 1.6%, 1.7% and 1.7–1.8% in Siza 3, respectively. The activities of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco), cytosolic fructose-1,6-bisphosphatase (cy-FBPase), sucrose phosphate synthase (SPS), sucrose synthase (SuSy) and amylase activities increased by K application, whereas soluble acid invertase (SAI) activity decreased. SPS and SuSy activities in Siza 3 were more sensitive than that in Simian 3. Correlation analysis revealed that higher Pn, sucrose transformation rate and SPS activity in LSCB were necessary to improve boll biomass, but the accumulation of sucrose in LSCB was not beneficial to boll biomass. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Potassium (K) is one of the major mineral elements for normal plant growth and development, and plays a vital role in many physiological processes, including maintenance of charge balances, tissue turgor pressure, electrogenic transport processes and photosynthesis, regulation of stomata movement, activation of numerous enzymes and in protein synthesis (Dobermann, 2001; Oosterhuis et al., 2014). In recent years, K deficiencies have been increasing

Abbreviations: CV(%), coefficient of variance; Pn, net photosynthetic rate; Gs, stomatal conductance; Rubisco, ribulose-1,5-bisphosphate carboxylase-oxygenase; cy-FBPase, cytosolic fructose-1,6-bisphosphatase; DPA, days post anthesis; LSCB, leaf subtending the cotton boll or the subtending leaf; SLW, specific leaf weight, mean the weight per cm2 leaf; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; SAI, soluble acid invertase. ∗ Corresponding author. Tel.: +86 25 84396813; fax: +86 25 84396813. E-mail address: [email protected] (Z. Zhou). http://dx.doi.org/10.1016/j.fcr.2015.04.017 0378-4290/© 2015 Elsevier B.V. All rights reserved.

across 30 provinces in China, which will limit crop growth and development and represents a significant threat to China’s future crop production. To rectify this deficiency of K will require an increase in K fertilizer use of more than 8% per year (Sheldrick et al., 2003). Cotton (Gossypium hirsutum L.) has a large demand for K and appears to be more sensitive to low K availability than other crops (Cassman et al., 1989; Oosterhuis, 2001). K deficiency decreases seed cotton yield and lint yield (Pettigrew, 1999; Gormus and Yucel, 2002; Read et al., 2006), attributable to reduced boll weight (Gormus, 2002), lower boll number (Li et al., 2012) and lower lint percentage (Pettigrew, 1999). K deficiency negatively affects cotton fiber quality, by decreasing fiber length (Cassman et al., 1990), uniformity ratio (Pettigrew et al., 1996), fiber strength (Cassman et al., 1990; Minton and Ebelhar, 1991) and micronaire (Pettigrew et al., 2005). Some studies have shown that K deficiency also negatively affected cotton photosynthesis (Bednarz et al., 1998; Pervez et al., 2004), biomass production (Zhao et al., 2001), altered biomass

W. Hu et al. / Field Crops Research 179 (2015) 120–131

partitioning (Reddy and Zhao, 2005; Makhdum et al., 2007) and morphological indices (Gerardeaux et al., 2009b). K deficiency significantly reduced dry matter partitioning to reproductive organs (Pettigrew et al., 2005; Makhdum et al., 2007) and increased dry matter partitioning to the leaf (Gerardeaux et al., 2009a; Wang et al., 2012). K deficiency increased carbohydrate concentrations in the cotton leaf (Bednarz and Oosterhuis, 1999), which may be associated with inhibition of phloem loading (Marschner et al., 1996; Zhao et al., 2001), such that the K disorder may cause an imbalance of source and sink (Wright, 1999). In cotton, the leaf subtending the cotton boll (LSCB) plays a vital role contributing to boll development as it is the main source of carbohydrate for the boll, supplying 60–87% of the total photoassimilate requirements (Constable and Rawson, 1980; Wullschleger and Oosterhuis, 1990; Liu et al., 2013). Sucrose and starch are the main products of photosynthesis in most plants including cotton, although sucrose is the primary photosynthate transported from source to sink tissues and can be broken down into hexoses, which provide carbon and energy for plant growth. Starch as a temporarily stored carbohydrate can be converted into sucrose (Gandin et al., 2009). Previous studies have suggested that under K deficiency, soybean (Glycine max) (Huber, 1984) leaves had significantly higher hexose, sucrose and starch contents. In contrary, starch content in lemon (Citrus volkameriana Ten. & Pasq) leaves (Lavon et al., 1995) and sucrose content in maize (Zea mays L.) leaves (Pretorius et al., 1999) were reduced under K deficiency. There are many important enzymes involved in carbohydrate metabolism processes. Rubisco is the key and rate-limiting enzyme in the Calvin cycle, SPS (sucrose phosphate synthase, E.C. 2.4.1.14), SuSy (sucrose synthase, E.C. 2.4.1.13) and acid invertase are the main enzymes that control sucrose accumulation and degradation (Hendrix and Huber, 1986). The penultimate step in sucrose synthesis is catalyzed by SPS and the first committed step is catalyzed by cy-FBPase (cytosolicfructose-1,6-bisphosphatase, E.C. 3.1.3.11) (Liu et al., 2013). Amylase plays a important role in starch degradation (Hammond and Burton, 1983). Previous research has shown that all these enzymes were affected by K status. Rubisco activity decreased in alfalfa(Medicago sativa L.) (Peoples and Koch, 1979) leaves under K deficiency, but was hardly affected in mulberry (Morus alba L.) (Yamashita and Hikasa, 1988). SPS activity declined and acid invertase activity increased under K deficiency in soybean (Huber, 1984), but SPS activity was unaffected in maize (Pretorius et al., 1999). SuSy activity and amylase activity decreased in potato (Solanum tuberosum) under K deficiency (Lindhauer and De Fekete, 1990). In contrast, amylase activity was significantly higher in K-deficient leaves of lemon (Lavon et al., 1995). Therefore, carbohydrate metabolism of different plants has different responses to K deficiency. K deficiency has been shown to affect carbohydrate content in main-stem leaf of cotton (Bednarz and Oosterhuis, 1999) and partitioning to the reproductive components of cotton (Pettigrew et al., 2005), but the explanation of this is lacking. The objectives of our research were (1) to study the effect of K on changes of plant and boll biomass partitioning during boll forming stage on the basis of carbohydrate metabolism in LSCB; (2) to understand the relationships between boll biomass and carbohydrate metabolism of cotton in relation to K fertility; and (3) to

