Soil Potassium Deficiency Reduces Cotton Fiber Strength by Accelerating and Shortening Fiber Development

Low potassium (K)-induced premature senescence in cotton has been observed worldwide, but how it affects cotton fiber properties remain unclear. We hypothesized that K deficiency affects cotton fiber properties by causing disordered fiber development, which may in turn be caused by the induction of a carbohydrate acquisition difficulty. To investigate this issue, we employed a low-K-sensitive cotton cultivar Siza 3 and a low-K-tolerant cultivar Simian 3 and planted them in three regions of different K supply. Data concerning lint yield, Pn and main fiber properties were collected from three years of testing. Soil K deficiency significantly accelerated fiber cellulose accumulation and dehydration processes, which, together with previous findings, suggests that the low-K induced carbohydrate acquisition difficulty could cause disordered fiber development by stimulating the expression of functional proteins such as CDKA (cyclin-dependent kinase). As a result, fiber strength and lint weight were reduced by up to 7.8% and 2.1%, respectively. Additional quantitative analysis revealed that the degree of accelerated fiber development negatively correlated with fiber strength. According to the results of this study, it is feasible to address the effects of soil K deficiency on fiber properties using existing cultivation strategies to prevent premature senescence of cotton plants.

. Responses of cotton lint yield to K application rates (A), and evaluation of cotton plant K + status by modeling the dynamic change of cotton plant critical K + concentration (B). Data (A,B) were collected in the preliminary experiment (2011), in which cotton was planted at two sites: Dafeng and Nanjing in Jiangsu Province, China. The agrotype of the Dafeng test site is coastal saline soil, with a soil soluble K + concentration of 161 mg kg −1 before planting in 2011. Details of the Nanjing test site from 2011 to 2013 were described in the Methods. The legend in (B) represents the K application rate (kg K 2 O ha −1 ). Here, we showed only the data for cv. Siza 3 as a representative.
After excluding fiber micronaire because of its minor response to K deficiency, we plotted detailed data point (five replicates) of fiber length (Fig. 4A) and fiber strength (Fig. 4B). The middle (8 th ) and higher (13 th ) FBs tended to yield longer and stronger cotton fibers that were more sensitive to impairment when suffering from severe K deficiency (K0). Both FB 8 and FB 13 were sufficiently sensitive to indicate the influence of K deficiency on fiber development. However, FB 8 had the advantages of being more representative and stable. Therefore, we used FB 8 to perform a detailed study of the response of developing fibers to K deficiency.  The changes in fiber dry weight (Fig. 5A), fiber length (Fig. 5B) and fiber strength ( Fig. 5C) with days post anthesis (DPA) were fitted using equations (1), (2) and (3) in Methods, respectively. As seen in Fig. 5, the difference in fiber dry weight and strength were likely due to the earlier termination of growth. The patterns of K0 occurred ahead of those of K300 for fiber dry weight and fiber strength, but not for fiber length. A comparable phenomenon was observed in the BMP (cotton boll maturation period) on FB 8 Table 2. Changes in main cotton fiber properties (length, strength and micronaire) of the FB 8 when grown in three K application rates. Data followed by different letters (a, b, c) in one group indicate statistically significant differences at the p < 0.05 level based on ANOVA. The fiber properties above were measured from the FB 8 . Each number was the mean value of 5 samples.
However, the reduction of fiber length appeared to occur as a result of osmoregulation rather than developmental acceleration 12 .

Quantification of Cotton Fiber Physiological Age and its Relationship with Fiber Strength.
The changes in fiber cellulose content (Fig. 6A) and water content (Fig. 6B) during fiber development were fitted with the sigmoid equation (4) and the exponential equation (5) in Methods, respectively. There were small differences in the effects of different levels of K supply on the final values of both fiber cellulose and water content. Therefore, the difference in K treatments during fiber development could be used to indicate the delay or acceleration caused by K treatments at that time. For the current study, we defined K0 and K150 to have the same physiological age as K300 when they shared the same Y value (see the horizontal line "a" in Fig. 6A,B). Based on this definition, K0 and K150 could reach the same physiological age as K300 did at an earlier DPA. Correspondingly, at time point "t", the physiological ages of K0 and K150 were, respectively, "t 1 " and "t 2 " (see the vertical line "b" in Fig. 6). Thereafter, the vertical line "b" was moved gradually from 15 DPA to 55 DPA, and the dynamic changes in physiological age gaps, "t 1 -t" and "t 2 -t" (Fig. 6A,B), were drawn in Fig. 7. The Y value in Fig. 7 represents the physiological age, and "y 1 " and "y 2 " are the advanced physiological ages of K0 and K150 compared with K300. For example, the physiological age of K300 at "x 0 " was "x 0 ", but for K0 and K150, they were "x 0 + y 1 " and "x 0 + y 2 ", respectively. These gaps increased with DPA when they were computed based on fiber cellulose accumulation (Fig. 7A), but    . Line segments "y 1 " and "y 2 " demonstrate the fiber physiological age gaps from K0 to K300 and from K150 to K300, respectively at the time point "x 0 ". The results were calculated based on the fibers grown on the FB 8 .
Scientific RepoRts | 6:28856 | DOI: 10.1038/srep28856 The advanced physiological age compared with K300 at 39/38 DPA (2012/2013) had a negative impact on fiber strength; this impact was greater on cv. Siza 3 than on cv. Simian 3 (see the slopes of the regression lines in Fig. 8). Compared with K0, K150 had less impact on fiber physiological age. Calculations based on different physiological indices (e.g., fiber cellulose accumulation and fiber dehydration) revealed the same result. Interestingly, from Figs 7 and 8, we observed that the physiological age gap between K treatments could reach 10-15 days. However, the actual difference on BMP reached only 4-5 days (mentioned above). A possible explanation for this result is that the fiber physiological age gap is completely different from BMP. BMP has a direct relationship with capsule wall maturity, but not with fiber maturity. For example, when a cotton boll opens, its BMP is fixed, whereas the fiber properties continue to change 24 . This relationship helps establish a connection between the low-K-induced acceleration of fiber development and the corresponding reduction of fiber strength.

