K+ is one of the essential macronutrients for plant growth and development. Additionally, K plays an important role in the biophysical and biochemical cellular functions involved in osmoregulation, photosynthesis, electrical neutralization, transport of metabolites, and enzyme activation in living plant cells   . However, compared with nitrogen (N) and phosphorus (P), K is applied at a lower level  , only meeting 20% - 33% of growth and developmental needs in maize  . Since the late 1990s, there has a negative trend of K balance about 60 kg ha−1year−1 in intensive agricultural production condition due to improper fertilizing in China  . Currently, more than 2/3 large agricultural areas of China were reported to be deficient in K availability (less than 100 mg/kg) or severe deficiency (less than 50 mg/kg), which severely restricts the growth and production of crops  . Therefore, it is of great significance to excavate potassium efficient genotype crops and alleviate the status of effective potassium in soil by mobilizing its own biological potential and improving the utilization of soil potassium.
Maize is the largest grain crop in China and high demand for potash, especially in the northeast area. Root system is the main organ for nutrient uptake. And ideal root morphology and activity were great significance to nutrient absorption from soil, which play an important role in the growth and development for crops  . The morphology and structure of root system were adjusted for absorption of surrounding nutrients under nutrient stress   . For example, K-efficient genotypes in rice often have larger root system than K-inefficient genotypes, which showed well-developed root, stronger absorption and large interface between root and soil   . Higher root activity could permit crops to acquire largely nutrient from soil, especially under abiotic stress. The root in different diameters was great differences in capacity for nutrient uptake, stronger in fine roots than coarse roots  . Various nutrient elements in plants were interrelationship with potassium, such as N, P, Na and Ca, etc.  . Studies have shown that different genotypes rice accumulation of potassium and dry matter was depressed under low K stress, but significantly increased the potassium use efficiency and Na+ accumulation, eventually increased the ratio of Na+ to K+  . Meanwhile, Ca2+ could promote the absorption of K+ by regulating the potassium channel protein AKT1    . It is well-known that seedling stage is a sensitive period for maize to be deficient in potassium. The objectives of this study were to investigate the effects of K deficiency on root configuration and nutrient absorption at seedling stage and to explore the different response to K deficiency in 90-21-3 and D937 on root morphology, root activity and nutrient absorption, in order to provide theoretical basis and further clarify the physiological mechanism to K deficiency.
2. Materials and Methods
Plant material and experimental design
Maize seeds (Zea mays. L.) of 90-21-3 (Tolerance to potassium deficiency) and D937 (Sensitive to potassium deficiency) were screened out by field, pool and hydroponics experiment for years in low potassium soil of Liaozhong county, China  . The genotype 90-21-3 showed no symptoms of K deficiency, but leaf margin chlorosis was significantly performed in D937 at the seedling stage (Figure 1).
The study was performed using the hydroponic planting method in Shenyang Agricultural University, Liaoning province, China. Full and uniform seeds were disinfected in 7% NaClO3 solution and washed for 3 times with deionized water before sowing. The seeds were primarily germinated in silica sand on July 12, 2016 and carefully transplanted into an opaque plastic box (50 cm × 35 cm × 15 cm) containing 22 L nutrient solution until development of two visible leaves (July, 18) without endosperm, in a rainproof greenhouse under a natural environment. The nutrient solution was 1/2 Hoagland’s nutrient solution and Arnon microelements modified from Cao et al. (2007)  , and containing: 2000 μmol Ca(NO3)2, 2500 μmol KCl, 1000 μmol MgSO4, 500 μmol NH4H2PO4, 100 μmol EDTA-Fe, 115 μmol H3BO3, 22.5 μmol MnCl2, 0.16 μmol CuSO4, 0.75 μmol ZnSO4, and 0.182 μmol (NH4)6Mo7O24. The pH of the nutrient solution was adjusted to 6.0 by addition of 0.1 mol L−1 NaOH or HCl and renewed once every seven days. Air was pumped into the culture solution to provide oxygen for roots, at a rate of 40 min h−1. For K starvation, 7-day-old plants were transferred for 1, 3, 5, or 7 days to a K-deficient nutrient solution. To maintain the stability of K+ supply during plant growth, the K+ concentration in culture solution was monitored daily and replenished to 2.5 mmol L−1.