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identify sensitive enzymes to K in carbohydrate metabolism for the two cultivars with different low-k sensitivity. 2. Materials and methods 2.1. Plant material Two cotton cultivars, Simian 3 (low-K tolerant) and Siza 3 (low-K sensitive) were selected in this study based on the variance of yield and fiber quality of 12 diverse cultivars, predominantly grown in the lower Yangtze region in China (Yang et al., 2014). Simian 3, was developed by the Siyang Original Seed Farm of Jiangsu Province, and Siza 3, was developed by the Cotton Research Group, Suqian Academy of Agricultural Sciences. 2.2. Experimental design Field experiments were conducted in the summer seasons of 2012 and 2013 at the Pailou experimental station of Nanjing Agricultural University, located at Nanjing, China (118◦ 50 E, 32◦ 02 N). The 2012 and 2013 field sites were located adjacently. The soil type was clay, mixed, thermic, Typic alfisols (udalfs; FAO luvisol) with a slightly acid pH of 6.7, the soil samples were collected at a 20 cm depth before sowing cotton, and the soil nutrient contents are listed in Table 1. Seeds were planted in a nursery bed on 23 April 2012 and 30 April 2013, and transplanted into the field when the cotton seedling had three true leaves. The experiment was performed in a randomized complete design with three replications. Plot size was 13 m long and 6.6 m wide, with 0.85 m between rows and 0.35 m between plants in the row. A uniform fertilizer application of 120 kg P2 O5 ha−1 (at transplanting stage) and 240 kg N ha−1 (40% at transplanting and 60% at the flowering stage) was applied. The treatments consisted of three K fertilizer rates: (i) 0 kg K2 O ha−1 , as a control, (ii) 150 kg K2 O ha−1 (the recommended quantity of K under the soil available K in this experiment) (Xia et al., 2010), and (iii) 300 kg K2 O ha−1 using potassium sulphate. 2.3. Sampling and processing White flowers at the first node of fruiting branches 7–8th of all plant were tagged with plastic tags that were used for noting the flowering date. The total time for tagging all treatments was not more than three days, to ensure that the labeled flowers had equivalent metabolic and developmental ages for all treatments. These tagged bolls and their subtending leaves were sampled every 7 days from 10 to 45 days post anthesis (DPA) at 9:00–10:00 A.M. local time. The samples of leaves were washed with distilled water, and divided into two halves by cutting the main vein; one half was immediately placed in liquid nitrogen and stored in an ultralow temperature freezer (−80 ◦ C) until enzymatic measurement, and the other half was used to measure leaf area and dry weight for calculating specific leaf weight (SLW), and the dried leaf tissues were then used to determine carbohydrate metabolism and leaf K concentration using an atomic absorption spectrophotometer (SpectAA-50/55, Varian, Australia). When the bolls opened, 50 tagged bolls in each treatment were harvested for the measurements of boll biomass accumulation and partitioning in to capsule wall, seed and lint biomass.

Table 1 Soil organic matter, nitrogen, phosphorus and potassium contents at the experimental sites in 2012 and 2013. Year

Organic matter content (g kg−1 )

Total N content (g kg−1 )

Available N content (mg kg−1 )

Available P content (mg kg−1 )

Available K content (mg kg−1 )

