Experimental materials and treatments
The cotton cultivar, Siza 3 was planted in the experimental field of Jiangsu Academy of Agricultural Sciences (clay soil with low available K content) in Nanjing (32° 20′ N and 118° 52′ E) from 2012 to 2013. The seeds were sown on 25 April in nutrient bowls. On 5 June, healthy and uniform seedlings were selected for transplanting in the field. There treatments [CK, no crop residue and K fertilizer as control group one; K150, 150 kg·ha−1 K2O, which is the recommended quantity of K fertilizer in the Yangtze River cotton belt (Hu et al. 2015), was applied into the soil in the form of potassium sulfate before transplanting as control group two; and W9000, 9 000 kg·ha−1 wheat straw, which could supply about 150 kg·ha−1 of available K for cotton based on final cotton yield (Sui et al. 2015; Yu et al. 2016), was crushed and incorporated in 0∼10 cm soil before transplanting] were designed for this experiment. A randomized complete blocks design with three replications was used for this experiment. The area of each plot was 7 × 4 m2. The intra- and inter-row spacings were 0.3 m and 1.0 m, respectively. In addition, the placement of each plot was fixed in the 2 years. The nutrients in 0∼20 cm soil before transplanting in each year are showed in the Additional file 1: Table S1. The nutrient (N, P and K) analysis of the crop straw performed using the Kjeldahl method (Nelson and Sommers 1972), the molybdenum blue colorimetric method (Rodriguez et al. 1994), and the flame atomic absorption spectrophotometer method (Hu et al. 2015), respectively, showed that the nutrients provided by 9 000 kg wheat straw were 96 kg N, 39 kg P2O5 and 161 kg K2O in 2012 and 79 kg N, 37 kg P2O5 and 133 kg K2O in 2013. In this study, sufficient N (300 kg·ha−1) and P (150 kg·ha−1 P2O5) were applied to all plots during the growth season (Yu et al. 2016).
Three plants per plot were sampling at 80, 95, 110, 125, 140, 155 days after sowing (DAS) in 2012 and at 80, 95, 110, 125, 140 DAS in 2013 to measure the plant biomass. The plant samples were divided into vegetative organs (root, stem, fruiting branches, petiole and leaves) and reproductive organs including bolls, flowers, and buds. After the samples were oven-dried at 105 °C for 30 min and then at 80 °C to constant weight, the dry weight was recorded.
Sampling and processing
The functional leaf [the third (after tip pruning) or fourth (before tip pruning) from top to bottom of main-stem] was sampled at the peak flowering stage (PFS) on 15 July, 2012 and 18 July, 2013, at the boll-setting stage (BSS) on 15 August, 2012 and 15 August, 2013, and at the boll-opening stage (BOP) on 8 September 2012 and 10 September 2013. The sampled leaves were cleaned with deionized water. Then, the main veins of the leaves were abandoned. Half of the leaves to be used for the measurement of enzyme activities, were quick-frozen using liquid nitrogen before being stored at − 80 °C in an ultralow temperature freezer. The remaining leaves to be used for determining the concentrations of N, P and K and the contents of substance related to C and N metabolism, were dried in an 80 °C oven.
Net photosynthetic rate (P
The Pn of functional leaf was measured between 9:00 and 11:00 using a Li-6400 gas exchange measuring system (Li-COR, Lincoln, NE, USA) in three replications. The leaf chamber condition was set at relative humidity of (65 ± 5)%, leaf temperature of (32 ± 2) °C, CO2 concentration of (380 ± 5) µmol·mol−1, and quantum flux of 1 500 μmol·m−2·s−1.
Leaf C, N, P and K contents
Leaf C content was assayed with the potassium dichromate wet digestion method according to Zhang et al. (2014). A H2SO4-H2O2 solution was used to digest the leaf tissues. Then, the contents of N, P and K in H2SO4-H2O2 solution were analyzed with the Kjeldahl method (Nelson and Sommers 1972), the molybdenum blue colorimetric method (Rodriguez et al. 1994), and the flame atomic absorption spectrophotometer method (Hu et al. 2015).