Four uniformed seedlings each treatment were harvested separately at each time point as described above and washed with deionized water. And roots and shoots were detached from the upper crown root, and baked at 105˚C for 15 min and then dried at 70˚C to constant weight. The root to shoot ratio (R/S) was based on ratio of root dry weight to shoot dry weight.
Root activity determination
Root activity measurement was performed according to Luo et al. (2016) with
Figure 1. Comparison of leaf blade between 90-21-3 and D937 under K deficiency at seedling stage.
triphenyl tetrazolium chloride (TTC) method  . Approximately 0.3 g roots were placed in tubes filled with 5 mL of 0.4% TTC and 5 mL of phosphate buffer (1/15 mol/L, pH 7.0), keep at 37˚C for 1 h, then add 2 mL sulfuric acid (1 mol/L) into the tubes to terminate reaction. The red triphenyl formazan (TTF) was extracted by ethyl acetate, and the absorbance was measured at 485 nm with an UV-spectrophotometer (Hitachi UV-1800, Japan). The activity was calculated by the standard curve prepared with known TTF concentrations.
Four uniformed seedlings each treatment were scanned with an EPSON Transparency unit and then analyzed with WinRhizo Program (Regent Instruments Inc. Canada). The root sample was placed in a glass rectangle sink (300 mm × 200 mm × 20 mm) with about 8 - 10 mm deep layer of water to extend fully and minimize root overlap for capturing image. Total root length, total surface area, total volume and average diameter were acquired by analysis of root image. Fine roots are commonly considered be stronger capacity to nutrient and/or water than coarse roots. Referred to the results of previous studies on maize seedling  and based on average diameter of the experiment, we classified total roots into 3 groups: fine roots with average diameter less than 0.4 mm, middle roots with average diameter between 0.4 mm and 0.8 mm, and coarse roots with average diameter larger than 0.8 mm.
The dry power of root and shoot was digested by H2SO4 at 380˚C for 1.5 h, and then clarified with drops of 30% H2O2. The clarified sulphate acid was cooled to room temperature and dilute with deionized water to 100 ml. N was measured by Kjeldahl method, and P was measured by vanadium molybdate yellow colorimetric method according to Bao (2005)  . K, Na, and Ca, analyses were conducted after digestion by HNO3-H2O2 using a flame photometer (Sherwood M410, UK).
All dates were compared with SPSS18.0 software (SPSS Inc., Chicago, IL, USA). Differences between treatments were considered significant at a 0.05 level of probability according to least significant difference (LSD) tests. The figures were plotted using Origin version 9.0 software. The data are presented as means ± SD.
3.1. Plant Performance
The accumulation of dry matter of 90-21-3 and D937 was reduced after K starvation, and more decrease in shoot than root (Figure 2). Compared with the controls, the total dry weight of 90-21-3 was significantly decreased 12.62%, and 27.83% in D937. The root and shoot weight were in no difference after 1 d and 3 d of K deficiency, but significantly decreased at 5 d and 7 d. The 90-21-3 was significantly decreased by 12.09% and 13.15% in shoot, 5.36% and 7.90% in root
Figure 2. Effect of potassium deficiency on dry weight of different maize genotypes.
Note: *represent significant differences for the same genotype between K deficiency and CK at 5% level. Each figure on the histogram was presented the R/S. Values are expressed as means of 4 replicates and the bars indicate the standard errors, the same as below.
at 5 d and 7 d of K deficiency. The D937 was significantly decreased by 24.68% and 25.59% in root of D937, 28.17% and 26.54% in shoot at 5 d and 7 d of K deficiency. But, the R/S of both was tendency to increase with time of K deficiency. The ratio was significantly increased by 20.07% and 23.23% after 5 d and 7 d of K deficiency, while no difference in D937.