2012 2013

15.9 17.1

0.9 1.1

69.8 77.3

23.6 18.1

86.3 91.8

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2.4. Photosynthetic parameters Photosynthetic parameters of the LSCB at the first node of the 7–8th fruiting branches were measured with a portable photosynthesis system (Li-6400, Li-COR, Lincoln, NE, USA) at 10, 17, 24, 31, 38, 45 DPA. All the leaves were tagged after the first measurement in order to measure same leaves every time. Photosynthetic parameters were measured from 9:00 to 11:00 A.M. at 32 ± 2 ◦ C leaf temperature, 65 ± 5% relative humidity, 380 ± 5 ␮mol mol−1 CO2 and 1500 ␮mol m−2 s−1 photosynthetic photon flux density. 2.5. Carbohydrate analysis Dried leaf tissues were ground to pass a 1 mm sieve. The powdered sample (0.1 g) was placed in 5 mL of 80% (v/v) ethanol and then incubated in a water bath at 80 ◦ C for 30 min. Next, the mixture was centrifuged at 4000 rpm for 5 min. The above process was repeated three times. The three supernatants were collected and diluted with 80% ethanol to 25 mL. The resulting extract was frozen at −20 ◦ C until analysis. Leaf nonstructural carbohydrate (hexose and sucrose) were assayed according to previously described by Hendrix (1993). The ethanol-insoluble residue was saved for starch extraction according to Liu et al. (2013). First, ethanol was evaporated to dryness. Starch in the residue was released in boiling with 2 mL distilled water for 15 min and cooled to normal temperature. Then, leaf starch was hydrolyzed with 9.2 mol L−1 HClO4 (2 mL) for 15 min. Distilled water (4 mL) was added into the samples, and the samples centrifuged at 4000 rpm for 10 min. The residue was extracted once more using 4.6 mol L−1 HClO4 (2 mL). The supernatants were combined and diluted with distilled water to 25 mL. The starch concentration was measured by spectrophotometer (UV-2450, Shimadzu, Japan) at A620 nm using an anthrone reagent (Morris, 1948) and glucose was used to prepare a standard curve. 2.6. Enzyme extraction and analysis All enzymes were extracted at 0 to 5 ◦ C. The enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase, E.C. 4.1.1.39) was extracted from frozen leaf samples as described by Liu et al. (2013) with some modification. The frozen leaf sample (0.2 g) was ground using a chilled mortar containing 3 mL cooled extraction buffer (50 mM Tris–HCl [pH 7.5], 1 mM EDTA, 1 mM MgCl2 , 12.5%(v/v) of glycerin, 10% of polyvinylpyrrolidone (PVP), and 10 mM ␤-mercaptoethanol). The homogenate was filtered through eight layers of cheesecloth and centrifuged at 15,000 × g for 15 min at 4 ◦ C. The supernatant was used for intial Ruisco activity assay. Leaf cy-FBPase (cytosolicfructose-1,6-bisphosphatase, E.C. 3.1.3.11) was extracted and assayed according to a modified method of Kuai et al. (2014). The extraction buffer contained 100 mM Tris–HCl (pH 8.2), 1 mM EDTA, 50 mL/L glycerinand and 15 mM ␤-mercaptoethanol. Cy-FBPase activity was assayed in a total volume of 1 mL reaction mixture (100 mM Tris–HCl (pH 8.2), 0.5 mM EDTA, 5 mM MgCl2 , 0.5 mM NADP, 2 units phosphoglucoisomerase (PGI, Sigma), 1 unit glucose-6-phosphate dehydrogenase). The reaction was activated by adding 60 mM FBP (Sigma) at 25 ◦ C. The Leaf cy-FBPase activity was measured by spectrophotometer (UV-2450, Shimadzu, Japan) at A340 nm. The enzymes SPS (sucrose phosphate synthase, E.C. 2.4.1.14) and SuSy (sucrose synthase, E.C. 2.4.1.13) were extracted from frozen leaf samples as described previously (Huber and Israel, 1982) for measurement of fructose 6-P or fructose-dependent formation of

sucrose from UDP-Glucose. The reaction mixture of SPS contained 50 mM UDP-glucose, 50 mM fructose-6-P, 50 mM extraction buffer, 10 mM MgCl2 and 200 ␮L of extract in a total volume of 550 ␮L. The reaction was initiated by incubating the enzyme extract at 30 ◦ C for 30 min. The reaction was stopped using 100 ␮L of 2 mol L−1 NaOH at 100 ◦ C for 10 min to destroy unreacted hexoses and hexose phosphates. Then, the solution was cooled and mixed with 1 mL of 0.1% (w/v) resorcin in 95% (v/v) ethanol and 3.5 mL of 30% (w/v) HCl before being incubated at 80 ◦ C for 10 min. Sucrose content was calculated from a standard curve measured at A480 nm. The reaction of SuSy was similar to SPS except fructose-dependent was substituted for fructose 6-P. The enzyme SAI (soluble acid invertase activity, EC 3.2.1.26) was extracted and assayed by using a modified method of Hanft and Jones (1986). SAI was extracted by grinding 1 g frozen leaf sample in 10 mL of 0.2 M citrate phosphate buffer (pH 8.0) in a chilled mortar at 4 ◦ C. Then, the crude enzyme extract was centrifuged at 1500 × g for 30 min at 4 ◦ C. The supernatant was dialyzed overnight against 10 mm citrate phosphate buffer (ten times the volume of the combined sample extracts) at 4 ◦ C. The dialyzed extracts were diluted 5 to 10 fold depending on the expected acid invertase activity of the sample. The reaction mixture contained 100 ␮L of the diluted extract, 300 ␮L of 0.2 M sodium acetate (pH 4.8) and 100 ␮L of 50 mM sucrose, then the assayed mixture was immediately incubated at 37 ◦ C for 30 min, 0.5 mL distilled water was added to the assayed mixture and the glucose was quantified using Nelson’s test (Nelson, 1944). SAI activity was expressed as ␮M glucose/g fresh weight·hour. The enzyme amylase was extracted according to Lavon et al. (1995), The extraction buffer contained 20 mM tris-maleate (pH 6.2), 0.04% Tween-20 and 5 mM CaCl2 , using a 1 leaf sample: 10 buffer ratio. The extract was filtered through eight layers of cheesecloth and centrifuged at 40,000 × g for 10 min at 4 ◦ C. Supernatants were dialyzed overnight against the same buffer except Tween20. Amylase activity was performed as described by Hammond and Burton (1983). The assayed mixture contained 1 mL 5% soluble starch solution and 1 mL dialysate. The mixture was incubated at 37 ◦ C for 45 min. The reducing sugars released were determined using Nelson’s test (Nelson, 1944).

