Changes in Leaf Structure and Chemical Compositions Investigated by FTIR Are Correlated with Di ﬀ erent Low Potassium Adaptation of Two Cotton Genotypes

: Potassium (K) is an essential macronutrient for plant growth and development. K deﬁciency seriously a ﬀ ects protein and carbohydrate synthesis in the leaves of plants. The present study was carried out with two cotton genotypes with low K tolerance to investigate the di ﬀ erent changes on chemical composition and structure in leaves of K-e ﬃ cient cotton genotypes under low K stress by using Fourier transform infrared spectroscopy (FTIR) technology. The results showed that K deﬁciency decreased the leaf photosynthetic pigments in both genotypes, but signiﬁcant observations were noted in K-e ﬃ cient genotype 103. FTIR spectra and semiquantitative analysis revealed that the cell membrane permeability, cell wall pectin, protein, and polysaccharides of leaves were greatly inﬂuenced by K deﬁciency, and the changes were more signiﬁcant in the leaf of genotype 122, indicating a better adaptation to low K in genotype 103. The results of this study revealed that the di ﬀ erence of low K adaptation of these two cotton genotypes might be related to maintaining cell wall integrity and carbohydrate transport in cells. These di ﬀ erent compositional and structural changes in the leaves of the two cotton genotypes under K-deﬁcient level gain a new physiological mechanism of K e ﬃ ciency in cotton.


Introduction
Potassium (K) is an essential mineral nutrient for normal growth and development of higher plants [1]. It is widely accepted that K plays a vital role in activating the enzyme system, maintaining photosynthesis, and promoting translocation of photosynthate [2]. A high concentration of K + is required for optimal protein synthesis and photosynthesis [3]. Although the earth's crust contains 2.6% K, the majority of K + in soils is either dehydrated or attached to oxygen atoms and is consequently unavailable for plants. Therefore, K deficiency has become a global issue [4,5]. Chemical fertilizer is mandatory to ensure an adequate supply of available K to crops in intensive agriculture. The K fertilizer demand in the world is projected to further increase from 23.8 million tons (Mt) in 2011 to 27.1 Mt in 2015 due to the targeted increase in global agricultural production [6]. According to the data of the Food and Agriculture Organization of the United Nations (FAO), the global K fertilizer demand has reached 68 (Mt) in 2019. China has a shortage of K resources, and K deficiency has severely limited the was applied at a quarter of the strength for the first week then to half of the strength for the second week. The culture solution was aerated for 20 min at an interval of 4 hours and renewed every week. The experiment had a completely randomized design with four treatments (2 K levels × 2 varieties), each treatment consisted of five buckets (five replications), and the three seedlings cultured in the same bucket were defined as one replication. Table 1. Nutrients and their contents in the nutrient solution.

Determination of SPAD Value and Photosynthetic Pigment Contents
Plant samples were collected and processed at the fourth week of K treatments, and then the SPAD (Soil and plant analyzer develotrnent) value was recorded by chlorophyll meter (SPAD-502 Plus, Konica Minolta Sensing, INC., Tokyo, Japan) to qualitatively reflect photosynthetic pigment level in leaves. Leaf photosynthetic pigment contents were measured according to method of Lichtenthaler and Wellburn [25].

Composition and Structure Analysis of Functional Leaves by FTIR
Leaf samples were dried to a constant weight (all the water in leaves was removed until the dry weight of leaves would not decrease with additional drying) at 75 • C and then ground to a fine powder. Then, the powder was mixed uniformly with KBr (1:100 m/m) and pressed into tablets for FTIR spectroscopy analysis. Infrared (IR) spectra in the range of 4000-400 cm −1 were recorded by a VERTEX 70 spectrometer with a resolution of 4 cm −1 and 32 scans per sample. The wavelengths between 1800 and 800 cm −1 , containing characteristic peaks for polysaccharides, amide, and ester, were selected to monitor the chemical information of leaves. All spectra were normalized and baseline was corrected by OPUS management software.

Statistical Analysis
Data analysis was performed and graphs were developed by SPSS software (IBM SPSS Statistics 20) and Origin 8.6 (OriginLab Corporation, Northampton, MA, USA). Significant differences (p < 0.05) between different treatments were determined by ANOVA followed by t-test, and significant differences (p < 0.05) within each group were indicated by different lower case letters (a, b, c, and d).