Discussion
There are two types of K-deficient disorders relevant to cotton. In type I, K deficiency syndrome first affects older leaves, which is called the classical K deficiency. This disorder usually occurs on soil with strongly fixed K 1 or inherently K-deficient soil 25 . Type II is called K deficiency-induced premature senescence, in which K-deficient signs appear on younger rather than older leaves 26 , and which predominantly occurs on soil with a relatively high levels of available K 2 . Compared with type I, the type II disorder is caused by an intrinsic imbalance between K + supply and K + demand, which commonly occurs in conditions of relatively high boll load 2,26 . For the current study, we suspect type II K deficiency because of the large number of specks found on younger leaves (Fig. 9) and the greater reduction of fiber strength among late-season bolls (Fig. 4). However, there was an apparent difference between previous studies and ours in that boll loads of K-deficient plants in our experiment were not larger than those of the controls. Specifically, the boll loads of severely K-deficient plants in our study (K0) were 8.  Table 1). Therefore, reasons other than boll load could have caused the premature senescence observed in this study. Potential causes could include environmental factors, endogenous hormones, disease, mineral element imbalances or soil construction 22 .
A number of studies have indicated that severe K deficiency could significantly reduce cotton leaf photosynthetic capacity 27,28 and reduce phloem loading speeds 29,30 . As 60-87% of the total photoassimilate for cotton boll development comes from the subtending leaves 20,31 , a strong connection between carbohydrate starvation 32,33 and accelerated fiber development exists. In fact, previous studies have already shown that plant carbohydrate supply barriers could induce premature development of many organs 34 . Based on our results, we determined that the carbohydrate supply barrier could also lead to acceleration of fiber development (Figs 5 and 6).
The reduction of fiber strength in our research (Fig. 8) is potentially caused by accelerated fiber development (Fig. 5B), which was presumably induced by the carbohydrate acquisition difficulty 33,34 . Studies have indicated that CDKA (cyclin-dependent kinase, also known as kinase p34 cdc2 ) could regulate plant cell cycles 34,35 and that difficulty obtaining sufficient carbohydrates for plant development could stimulate the generation of CDKA 34 . Previous research has reported a positive correlation between CDKA activity and the cell growth rate in Arabidopsis roots 35 as a result of comparing 18 ecotypes. Similar results have also been found among maize leaf cells suffering from water stress 34 . Therefore, CDKA was a potential factor affecting cotton fiber cells and was activated by the low-K-induced carbohydrate acquisition difficulty, which reduced fiber strength by accelerating fiber development.
Carbohydrate acquisition difficulty could also be a cause of reductions in cotton lint yield. A recent study of cotton demonstrated a positive correlation between boll weight and sucrose transport capacity from leaf to boll 20 . As a result, the carbohydrate acquisition difficulty induced by K deficiency [36][37][38] could lead to cotton yield reductions of as much as 20% 4 . An interesting phenomenon observed in the current study was that the effect of different K conditions on fiber dry weight accumulation (Fig. 5A) was very similar to the effect on fiber strength formation (Fig. 5C), in that the K0 conditions resulted in earlier changes than the other conditions. We speculate that K deficiency might inhibit cotton lint yield in terms of fiber weight per ovule (Fig. 5A) through a pathway similar to the one affecting fiber strength. This point is worthy of further investigation.
Two hypotheses have been proposed to explain why some cotton varieties are more prone to premature senescence: (1) relatively higher boll loads and higher yield potential require amounts of K and P that their root systems cannot provide 26,39 ; and (2) the lower net P n and carbon assimilation, along with higher nitrogen assimilation and reactive oxygen species (ROS) accumulation in the leaves, make them more sensitive to K deficiency 40 .
The results from the current study (Fig. 8) showed that cv. Siza 3 exhibited a greater reduction of fiber strength than cv. Simian 3, despite similarly advanced physiological age (K0). According to our investigations of boll load (Table 1) and leaf Pn (Fig. 3), the latter hypothesis seems more appropriate, which means that metabolic rather than supply features are more strongly related to the low-K-induced acceleration of fiber development. Previous studies showed that some cotton cultivars could achieve the same biomass as others under K-sufficient conditions, but not under K-deficient conditions 40,41 . Data from our research similarly found that the low-K-sensitive cv. Siza 3 had consistently greater CV of lint yield than the low-K-tolerant cv. Simian 3 (Table 1). These results suggest that the low-K-sensitive cultivars are prone to show sensibility only under K-deficient conditions. Up to this point, a series of field management strategies has been established to address the premature senescence caused by K deficiency. These strategies have included features such as reducing the occurrence of waterlogging by employing more appropriate irrigation and field layout schemes 39 , minimizing soil compaction by conducting tillage operations 35 , and implementing a late planting production system (LPPS) 42 . The significance of our current study is that we have established a physiological connection between the low-K-induced carbohydrate acquisition difficulty and fiber strength and have made it easy and feasible to cope with the inhibition on fiber development using strategies already employed to defend against cotton plant senescence.