2.7. Biomass accumulation and partitioning Plant dry weight was measured on three plants at 45 DPA in 2012 and 2013. On the sample date, the weights of root, stem (and branches), leaves and reproductive organs (floral buds, flowers, and bolls) were measured after detachment and drying to constant weight at 80 ◦ C.

2.8. Statistical assays All data were subjected to an analysis of variance with SPSS statistic package Version 17.0 (Liu et al., 2013), the difference between mean values were determined using the least significant difference (LSD, P = 0.05) test. The coefficient of variation (CV, %) was calculated as the ratio of the standard deviation to the mean. The maximum sucrose content and the minimum sucrose content of LSCB indicate the amount of available sucrose and the residual sucrose content, respectively. Thus, sucrose transformation rate of LSCB can be calculated by the expression of 100 × [(maximum sucrose content − minimum sucrose content)/maximum sucrose content] (Liu et al., 2013; Kuai et al., 2014). In order to assess physiological responses to K, all parameters were expressed in relative terms as a percentage of the treatment receiving 300 kg K2 O ha−1 , which refers to the method of Reddy and Zhao (2005).

W. Hu et al. / Field Crops Research 179 (2015) 120–131

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Table 2 Dry matter of root, steam, leaf and reproductive organs for two cultivars Simian 3 and Siza 3 at 45 DPA in 2012 and 2013. Year

Cultivar

K rate (kg K2 O ha−1 )

Root (g plant−1 )

Stem (g plant−1 )

Leaf (g plant−1 )

Reproductive organs (g plant−1 )

Total biomass (g plant−1 )

Leaf/total biomass ratio (%)

Leaf/reproductive organs ratio (%)

2012

Simian 3

0 150 300 LSD0.05 0 150 300 LSD0.05

19.00aA 18.50a 18.50a NSB 17.50a 18.00a 19.50a NS

34.82b 40.04a 42.31a

34.83b 36.87ab 38.63a

80.57b 88.44a 89.68a

169.21b 183.85a 189.13a

*

*

*

*

35.11a 34.33a 35.78a NS

32.81b 40.34a 41.69a

81.58b 105.51a 111.77a

166.99c 198.18b 208.74a

43.22a 41.69a 43.08a NS 40.21a 38.24ab 37.30b

*

**

**

19.99a 20.05a 20.43a NS 19.64a 20.36a 19.97a NS

0 150 300 LSD0.05 0 150 300 LSD0.05

20.32a 20.26a 20.71a NS 17.04a 19.65a 19.50a NS

41.19a 41.32a 43.97a NS 39.39a 38.36a 40.80a NS

35.81b 40.01a 40.99a

77.98b 88.91a 90.72a

175.29b 190.49a 196.38a

20.43a 21.00a 20.87a NS 21.04a 21.28a 20.72a NS

45.92a 45.00a 45.18a NS 44.12a 42.73ab 40.90b

Siza 3

2013

Simian 3

Siza 3

A B

*

*

*

37.98b 42.67a 43.65a

86.08c 99.88b 106.71a

180.50c 200.56b 210.67a

*

**

**

*

*

Values followed by a different letter within the same column are significantly different at P = 0.05 probability level. Each value represents the mean of three replications. NS means non-significant differences; * and ** means significant differences at 0.05 and 0.01 probability levels, respectively.

3. Results

distribution rate to boll (capsule wall, seed and lint) biomass, and increased the proportion of lint biomass and lint:seed ratio, but decreased the proportion of seed biomass in both years.

3.1. Biomass partitioning in cotton The weights of root and stem were not affected by K application (Table 2). With the increased K rates, leaf, reproductive organs and total biomass were increased significantly. The 150 and 300 kg K2 O ha−1 applications improved leaf, reproductive organs and total biomass by 8.2–14.5%, 6.6–16.3% and 8.0–12.0% in Simian 3, and by 11.0–27.1%, 15.8–37.0% and 10.0–25.0% in Siza 3 for the 2 years. The leaf/total biomass ratio were not significantly affected by K application. However, K application decreased the leaf/reproductive organs ratio, indicating that K application was of benefit for reproductive organs biomass.

3.3. Leaf K concentration Overall, K concentrations in LSCB were considerably affected by K fertilizer supply (Fig. 1). The leaf K concentrations of the 0, 150, and 300 kg K2 O ha−1 treatments declined rapidly from 10 DPA to 31 DPA, and decreased slightly at 31 DPA to 45 DPA. As the amount of K application increased, leaf K concentrations increased at every sample time. When leaf K concentrations were averaged across all sample times, the 150 and 300 kg K2 O ha−1 treatments for Simian 3 had 37.4%, 77.4% higher leaf K in 2012 and 22.8%, 51.8% higher leaf K in 2013, and for Siza 3, leaf K concentrations increased by 27.2%, 51.1% in 2012 and by 27.4%, 51.4% in 2013.