Plant Growth and Symptoms of Functional Leaves under Different K Treatments
As shown in Figure 1A, functional leaves of the two cotton genotypes were green and healthy under K-normal conditions, while under K-deficient conditions, leaf chlorosis were observed in both cotton genotypes. Moreover, the leaves of K-inefficiency genotype 122 had more severe yellowing symptoms than that of K-efficiency genotype 103. Furthermore, the two cotton genotypes treated Agronomy 2020, 10, 479 4 of 10 with adequate K have similar dry weight of root, stem, and leaf, and although K deficiency impeded dry mass accumulation on different plant parts at a significant level (p < 0.05), the inhibition was more obvious in genotype 122 ( Figure 1B). The results indicated that genotype 122 is more sensitive to K deficiency.
Agronomy 2020, 10, x FOR PEER REVIEW 4 of 10 obvious in genotype 122 ( Figure 1B). The results indicated that genotype 122 is more sensitive to K deficiency. The SPAD values of functional leaves were decreased in both genotypes under K deficiency; however, such effects were more obvious in the 122K1 treatment. Furthermore, there was no significant difference in SPAD values of functional leaves in the two genotypes under normal K supply ( Figure 2A). Similarly, the photosynthetic pigment contents of functional leaves under different K conditions suggested that low K treatment remarkably decreased the contents of chla, chlb, chl(a+b), and carotenoid in functional leaves, especially in leaves of K-inefficient genotype 122 ( Figure 2B). The results of SPAD and photosynthetic pigments are consistent with the pattern of Kdeficient symptoms revealed by Figure 1A.  The SPAD values of functional leaves were decreased in both genotypes under K deficiency; however, such effects were more obvious in the 122K1 treatment. Furthermore, there was no significant difference in SPAD values of functional leaves in the two genotypes under normal K supply ( Figure 2A). Similarly, the photosynthetic pigment contents of functional leaves under different K conditions suggested that low K treatment remarkably decreased the contents of chla, chlb, chl(a+b), and carotenoid in functional leaves, especially in leaves of K-inefficient genotype 122 ( Figure 2B). The results of SPAD and photosynthetic pigments are consistent with the pattern of K-deficient symptoms revealed by Figure 1A.
Agronomy 2020, 10, x FOR PEER REVIEW 4 of 10 obvious in genotype 122 ( Figure 1B). The results indicated that genotype 122 is more sensitive to K deficiency. The SPAD values of functional leaves were decreased in both genotypes under K deficiency; however, such effects were more obvious in the 122K1 treatment. Furthermore, there was no significant difference in SPAD values of functional leaves in the two genotypes under normal K supply ( Figure 2A). Similarly, the photosynthetic pigment contents of functional leaves under different K conditions suggested that low K treatment remarkably decreased the contents of chla, chlb, chl(a+b), and carotenoid in functional leaves, especially in leaves of K-inefficient genotype 122 ( Figure 2B). The results of SPAD and photosynthetic pigments are consistent with the pattern of Kdeficient symptoms revealed by Figure 1A.