Test Site Description, Experimental Design and Crop Management. The low-K-tolerant cotton
cultivar Simian 3 and low-K-sensitive cotton cultivar Siza 3 were obtained by comparing 12 ecotypes (Gossypium hirsutum L.) 43 for study. Cluster analysis was used to classify these ecotypes by considering boll number, lint weight per boll, fiber length and fiber strength. Afterwards, cv. Simian 3 and cv. Siza 3 were planted in a purpose-built test field (yellow-brown loam) at the Pailou test site (Nanjing Agricultural University, Jiangsu, China) for three years (2011, 2012 and 2013), with the constant density of 33,600 plants ha −1 . The 2011 planting was a preliminary experiment, in which the soil soluble K concentration (0-40 cm) before planting was 115 mg kg −1 , not consistent with severe K deficiency. Accordingly, limited data of cotton fiber properties in 2011 was obtained. However, the plantings in 2012 and 2013 were successfully prepared with regular K depletion before each year's planting. One season of wheat and one season of peas were planted for K depletion. The soil soluble K concentrations (0-40 cm) before planting in 2012 and 2013 were 92 and 86 mg kg −1 , respectively. There were three individual fields for each year's experiment, which eliminated fertilizer residual effects.
K treatments of 0, 150 and 300 kg K 2 O ha −1 (K0, K150 and K300) were arranged in a randomized block design with 3 field replicates. The K0 treatment combined with K-deficient soil created severely K-deficient conditions for cotton growth, whereas the 150 and 300 kg K 2 O ha −1 conditions induced mildly K-deficient and K-sufficient conditions, respectively 36,44 . Each test plot was 6.6 × 13 meters with 16 rows. K fertilizer (potassium sulfate, in which K 2 O content is 50%) was applied to holes at the start of cotton flowering. N fertilization (240 kg N ha −1 ), P fertilization (120 kg P 2 O 5 ha −1 ), irrigation and pest control were performed in accordance with recommended practices. No symptoms indicating inappropriate N supply, water stress or pest pressure negatively influenced cotton growth. For the reason mentioned above, the 2011 dataset was a preliminary experiment, did not include dynamic soil soluble K + data. Soil soluble K + was determined following the method described in Yang et al. 12  , after which the K + concentrations were measured using the same procedure as soil soluble K + concentration measurements 12 .

Soil
Cotton boll age was determined by tagging the fully opened white flower (0 DPA). The first fruit-bearing sympodial branch was defined as the 1 st FB. Generally, according to each plant's growth habits and the climate in Nanjing, China, the two cultivars would have 14 to 16 FBs before artificial topping. Therefore, the 3 rd , 8 th and 13 th FBs were tagged as representatives of the lower, middle and upper FBs, respectively, of the cotton plant. For mature fiber sampling, cotton fibers grown on each FB were picked one week after boll opening; for immature sampling, 15, 17, 20, 24, 31, 38 and 45 DPA samples of bolls grown on the FB 8 were collected.
Mature fiber properties, including fiber length, strength and micronaire, were determined using the cotton fiber quality measurement system USTER HVI MF100 (Uster Technologies, Switzerland). The measurement was performed in a standard testing room with constant temperature (20 ± 2 °C) and humidity (65 ± 2%). Fiber samples were placed in this room for 48 h before measurement.
The length measurement of immature fibers was based on Yang et al. 12 . Prior to the strength measurement of immature fibers, they were preprocessed in an oven dryer at 60 °C for 0.5 h and then at 40 °C for 2 days; they were then placed into a standard testing room with constant temperature (20 ± 2 °C) and humidity (65 ± 2%) for 48 h. A Functional Fiber-bundle Tensile Tester (KX-154, Shanghai Kangxin Photoelectric Instrument Co., Ltd., China) was used for the strength measurement of immature fibers.
Fresh fiber water content was calculated from the difference in weight before and after the drying procedure.
Fiber Cellulose Concentration Analyses. Fibers recycled from strength measurements were digested using acetic-nitric acid. The determination of cellulose concentration was based on the anthrone colorimetry method 46 .