3.2. Boll biomass accumulation and partitioning Boll biomass was improved by K application. For Simian 3, the 300 kg K2 O ha−1 treatment had 16.4% and 13.8% more boll biomass in 2012 and 2013, respectively, than the no-K treatment (Table 3). For Siza 3, boll biomass from 300 kg K2 O ha−1 was increased 22.0% and 22.3% in 2012 and 2013, respectively. K application also improved capsule wall, seed and lint biomass, and affected the

3.4. Changes of photosynthetic parameters and carbohydrate contents in LSCB Photosynthesis in LSCB decreased with DPA and decreased more rapidly in the no-K application treatment (Fig. 2). A general trend for Pn to decrease with lower K application rate was observed from 24

Table 3 Effect of potassium application on capsule wall, seed, lint and all total biomass. Year

2012

Cultivar

Simian 3

Siza 3

2013

Simian 3

Siza 3

A B

K rate (kg K2 O ha−1 )

Boll biomass(g)

Proportion (%)

Capsule wall

Seed

Lint

total

Capsule wall

Seed

Lint

Lint/Seed ratio

A

0 150 300 CVB % 0 150 300 CV%

1.4b 1.6ab 1.8a 12.5 1.5b 1.7a 1.8a 9.2

2.7a 2.8a 2.9a 3.6 2.8c 3.1b 3.2a 3.9

1.4b 1.6ab 1.7a 9.8 1.6c 2.0b 2.2a 13.5

5.5b 6.0a 6.4a 7.6 5.9c 6.8b 7.2a 10.0

25.4b 26.7ab 28.1a 5.0 24.8a 24.4a 24.5a 1.3

49.1a 46.7ab 45.3b 4.1 47.3a 46.2ab 44.7b 4.4

25.5a 26.7a 26.6a 2.6 27.9c 29.3b 30.8a 5.8

51.9b 57.1a 58.6a 6.4 58.9c 63.4b 68.9a 10.1

0 150 300 CV% 0 150 300 CV%

1.5b 1.9a 1.7ab 11.8 1.7b 2.2a 2.2a 14.2

2.7b 3.1a 2.9ab 6.9 3.0b 3.3a 3.3a 5.4

1.6b 2.0a 2.0a 12.4 2.2b 2.7a 2.8a 12.6

5.8c 7.0a 6.6b 9.4 6.9b 8.2a 8.3a 10.0

25.9a 27.1a 25.8a 2.8 24.6b 26.8a 26.5a 4.6

46.6a 44.3b 43.9b 3.2 43.5a 40.2b 39.7b 5.0

27.6b 28.6b 30.3a 4.7 31.8b 32.9ab 33.8a 2.9

59.3c 64.5b 69.0a 7.5 73.3c 81.8b 85.2a 7.6

Values followed by the different letters within the same column are significantly different at P = 0.05 probability level. Each data represents the mean of three replications. CV, coefficient of variation.

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Fig. 1. Changes of leaf K concentration for three potassium treatments in 2012 and 2013. The data are the means for three replications ± SD.

DPA in 2012 and from 17 DPA in 2013. The 150 and 300 kg K2 O ha−1 applications improved Pn by 9.1–12.3% and 15.4–19.9% in Simian 3 and by 12.4–15.6% and 25.1–29.5% in Siza 3 for the 2 years. The trend of Gs was similar with that of the Pn in LSCB. Gs decreased with DPA, and increased with K fertilizer application (Fig. 2). Sucrose content in LSCB declined with DPA (Fig. 3). The result was consistent with the same trend of reported by Liu et al. (2013). Sucrose content also declined with increasing of K application rate and the difference of sucrose content among the three K treatments

increased with DPA, and this difference was greater in Siza 3 than that in Simian 3. Hexose content showed a similar trend in that hexose content declined with DPA during boll development and was reduced in K application treatments (Fig. 3). Starch is a temporarily stored carbohydrate in leaves. When photosynthesis is limited, starch breaks down in cotton leaves and provides a supply of carbohydrate for energy and fiber formation (Chang, 1980). In our study, it was found that K application decreased the starch content in cotton leaf (Fig. 3). There were some

Fig. 2. Changes of Photosunthesis (Pn) and stomatal conductance (Gs) in the LSCB for three potassium treatments in 2012 and 2013. The data are the means for three replications ± SD.

W. Hu et al. / Field Crops Research 179 (2015) 120–131

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Fig. 3. Changes of carbohydrates content in the LSCB for three potassium treatments in 2012 and 2013. The data are the means for three replications ± SD.

differences in starch content between 2012 and 2013; in 2012, starch content peaked at 38 DPA, but increased with DPA in 2013. Average starch content across DPA decreased by 12.1% and 17.8% in 150 and 300 kg K2 O ha−1 treatments, respectively, for Simian 3, and by 9.2% and 16.9% for Siza 3 in 2012. In 2013, the average contents of starch across DPA decreased by 10.3% and 14.9% in the 150 and 300 kg K2 O ha−1 treatments for Simian 3 and by 9.5% and 17.4% for Siza 3, respectively. In Table 4, increasing K application decreased the minimum sucrose contents compared with the untreated control, and significant differences were observed. Sucrose transportation rate of LSCB was significantly higher in K treatments for Simian 3, increased by 36.8% and 57.7% for the 150 and 300 kg ha−1 treatments in 2012, and by 27.3% and 42.7% in 2013, respectively. Compared with Simian 3, Siza 3 had the same trend in K treatments but a higher increment (53.9% and 92.6% increment for 150 and 300 kg ha−1 in

2012, and 22.1% and 50.9% increment for 150 and 300 kg ha−1 in 2013, respectively). In addition, the CV of the sucrose transformation rate in LSCB for Siza 3 was higher than that of Simian 3. The sucrose:starch ratio in K treated leaves increased. SLW was reduced by 6–28% for Simian 3 in the K application treatments (150 and/or 300 kg K2 O ha−1 ) and by 14–34% for Siza 3, compared to the no-K treatment. An exponential equation (Y = a × e−x/b + c, R2 = 0.8827–0.8517** , P < 0.01) could be used to reflect the relationship between leaf K concentration and the relative value of Pn (Fig. 4), where x is leaf K concentration, Y is the relative value of Pn, a, b and c are constants. The exponential equations showed that Pn was impaired significantly in Simian 3 when leaf K was lower than 1.2%, and the critical leaf K level for Siza 3 was 1.4%. The relative content of hexose, sucrose, and starch content showed a close fit with leaf K concentrations (R2 = 0.7574–0.9323** , P < 0.01, Fig. 4) using