3.2. Root Configuration of Maize Seedlings
The total length and total surface area of the root system are closely related to the absorption capacity of the root system  . Compared with the control, the total root length, total surface area, total volume and average diameter of 90-21-3 and D937 were decreased under K deficiency treatments (Table 1). After 5 d of K deficiency, the root length and surface area were decreased by 3.76% and 3.07%, but significantly decreased by 19.90% and 13.41% in average diameter and total volume. The total length, total surface area and total volume of D937 were significantly reduced by 10.53%, 11.25% and 14.06%, respectively. Compared with the controls, the 90-21-3 was significantly decreased by 9.36%, 6.96%, 25.22% and 14.05% in root length, surface area, average diameter and root volume at 7 d of K deficiency, respectively, and 19.21%, 17.23%, 11.69% significantly decreased in root length, surface area and root volume of D937 although no difference in average diameter.
3.3. Growth of Seedling Roots with Different Diameters
3.3.1. Root Length
The root in different diameters was great differences in capacity for nutrient uptake, stronger in fine roots than coarse roots  . The fine root length was the largest proportion in the total root length, but decreased in fine root, middle
Table 1. Varieties of K deficiency on root parameters of different maize genotypes.
Note: Values are expressed as means ± SE with 4 replicates. Significant differences (p < 0.05) between days and treatments are indicated by different letters.
Figure 3. Varieties of K deficiency on root length among genotypes with different root diameter ranges.
Note: *represent significant differences for the same genotype between K deficiency and CK at 5% level.
root and coarse root after treatment of K deficiency (Figure 3). Compared with the control, 90-21-3 was slightly decreased after 7 d of K deficiency, and no difference in middle and coarse root. The fine root and middle root of D937 were significantly decreased by 17.65% and 33.81% after 7 d of K deficiency, but no affect on coarse root.
3.3.2. Root Surface Area
The fine and coarse root surface area of 90-21-3 and D937 were main part in total root surface area (Figure 4). The 90-21-3 was significantly increased 8.96% after 3 d of K deficiency, but significantly decreased of 9.05% in D937. After 7 d of K deficiency, the surface area of D937 was decreased 24.15%, 31.17% and 13.33% in the three types root, respectively, and little affect on 90-21-3.
Figure 4. Varieties of K deficiency on root surface area among genotypes with different root diameter ranges.
Figure 5. Varieties of potassium deficiency on root volume among genotypes with different root diameter ranges.
3.3.3. Root Volume
As shown in Figure 5, the coarse root volume of 90-21-3 and D937 was larger ratio in the total volume of the root than fine root and middle root. The D937 were significantly decreased by 28.99% and 28.52% in fine and middle root, respectively, and little decreased in 90-21-3 until 7 d of K deficiency. And the coarse root of 90-21-3 and D937 were significantly decreased by 14.29% and 11.53% in comparison to the controls.
3.4. Root Activity
The dynamic changes of root system were shown in Figure 6. The activity of 90-21-3 and D937 is relatively stable in the controls, but significantly varied with time of K deficiency treatments. Compared with the controls, the activity of 90-21-3 was significantly enhanced by 19.09%, 18.21%, 46.04% and 67.30% after 1 d, 3 d, 5 d, or 7 d of K deficiency. Differed from 90-21-3 under K deficiency treatment, the activity of D937 were significantly decreased by 22.17% and 35.24% at 1 d and 3 d, and then significantly increased by 28.69% and 29.24% at 5 d and 7 d in comparison to the controls.
Figure 6. Varieties of potassium deficiency on root activity of different maize genotypes.
Figure 7. Effect of potassium deficiency on N content of different maize genotypes.
3.5. Nutrient Uptake of Maize Seedling
N content As shown in Figure 7, the N content in the root of 90-21-3 and D937 roots was relatively stable with time of K deficiency, but significantly increased in shoots. After 5 d of K deficiency, the N in shoot of 90-21-3 was largely accumulated in 90-21-3. Compared with controls, more than 30.87% and 25.60% N were accumulated in 90-21-3 and D937 after 7 d of K deficiency.
P content As shown in Figure 8, the P in root of 90-21-3 and D937 were significantly affected by K deficiency, and no difference in shoots. The P in roots of 90-21-3 was largely accumulated, 27.44% and 30.48% after 1 d and 3 d of K deficiency, inversely, significantly decreased of 17.99% and 16.62% at 5 d and 7 d in comparison to the control. The D937 were slightly increased at 1 d and 3 d of K deficiency, but significantly reduced by 34.39% and14.61% after 5 d and 7 d of K deficiency, respectively.