FTIR Spectral Analysis of the Functional Leaves in the Two Cotton Genotypes
The absorbances of chemical bands vibration in the functional leaves ranged between 4000-400 cm −1 , and results showed that most of the relative absorbance was decreased by low K treatment ( Figure 3A). However, the significant differences on location and the relative absorbance of vibrations induced by low K were mainly manifested in the region of 1800-800 cm −1 (Figure 3B), indicating differences in the structure and composition of the function of leaves between control and low-K cotton.
1735 cm −1 corresponds to C=O stretching vibration and is mainly related to alkyl-esters in lipid membrane and cell walls pectin. K deficiency caused lower intensity at 1735 cm −1 in spectra of the two genotypes, which indicated that low-K stress destroyed the cell membrane permeability and decreased the pectin content in functional leaves. The peaks at 1650, 1550, and 1244 cm −1 correspond to amide I, amide II, and amide III, respectively, and vibration located at 1436 cm −1 originates from C−N stretching of protein. The weakened intensity of these peaks showed that K starvation destroyed the protein structure and decreased protein content in functional leaves of genotypes 103 and 122. Additionally, K deficiency resulted in the obvious shift from 1652 to 1641 cm −1 , and a missing peak around at 1386 cm −1 was found in the functional leaves of K-inefficient genotype 122. Spectra of low-K leaf had a lower relative absorbance at 1384 cm −1 , which is characteristic of the CH3 stretching vibration of cellulose [26]. The changes in absorption bands located at 1100-1000 cm −1 between FTIR spectra of normal K treatment and low K treatment were more significant in K-efficient genotype 103, suggesting a remarkable decrease of carbohydrates, which might be induced by the weakened photosynthesis in leaves of genotype 103. The results indicated that K deficiency has a more significant effect on protein structure and cellulose in the functional leaves, while it does not decrease carbohydrates in K-inefficient genotype 122.   As shown in Figure 3B, relative absorbance corresponding to characteristic peaks of the normal functional leaves was much higher than that in K-deficient cotton genotype 103 and 122. Table 2 lists the assignment of absorption bands to their major chemical components. The peak located around 1735 cm −1 corresponds to C=O stretching vibration and is mainly related to alkyl-esters in lipid membrane and cell walls pectin. K deficiency caused lower intensity at 1735 cm −1 in spectra of the two genotypes, which indicated that low-K stress destroyed the cell membrane permeability and decreased the pectin content in functional leaves. The peaks at 1650, 1550, and 1244 cm −1 correspond to amide I, amide II, and amide III, respectively, and vibration located at 1436 cm −1 originates from C−N stretching of protein. The weakened intensity of these peaks showed that K starvation destroyed the protein structure and decreased protein content in functional leaves of genotypes 103 and 122. Additionally, K deficiency resulted in the obvious shift from 1652 to 1641 cm −1 , and a missing peak around at 1386 cm −1 was found in the functional leaves of K-inefficient genotype 122. Spectra of low-K leaf had a lower relative absorbance at 1384 cm −1 , which is characteristic of the CH 3 stretching vibration of cellulose [26]. The changes in absorption bands located at 1100-1000 cm −1 between FTIR spectra of normal K treatment and low K treatment were more significant in K-efficient genotype 103, suggesting a remarkable decrease of carbohydrates, which might be induced by the weakened photosynthesis in leaves of genotype 103. The results indicated that K deficiency has a more significant effect on protein structure and cellulose in the functional leaves, while it does not decrease carbohydrates in K-inefficient genotype 122. The result of principal components analysis showed that approximately 70% of total sample variability was incorporated in the first and second principal components (PC1 and PC2), accounting for 71% and 14% of the composition variability, respectively. Plots of PC score can reveal clustering in the data set. The plots of the PC1 versus PC2 scores suggested that, regardless of K-efficient genotypes, the leaf compositions and structure of K-deficient and K-normal cotton were generally resolved from each other by PC1 (71%), while an overlap between the two treatments was observed in PC2 (14%). In addition, the two genotypes under low K condition were resolved from each other by PC2 (14%) ( Figure 4A). In combination with the loading corresponding to PC1 and PC2 ( Figure 4B), differences in PC1 were mainly attributed to changes in proteins, and differences in PC2 were associated with the changes of polysaccharides. The result of principal components analysis showed that approximately 70% of total sample variability was incorporated in the first and second principal components (PC1 and PC2), accounting for 71% and 14% of the composition variability, respectively. Plots of PC score can reveal clustering in the data set. The plots of the PC1 versus PC2 scores suggested that, regardless of K-efficient genotypes, the leaf compositions and structure of K-deficient and K-normal cotton were generally resolved from each other by PC1 (71%), while an overlap between the two treatments was observed in PC2 (14%). In addition, the two genotypes under low K condition were resolved from each other by PC2 (14%) ( Figure 4A). In combination with the loading corresponding to PC1 and PC2 ( Figure  4B), differences in PC1 were mainly attributed to changes in proteins, and differences in PC2 were associated with the changes of polysaccharides.

Semiquantitative Analyses of the Main Absorption Bands in Functional Leaves under Low K
Semiquantitative analysis is a common method in FTIR to eliminate the differences caused by sample quantity and indicate the repeatability of the spectra in one treatment. The semiquantitative analysis also can be calculated to analyze the changes in main absorption bands corresponding to functional group from chemical composition in the functional leaf under low K. The results suggest that the intensity of peaks at 1433, 1407, and 1244 cm −1 and 1076, and 1037 cm −1 attributed to proteins and carbohydrates, respectively were significantly changed by K deprivation in genotype 103 and l22, and the intensity of another peak located at 1101 cm −1 was remarkably increased in genotype 122 under low K treatment (Table 3). In order to further analyze the differences caused by low K treatment between these two genotypes, variations (%) were calculated and the results indicated that

Semiquantitative Analyses of the Main Absorption Bands in Functional Leaves under Low K
Semiquantitative analysis is a common method in FTIR to eliminate the differences caused by sample quantity and indicate the repeatability of the spectra in one treatment. The semiquantitative analysis also can be calculated to analyze the changes in main absorption bands corresponding to functional group from chemical composition in the functional leaf under low K. The results suggest that the intensity of peaks at 1433, 1407, and 1244 cm −1 and 1076, and 1037 cm −1 attributed to proteins and carbohydrates, respectively were significantly changed by K deprivation in genotype 103 and l22, and the intensity of another peak located at 1101 cm −1 was remarkably increased in genotype 122 under low K treatment (Table 3). In order to further analyze the differences caused by low K treatment between these two genotypes, variations (%) were calculated and the results indicated that differences in vibrations attributed to polysaccharides were more significant in K-inefficient cotton genotype 122. × 100%, where A L is relative absorbance of low K condition, and A N is relative absorbance of normal K condition.