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Table 4 Maximum and minimum sucrose content, sucrose transformation rate (Tr), sucrose/starch ratio, SLW in the LSCB of cultivars Simian 3 and Siza 3 for the three potassium treatments in 2012 and 2013. K rate (kg K2 O ha−1 )

Maximum sucrose content

Minimum sucrose content

TrA

Simian 3

Siza 3

Simian 3

Siza 3

Simian 3

2012 0 150 300 CVD %

23.47aC 23.05a 23.80a 1.6

26.89a 27.19a 27.64a 1.4

16.53a 13.72b 12.70b 13.9

20.02a 16.51b 14.05c 17.8

2013 0 150 300 CV %

22.27a 23.24a 22.61a 2.2

26.23a 25.52a 26.27a 1.6

14.15a 12.45b 10.84c 13.3

16.90a 14.43b 12.17c 16.3

A B C D

Sucrose/starch ratio

SLWB

Siza 3

Simian 3

Siza 3

Simian 3

Siza 3

29.59c 40.49b 46.65a 22.2

25.53c 39.28b 49.17a 31.3

1.44a 1.53ab 1.58b 4.3

1.84a 1.85a 1.89a 1.2

60.5a 56.8a 49.5b 10.1

58.5a 48.7b 43.1c 15.6

36.47c 46.44b 52.06a 17.5

35.56c 43.44b 53.68a 20.5

1.24a 1.29a 1.29a 2.5

1.46a 1.47a 1.52a 1.7

57.2a 50.1b 49.3b 8.3

59.4a 42.4b 39.5b 22.8

Tr, sucrose transformation rate. SLW, specific leaf weight. Values followed by the different letters within the same column are significantly different at P = 0.05 probability level. Each data represents the mean of three replications. CV, coefficient of variation.

another exponential equation of Y = a × eb/(x−c) , where Y is the relative content of these carbohydrate contents. These equations in Fig. 4 showed that the leaf critical K levels for hexose, sucrose and starch contents in Simian 3 were 1.1%, 1.3% and 1.2–1.4%, whereas in Siza 3, the leaf critical K levels were 1.6%, 1.7% and 1.7–1.8%, respectively. 3.5. Changes of carbohydrate-metabolizing enzymes activities in LSCB Initial Rubisco activity reflects the level of actual Rubisco activity in leaves. In 2012, Initial Rubisco activity in LSCB increased from 10 DPA, and reached peak values at 17 DPA, then

subsequently decreased in all the three K treatments (Fig. 5), whereas in 2013, Initial Rubisco activity in LSCB was reduced with DPA. As the amount of K application increased, initial Rubisco activity in LSCB was enhanced; average initial Rubisco activity increased by 13.3–30.2% for Simain 3 and by 15.1–44.0% for Siza 3 in the two years. The trend of cy-FBPase activity in LSCB (Fig. 6) was similar to that of initial Rubisco activity, the levels of cy-FBPase activity in LSCB peaked at 17 DPA in 2012, but declined with DPA in 2013. cy-FBPase activity in K application treatments (150 and 300 kg K2 O ha−1 ) was significantly higher than that in the 0 kg K2 O ha−1 treatment (P < 0.05). Compared with Smian 3, cy-FBPase activity of Siza 3 had significant differences between 150 and 300 kg K2 O ha−1 . In

Fig. 4. Relationships between relative value of photosynthesis (Pn), relative content of hexose, sucrose or starch and leaf K concentration in the LSCB. The solid and dotted lines represent the exponential regression lines of Simian 3 and Siza 3, respectively.

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Fig. 5. Changes of initial Rubisco activity in LSCB for three potassium treatments in 2012 and 2013. The data are the means for three replications ± SD.

Fig. 6. Changes of cy-FBPase activity in theLSCB during boll development for three potassium treatments in 2012 and 2013. The data are the means for three replications ± SD.

addition, there was a significant positive correlation between cyFBPase activity and sucrose content in LSCB (R2 = 0.401–0.642** ) (Fig. 7), with the slope of the fitted line in Siza 3 being higher than that in Simian 3 for the both years.

Fig. 7. Relationship between sucrose content (mg g−1 DW) and cy-FBPase activity (␮mol min−1 g−1 FW). The solid and dotted lines represent Simian 3 and Siza 3, respectively.