Carbohydrates and N compounds
Leaf tissues (0.1 g) and 80% (v/v) ethanol (5 mL) were placed into a 10 mL centrifuge tube. Then, the tube was heated for 30 min in an 80 °C water bath before centrifuging for 5 min at 4 000 r·min –1. The solid at the bottom of centrifuge tube was extracted twice more using 5 mL 80% (v/v) ethanol. After each centrifugation, the supernatant was collected. The final volume of the supernatant was fixed at 25 mL by adding 80% (v/v) ethanol. The anthrone colorimetric method was used for the assay of soluble sugar content and the resorcinol chromogenic method was used for the measurement of sucrose content in the final supernatant (Hendrix 1993). The final insoluble residue mentioned above was used for starch measurement. The starch in the residue was degraded to glucose using the perchloric acid decomposition method described previously (Hu et al. 2015). Then, the glucose content was assayed using the anthrone reagent (Morris 1948).
Nitrate content was extracted according to Ruiz and Romero (2002). Millipore-filtered water (10 mL) was used to extract nitrate in the dried leaves (0.2 g); 100 μL extract and 0.2 mL salicylic acid (10%) were added into a tube to wait for 20 min. Then, 4.75 mL NaOH (8%) was pipetted into the tube to wait for 30 min. The nitrate was calculated after measuring the absorbance of the mixture at 410 nm. The extraction of carbohydrates described above were used for assaying the free amino acid content using the ninhydrin method according to Yemm et al. (1955).
The crude enzyme solution was extracted according to Huber and Israel (1982). SPS (E.C. 18.104.22.168) activity was quantified according to the method of Hu et al. (2015); 200 μL crude enzyme solution and 350 μL reaction solution prepared with extraction buffer, fructose-6-P (50 mmol·L –1), MgCl2 (10 mmol·L –1) and UDP-glucose (50 mmol·L –1) were incubated for 30 min at 30 °C before adding 2 mol·L−1 NaOH (100 μL). After cooling, the mixture was heated again at 80 °C with 0.1% (w/v) resorcin (1 mL) which was prepared with 95% (v/v) ethanol and 30% (w/v) HCl (3.5 mL) for 10 min. The sucrose in the reaction mixture was measured at the wavelength of 480 nm. The assay method of SuSy (E.C. 22.214.171.124) activity was same as the method described above for SPS except that fructose 6-P was replaced with D-fructose.
For acid invertase (E.C. 126.96.36.199) activity assay, the crude enzyme solution of 100 μL was incubated at 30 °C with 2.2 mL acetic acid-NaOH (200 mmol·L –1, pH 5.0) and 200 μL sucrose (1 mol·L –1) for 30 min. After adding 1 mL 3,5-dinitro salicylic acid, the mixture was boiled for 5 min. The glucose in the reaction mixture was determined at the wavelength of 540 nm (Hu et al. 2019a). The assay method of alkaline invertase activity was similar to that of acid invertase except that sodium acetate-acetic acid (100 mmol·L –1, pH 7.5) was used to replace acetic acid-NaOH.
The statistical analysis software SPSS (ver. 22.0, IBM, USA) was used for the analysis of variance (ANOVA) and means were compared using the LSD test at P = 0.05. The mapping software Origin (Pro 8.0, OriginLab, USA) was chosen to make the figures. Yang et al. (2011) reported that the accumulation process of reproductive organ biomass can be fitted by a logistic formula as:
where a and b are constants; Bio and Biom are the reproductive organ biomass at DAS, and the theoretical maximal biomass of reproductive organs, respectively. The initiation DAS (DAS1) and termination DAS (DAS2) of rapid accumulation of reproductive organ biomass, maximum accumulation rate of reproductive organ biomass (Vm) and occurrence time of Vm (DASm) were derived from Eq. (1) and estimated according to Eqs. (2)–(5):
The duration of rapid-accumulation period of reproductive organ biomass (T) is equal to DAS2 minus DAS1 in Eq. (6). The average accumulation rate of reproductive organ biomass during T (VT) was calculated according to Eq. (7):