Discussion
Potassium is an essential macronutrient that participates in the activation of various enzymes and protein synthesis in plants and promotes transport of soluble substances in the xylem and phloem [27]. The accumulation of amino acids and amides in plants is one of the typical K-deficient symptoms [28]. A K deficiency has been proposed to limit protein synthesis by hindering the conversion process of amino acids to polypeptide [29]. Helal and Mengel [30] found that K supply enhances nitrogen (N) uptake and N incorporation into proteins. Significant changes were also observed in the protein concentration and leaf structure of K-starved cotton [31]. Our FTIR spectra showed the decreased intensities of vibrations in 1650, 1550, 1436, and 1244 cm −1 were associated with protein and its structure ( Table 2), implying that K deficiency stress seriously influenced the protein synthesis and structure of both cotton genotypes. It has been reported that the disordered protein structure might be associated with the secondary structure of proteins from α helix into β fold [32].
In addition, the intensities at 1735 and 1384 cm −1 were also significantly decreased by K deficiency, especially the intensity at 1384 cm −1 in K-inefficient genotype 122 (Table 2). Meanwhile, K deficiency weakened the vibration of the peak at 1384 cm −1 in K-inefficient genotype 122 while it had no obvious effect on the sharpness of this peak in K-efficient genotype 103. The different changes induced by low K fertilization indicated that K deficiency reduced contents of pectin and cellulose in the cell wall [20] of functional leaf of the two cotton genotypes, and the effect on cellulose content was greater in 122. The secondary cell wall mostly composed of cellulose, hemicellulose, and lignin has a special cell organization present between the plasma membrane and primary wall of plants, and the structural integrity of secondary cell wall is closely related to normal cell morphology [33]. The results of the present study suggest that a cell wall of genotype 122 was more seriously damaged than genotype 103 under K deficiency stress, resulting in low K adaptation ability.
A high K + concentration is also required for photosynthesis [34,35] and appropriate K supply can effectively improve plant productivity, while K deficiency leads to a decrease in chlorophyll content and photosynthetic rate [36]. Our results suggested K deficiency significantly decreased contents of four typical photosynthetic pigments of the functional leaf of genotype 103 and 122, especially of genotype 122 ( Figure 2B). Furthermore, the two cotton genotypes showed obvious K-deficiency symptoms of leaf chlorosis under low K conditions, and K-inefficient genotype 122 showed more severe symptoms than K-efficient genotype 103 ( Figure 1A). These results confirmed that K-efficient genotype 103 has the better adaptability to low K than genotype 122, as indicated in our previous study [18]. In this study, the two cotton genotypes significantly differed in carbohydrates synthesis and its transport under low-K stress (Table 3). Compared with normal-K treatment, genotype 122 revealed reduced carbohydrates transport than genotype 103. It is widely accepted that K deficiency can increase sucrose concentration in leaves. The translocation of photosynthates from leaf to root and other organs is mainly mediated by the phloem, and adequate K supply is known to play a crucial role in phloem translocation of assimilates [6]. It has been reported that sugar accumulation in leaves contributed to the replacement of osmotic molecules; however, inhibition of photosynthesis products transportation from leaf to root hindered root growth of plants [37]. In this study, the severe assimilation of polysaccharides in leaf and less carbohydrate transportation from leaf of genotype 122 might be related to its poor adaptation ability to low K.

Conclusions
The results of the present study indicated that K deficiency hampered the synthesis of photosynthetic pigments, and remarkably changed leaf structure and chemical compositions of the two different cotton genotypes. Potassium deficiency inhibited the synthesis of proteins and resulted in the alteration of protein structure. In addition, the FTIR spectra indicated reduced cellulose in the leaf of K-efficient genotype 103, and changes on polysaccharides were more significant in K-inefficient cotton genotype 122. Moreover, these changes induced by low K were more serious in the leaf of genotype 122 than genotype 103, implying that genotype 103 has a better adaptation to low K than genotype 122. Therefore, the different changes of structure and chemical compositions in leaf under low-K condition could be one of the key reasons for the difference of K efficiency between the two cotton genotypes.