SPS and SuSy play important roles in sucrose metabolism in vivo. Both SPS activity and SuSy activity in LSCB peaked at 31 DPA in 2012 and at 24 DPA in 2013 (Fig. 8). In 2012, SPS activity and SuSy activity in LSCB of the no-K treatment were significantly lower beginning 24 DPA in Simian 3 but beginning 10 DPA in Siza 3. In 2013, SPS activity and SuSy activity in LSCB had significant differences between noK and K-supply treatments beginning 24 DPA. SPS activity of 150 and 300 kg K2 O ha−1 had significant differences in both cultivars, whereas SuSy activity had significant differences between 150 and 300 kg K2 O ha−1 treatments only in Siza 3. SAI is one of the critical enzymes responsible for the hydrolysis of sucrose to fructose and glucose. Compared to no-K treatments, K application treatments had lower SAI activities, and SAI activity of 300 kg K2 O ha−1 was clearly lower than the SAI activity of 150 kg K2 O ha−1 (Fig. 9). The pattern of SAI in LSCB was similar in all treatments, increasing from 10 DPA, reaching their peak values at 24 DPA in 2012 and 31 DPA in 2013, and then declining significantly to 45 DPA. Amylase has an important role in the degradation of starch into maltose and glucose in the chloroplast. Amylase activity in LSCB increased at first and then declined with DPA, with the peak values observed at 17 DPA in 2012 for both cultivars, at 24 DPA for Simain 3 and at 17 DPA for Siza 3 in 2013 (Fig. 9). The mean amylase activity in LSCB of K application treatments increased by 5.47–16.29% in Simian 3, and by 11.65–21.19% in Siza 3 for the 2 years.

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Fig. 8. Changes of SPS activity and SS activity in LSCB during boll development for three potassium treatments in 2012 and 2013. The data are the means for three replications ± SD.

Further analysis of the relationship between the peak of enzyme activity and the leaf K concentration, showed that the peak SPS activity, peak SAI activity, and peak amylase activity with leaf K concentration could be expressed for both cultivars using a linear equation (r2 = 0.8310–0.9782** , Fig. 10). There was a positive correlation between peak SPS activity, peak amylase activity and leaf K concentrations, whereas, peak SAI activity showed a negative correlation with leaf K concentrations. The peak SuSy activity had different patterns for the two cultivars. Peak SuSy activity showed a positive correlation with leaf K concentration in Siza 3, but for Simian 3, with the increasing of leaf K concentration, the peak SuSy activity increased rapidly at first, and then levelled off.

3.6. Relationship among Pn, carbohydrate, carbohydrate-metabolizing enzymes in LSCB with boll biomass Relationship between boll biomass and carbohydrate, carbohydrate-metabolizing enzymes in LSCB showed that there were positive correlations between Pn, sucrose transformation

rate, SPS activity in LSCB and boll biomass (P < 0.05), a negative correlation between sucrose content and boll biomass (P < 0.05) in Simian 3 and Siza 3 (Table 5). Moreover, boll biomass also had a significant negative correlation with SAI (P < 0.01) in Siza 3.

4. Discussion Potassium is an essential macronutrient involved in numerous physiological processes (Oosterhuis et al., 2014) determining cotton growth and yield. Deficiency of potassium has been shown to affect carbohydrate metabolism in the leaf (Bednarz and Oosterhuis, 1999) and partitioning to the reproductive components of cotton (Pettigrew et al., 2005; Wang et al., 2012) but the explanation for this is lacking. In our study, through the changes of biomass partitioning (Table 2), K application treatments were characterized by the increased reproductive biomass and lower proportion of leaf/reproductive ratio. The LSCB appeared to retain fewer assimilates and translocate more of them to the reproductive sinks. As a result, less of the non-structural carbohydrate (such as

Table 5 Correlation coefficient among Pn, carbohydrates, carbohydrate metabolizing enzymes with boll biomass for two cultivars Simian 3 and Siza 3 in two years. cultivar Simian 3 Siza 3

Pn

Sucrose *

0.870 0.832*

−0.854 −0.897* *

Sucrose transformation rate *

0.878 0.848*

n = 6, R0.05 = 0.811, R0.01 = 0.917. * Significant difference at 0.05 probability level. ** Significant difference at 0.01 probability level.

Starch −0.267 0.074

Rubisco 0.617 0.516

cy-FBPase 0.554 0.477

SPS *

0.859 0.893*

SuSy

SAI

Amylase

0.435 −0.108

−0.762 −0.925**

0.057 0.632

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Fig. 9. Changes of SAI activity and amylase activity in the LSCB during boll development for three potassium treatments in 2012 and 2013. The data are the means for three replications ± SD.

sucrose, hexose and starch) in LSCB contributed to the decreased SLW (Table 4). More capsule wall, seed and lint biomass were found in K application treatments compared with the no-K application treatment (Table 3) in agreement with previous studies (Pettigrew and Meredith, 1997; Pettigrew, 2003). K application changed the distribution of biomass in the boll, with K treatments being characterized by a markedly higher proportion of lint biomass and lower proportion of seed biomass. Earlier studies showed that 60–87% of boll biomass was derived from the LSCB (Constable and Rawson, 1980; Wullschleger and Oosterhuis, 1990). In the present study, boll biomass had a significant correlation with Pn, sucrose content, sucrose transformation rate and SPS activity in LSCB (Table 5), indicating that photosynthetic carbohydrate metabolism in LSCB provides a good explanation of this relationship. According to previous studies, K deficiency induced accumulation of soluble sugars and starch in soybean leaves (Huber, 1984) and mulberry leaves (Yamashita and Hikasa, 1988), and our study supported these results. The average content of starch, sucrose, and hexose in LSCB was decreased by 9–18%, 6–15% and 5–17%, respectively, during boll development in K application treatments (Fig. 3). In contrast, large amounts of starch were accumulated in LSCB of no-K treatments in late stages of boll development (particularly at 31–45 DPA) (Fig. 3). This might be an indication that K deficiency affected the components of the non-structural carbohydrate (Zhao et al., 2001), as the trend of sucrose: starch in Table 4 confirmed this result. Sufficient non-structural carbohydrate (sucrose, hexose and starch) were stored and the boll biomass was still lower in the no-K treatments, which indicated that transferred sucrose from the LSCB to the cotton boll was restricted, which was confirmed