K content Compared with the control, the K-deficient treatment significantly reduced the content of K+ in the shoots and roots of the two inbred lines, and accumulated higher K in shoot than root (Figure 9). Under K deficiency, the K+ of 90-21-3 was significantly decreased by 12.37%, 38.34%, 48.34% and 48.25% in shoots, and reduction of 32.52%, 68.42%, 55.65%, 41.25% and 44.76% in roots. The D937 were significantly reduced by 15.60%, 40.32%, 51.06% and 52.64% in
Figure 8. Effect of potassium deficiency on P content of different maize genotypes.
Figure 9. Effect of potassium deficiency on K content of different maize genotypes.
shoots, and reduction of 44.76%, 54.19%, 54.41% and 24.39% in roots.
Na content As shown in Figure 10, Na+ was more accumulated in roots than shoot both treatments and controls. Compared with the controls, Na in shoots of 90-21-3 and D937 were in no difference, but significantly accumulated in root. Distinctly, Na+ in root of 90-21-3 significantly increased by 67.22%, 181.58% and 19.33% after 1 d, 3 d and 5 d of K deficiency in comparison to the controls. Compared with the controls, Na+ in D937 were significantly increased by 105.60% and 55.64 at 3 d and 5 d of K deficiency, but slightly decreased at 1 d.
Ca content As shown in Figure 11, the Ca2+ content of root were in no difference, and promoted in shoots under K deficiency treatments. Compared with the controls, the Ca2+ content in shoots of 90-21-3 was significantly increased by 18.92% and 48.28% after 5 d and 7 d of K deficiency, respectively, and little
Figure 10. Effect of potassium deficiency on Na content of different maize genotypes.
Figure 11. Effect of potassium deficiency on Ca content of different maize genotypes.
changed in shoot of D937 although little increase.
Potassium is a necessary nutrient element for crop growth and development. However, more than 90% - 98% is in the combination state although abundant in soil, useless for root system. Due to the irrational fertilization and compound index, the available potassium in soil is decreasing and more seriously, which resulted in K deficiency area around rhizosphere and inhibiting growth. However, root system has high plasticity by adjusting root configuration and activity to make adaptive changes to promoted ability of absorption for nutrients around the root.
Low potassium stress could reduce the activity of enzyme and nitrogen metabolism related enzymes, and influence the synthesis and accumulation of plant matter    . Hell and Mendel (2010) found that the transport of photosynthesis products from the leaves to the roots was hindered under K deficiency, resulting in a decrease in the accumulation of roots  . In the current study, the growth of two maize were inhibited, and more reduction in shoots than roots. The results were in accordance with study of Jordan-Meille and Pellerin (2008)  . The growth was more depressed with the time of K deficiency, and the R/S ratio was increased. It suggested that the growth was inhibited under K deficiency, but leaves in 90-21-3 could give priority to the distribution of photosynthetic products to the root system, which promoted the growth of the root system to a certain extent  .
Root is the major actively organ that absorption and synthesis, and its state and vitality level play an important role in crop growth and nutrient uptake  . The root configuration could be adjusted to response the stress for the absorption of nutrients. Crops could promote the absorption of K+ through different physiological mechanisms when limiting factor is K+. Epstein et al. (1963) found that the root of barley was promoted by increasing the high affinity potassium transporter under low potassium stress and promoting the transmembrane absorption of K+  . Hafsi et al. (2011) found that the root was able to promote the uptake of potassium by increasing length and surface  . It was found that root length, root surface area, root diameter and root volume of 90-21-3 and D937 were decreased under K deficiency. The results were same as Long et al. (2017) that studied for Fe deficiency  . In the study, largely reduction of total root length and surface in D937 were displayed under K deficiency stress, and hardly in 90-21-3. By analyzing the morphological of different diameters, the growth of fine root in D937 was inhibited by K deficiency, which showed a significant decrease in the length and surface area. The fine root is the main part for nutrient absorption, and easily to produce the high gradient, which could effectively promote the absorption of nutrients from rhizosphere  .