by the reduced sucrose transformation rate in the no-K treatment (Table 4). Therefore, the sucrose that could not be transferred in time was more likely to be accumulated and convert to other sugars. These findings might help explain the K affected non-structural carbohydrate distribution, the change of SLW and boll weight. Genotypic variation in sensitivity to K deficiency has been reported (Wang et al., 2012). In our study, the influence of K application on Pn, hexose content, sucrose content, starch content and sucrose transformation rate in LSCB was different between the two cultivars Simian 3 and Siza 3 (Figs. 2 and 3 and Table 4). The leaf critical K concentration depends upon the various physiological parameters (Reddy and Zhao, 2005). Our results showed that the leaf critical K concentrations during boll development were 1.2%, 1.1%, 1.3% and 1.2–1.4% for Pn, hexose content, sucrose content and starch content in Simian 3, respectively (Fig. 5), and 1.4%, 1.6%, 1.7% and 1.7–1.8% for Siza 3. These results showed that Siza 3 was more sensitive to low-K than Simian 3. Initial Rubisco activity and cy-FBPase activity have been shown to increase with K treatment in some crops (Peoples and Koch, 1979; Bednarz et al., 1998; Chen et al., 1998). In our study, the trend of initial Rubisco activity (Fig. 5) and cy-FBPase activity (Fig. 6) confirmed the previous reported results. SPS and SuSy activities in K treatments (150 and 300 kg K2 O ha−1 ) were up-regulated by 8–36% and 20–45%, respectively (Fig. 8), in agreement with earlier research regarding the response of SPS and SuSy activities to K application in soybean and maize (Huber, 1984; Cao and Zhao, 2009; Qu et al., 2011). The results showed that the enzyme activities (initial Rubisco, cy-FBPase, SPS and SuSy) related to sucrose synthesis were increased in cotton with K application. Amylase enzyme

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Fig. 10. Relationship between the peak values of SPS, SuSy, SAI or Amylase activities with leaf K concentration in the LSCB in 2012 and 2013. The solid and dotted lines represent Simian 3 and Siza 3, respectively.

was up-regulated and SAI was down-regulated in the K application treatments (Fig. 9), indicating that starch degradation was able to quickly provide more carbon for the synthesis of sucrose (Servaites et al., 1989) and sucrose decomposition was reduced in K-treated plants (Lavon et al., 1995). Therefore, the accumulation of sucrose should occur more in the leaves of K application treatments, however, our results did not support the theoretical prediction because sucrose content was lower in K treatments than that in no-K treatment (Fig. 3). Similar results have been reported showing that K affected photosynthate transport from the source to sink organs in bean (Cakmak et al., 1994) and soybean (Huber, 1984), and our results showing the trend of sucrose transformation rate supported this conclusion (Table 4). In our study, SPS and SuSy activities in LSCB of the two cultivars had different sensitivities to K. The average SPS activity in the K treatments (150 and/or 300 kg K2 O ha−1 ) increased by 8–28% compared to the control for Simian 3, and by 18–36% for Siza 3. The average SuSy activity increased by 20–29% for Simian 3 and by 24–45% for Siza 3. The peak SPS activity (Fig. 10) could be represented by a straight line equation with leaf K concentration. The slope for SPS activity in Siza 3 was larger than that of Simian 3, indicating that SPS activity is more susceptible to K in Siza 3. The peak SuSy activity increased rapidly with increased leaf K concentrations in Siza 3, but the peak SuSy activity of Simian 3 increased rapidly at first, and then increased only slightly. Therefore, SPS and SuSy were more sensitive to K in Siza 3, which is probably the main reason for the different sensitivity to low-K in the two cultivars. 5. Conclusions Based on the experiments in 2012 and 2013, our results clearly demonstrated that: (1) K application increased plant biomass and

enhanced boll biomass (capsule wall, seed and lint biomass) production through increased Pn, sucrose transformation rate and SPS activity. K application increased the proportion of lint biomass, decreased the proportion of seed biomass, but did not affect the proportion of capsule wall biomass. (2) Nonstructural carbohydrate (hexose, sucrose, starch) metabolism in LSCB was significantly affected by K application in both cultivars. Siza 3 required a high leaf critical K concentration to break down starch, transport sucrose and reduce hexose in LSCB compared to Simian 3. The difference may explain the reason why Siza 3 was more sensitive to low K than Simian 3. (3) K application affected the activities of nonstructural carbohydrate-metabolizing enzymes in LSCB during boll development. Enzymes activities (Initial Rubisco, cy-FBPase, SPS, SuSy) related to sucrose synthesis were increased in plants applied with K. In addition, the amylase enzyme was up-regulated and SAI activities were down-regulated, while the sucrose content was lower in LSCB because the sucrose transformation rate significantly increased, which was beneficial for boll biomass accumulation. (4) SPS and SuSy activities in LSCB were significantly different between Simian 3 and Siza 3, which is probably the main reason for the different sensitivity to low-K. These results have aided our understanding of the effects of K fertilizer on nonstructural carbohydrate metabolism in LSCB during boll development, and will promote the development of new cotton cultivars with stronger tolerance to the low K stress. Acknowledgments We are grateful for financial support from the National Natural Science Foundation of China (30971735, 31401327), China Agriculture Research System (CARS-18-20), Jiangsu Collaborative Innovation Center for Modern Crop Production (JCIC-MCP) and

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