The root activity is commonly regarded as importantly physiological metabolism of root system, indicating absorption ability of root system in soil. Due to higher root activity, crops still could obtain adequate nutrient to maintain development and growth in spite of nutrition deficiency in soil  . In the study, the root activity of 90-21-3 and D937 were promoted and higher activity in 90-21-3 than D937 under K deficiency. It suggested that 90-21-3 could be able to increase root activity and get more nutrients from rhizosphere.
Potassium was interrelationship with various nutrient elements in plants  . N is the main constituent of protein, as well as chlorophyll. The significantly synthetic process that nitrogen assimilation was impeded due to the reduction in the activities of important nitrogen metabolism enzymes by K deficiency, involved nitrate reductase, glutamate dehydrogenase, glutamate synthase, etc.  . However, Høgh-Jensen (2003) observed that little affect to accumulate for N under K deficiency  . But, Wang et al. reported that more accumulated in shoot and root of rice under K deficiency and increased N/K ratio  . In our study, the nitrogen was accumulated in shoots, and no effect on root. It may be the result of the “enrichment effect” caused by the inhibition of growth of shoots. Phosphorus is mainly in phospholipids and nuclear proteins, and composing of the protoplasm and nucleus. Meanwhile, it directly regulated the process of synthesis, transport and decomposition of carbohydrates in plants. In the present study, K deficiency resulted in significantly less P accumulation in root, but no affect on shoots. The results were same as the study of Zhang et al. (2015)  .
The concentration of K+ in cells was decreased by potassium deficiency, but other cations could be transferred into cytoplasm to promote osmotic potential   . The presence of Na+ and Ca2+ in cytoplasm is important in alleviating the effects of K deficiency. And the function of Na+ and Ca2+ substituted for K+ in process of osmotic regulation has been reported. In the present study, the K+ in roots and shoots was rapidly decreased. Compared with the controls, the K+ of roots in 90-21-3 was more sensitively decreased than D937, and insensitively in shoots than D937. It suggested that 90-21-3 could promote the transportation of the carbohydrate for growth and development by transferring more K+ from root system to shoot. The decrease of K+ concentration in root cells led to an increase in osmosis potential, resulting in osmotic stress. But, the accumulation of Na+ and Ca2+ could be beneficial to enhance cell osmotic regulation and stabilize cell structure and function in some non-specific functions  . In the present study, K deficiency promoted the accumulation of Na+ in the root of 90-21-3 and D937, and little effect on shoot. The accumulation of Na+ and Ca2+ in the vacuoles could effectively alleviate the symptoms of potassium deficiency, and not altered the activity of potassium specificity enzyme  . Distinctly, 90-21-3 was more sensitive to Na+ than D937 that rapidly accumulated, which compensate for the osmotic stress caused by the decrease of K+ in roots. Meanwhile, Ca2+ in shoots of 90-21-3 was largely accumulated under K deficiency. Ca2+ was able to regulate the CBL4-CIPK6 to activate K+ channel AKT2 to accumulate more K+ to leaves  . Therefore, the increased Ca2+ in shoots of 90-21-3 not only maintains the osmotic potential, but also promoted the transport and accumulation of K+ in shoots from root system.
In conclusion, K deficiency led to inhibit the growth of 90-21-3 and D937, more seriously in shoots than roots. The total root length and surface area, especially the fine root, were significantly reduced under K deficiency. Compared with D937, little reduction in root length and surface area in 90-21-3 was found under K deficiency, and significantly increased in root activity. K+ content in shoots and roots was significantly decreased due to the K deficiency, but accumulated Na+ in roots and Ca2+ in shoots. The 90-21-3 was able to increase the K+ in shoots that promoted the transport of carbohydrates to the root system. At the same time, the more accumulation of Na+ and Ca2+ in the root system not only effectively alleviated the osmotic stress caused by decreased K+, but also promoted the absorption of K+ in root. It may be a response mechanism for short-term K deficiency by maintaining large root system, increasing root activity and adjusting nutrient absorption.
The work was financially supported by the National Natural Science Foundation of China (31771725, 31301259) and the Science and Technology Project for Crop High Yield of China (2013BAD07B03).