Nitrogen Deficiency-Induced Decrease in Cytokinins Content Promotes Rice Seminal Root Growth by Promoting Root Meristem Cell Proliferation and Cell Elongation.

Rice (Oryza sativa L.) seedlings grown under nitrogen (N) deficiency conditions show a foraging response characterized by increased root length. However, the mechanism underlying this developmental plasticity is still poorly understood. In this study, the mechanism by which N deficiency influences rice seminal root growth was investigated. The results demonstrated that compared with the control (1 mM N) treatment, N deficiency treatments strongly promoted seminal root growth. However, the N deficiency-induced growth was negated by the application of zeatin, which is a type of cytokinin (CK). Moreover, the promotion of rice seminal root growth was correlated with a decrease in CK content, which was due to the N deficiency-mediated inhibition of CK biosynthesis through the down-regulation of CK biosynthesis genes and an enhancement of CK degradation through the up-regulation of CK degradation genes. In addition, the N deficiency-induced decrease in CK content not only enhanced the root meristem cell proliferation rate by increasing the meristem cell number via the down-regulation of OsIAA3 and up-regulation of root-expressed OsPLTs, but also promoted root cell elongation by up-regulating cell elongation-related genes, including root-specific OsXTHs and OsEXPs. Taken together, our data suggest that an N deficiency-induced decrease in CK content promotes the seminal root growth of rice seedlings by promoting root meristem cell proliferation and cell elongation.


Introduction
Rice (Oryza sativa L.) is one of the most important food crops in the world [1]. In the past 50 years, rice yield has steadily increased worldwide, partly owing to an increase in nitrogen (N) application. However, at present, the average recovery efficiency of N fertilizer (the percentage of fertilizer N recovered in aboveground plant biomass at the end of the cropping season) is only 33% at the field level [2]. High N input and low N use efficiency not only increase crop production costs but also cause severe environmental pollution [3,4]. Therefore, decreasing N application is an important goal of sustainable agriculture. However, decreasing N application may lead to N deficiency and affect rice root growth, and the underlying mechanism by which N deficiency affects rice root growth is still poorly understood.
Studies of crop responses to N deficiency have focused on the root [5,6], which is the plant organ that is most important for acquiring soil nutrients [7,8]. The developmental plasticity of root architecture is crucial for the acclimation of crops to unfavorable environments, including those that induce N stress. For example, a steeper and deeper root system more efficiently absorbs N in deep soil layers [9]. Root growth is influenced by several external and internal factors, including N availability and phytohormone homeostasis [10][11][12][13]. In general, a supraoptimal N supply inhibits root growth, and the decrease in root size can lead to decreased N uptake [14][15][16][17]. In contrast, N deficiency promotes root growth, and the increase in root size can improve N uptake ability [9,18]. Similarly, supraoptimal levels of the phytohormone cytokinin (CK) inhibit root growth [19], whereas a mild decrease in CK content promotes root growth [19][20][21]. These findings provide evidence that both N and CK are involved in mediating root growth.
CK regulates root growth in a dose-dependent manner [22]. We previously found that a threshold CK content is required for the rapid growth of rice seminal roots, but that supraoptimal CK levels inhibit growth [19]. Usually, the CK contents in roots cultured with high or moderate concentrations of N are supraoptimal for root growth, and thus a mild decrease in CK content promotes root growth. For example, a mild decrease in CK content achieved through overexpression of the CK degradation gene CYTOKININ OXIDASE DEHYDROGENASE (CKX) or mutation of the CK biosynthesis gene ISOPENTENYLTRANSFERASE (IPT) can promote primary root growth in Arabidopsis grown under moderate concentrations of N [20,21]. In contrast, without N application, the endogenous CK content in rice seedlings is optimal for growth of the seminal roots, and thus either a decrease or an increase in CK content leads to growth inhibition of the seminal root [19]. In addition, it has been reported that N treatment can increase CK content in roots [23]. These results suggest that N concentration is closely associated with CK content in the root. However, the mechanism by which the interaction between N and CK mediates rice root growth remains elusive.
Root growth is mainly determined by root meristem cell proliferation and root cell elongation [24][25][26]. The meristem cell proliferation rate is positively correlated with meristem cell number and meristem cell division activity [26]. The root meristem cell number is antagonistically regulated by many regulators, including PLETHORA (PLT) and SHORT HYPOCOTYL2/INDOLE-3-ACETIC ACID3 (SHY2/IAA3) [26,27]; and the meristem cell division activity is positively correlated with the transcription level of cyclin and cyclin-dependent protein kinase genes, such as CycB1;1 and CDKB2;1 [24,28]. PLT genes encode APETALA2 (AP2) transcription factors and are essential for root meristem maintenance [27]. In Arabidopsis, plt1plt2 double mutants show a severe reduction in root meristem cell number, while the ectopic overexpression of PLT2 leads to an increased number of meristematic cells and increased meristem size [27,29]. SHY2/IAA3 controls the root meristem cell number by promoting the mitotic-to-endocycle transition in the root, which in turn decreases the meristematic cell number and reduces the root meristem size [19,26]. Plants with a loss-of-function mutation in SHY2/IAA3 have a larger-than-usual meristem, whereas those with a gain-of-function mutation in SHY2/IAA3 have a smaller meristem than the wild type [25,30]. XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH) and EXPANSIN (EXP) proteins play important roles in mediating root cell elongation [31,32], and thus mutations in EXP or XTH genes have been found to result in short root cells and short roots. For example, the Arabidopsis atxth31 loss-of-function mutant has shorter root cells and shorter roots than the wild type [33], and the lengths of roots and root cells in OsEXPB2 RNA interference lines were significantly shorter than those in wild-type rice plants [34]. In rice, the transcription levels of OsXTH and OsEXP genes, such as OsXTH1, OsEXP3, and OsEXPB4, which are specifically expressed in the root, are closely correlated with the lengths of mature cells in rice seminal root [19]. CK has been shown to regulate the expression of the genes regulating root meristem cell number and root cell elongation [27,35]. Previous studies have reported that some hormone response elements, including the CK-response element (AGATT), exist in promoter regions of the root-expressed OsPLTs [35,36]. OsPLT1-6 can strongly respond to CK because there are more than seven AGATT elements in each promoter region [35]. In addition, it has been reported that CK can up-regulate OsIAA3 and down-regulate OsXTHs and OsEXPs [19]. However, up to now, it has been unclear whether N and CK collaboratively regulate root meristem cell number and cell elongation by modulating transcription levels of OsPLTs, OsIAA3, OsXTHs, and OsEXPs.
Rice seminal roots are the first roots to emerge from seeds after germination and are responsible for water uptake and nutrient absorption during seedling establishment [37]. In this study, the influence of N deficiency on CK metabolism in rice seminal roots was investigated. Furthermore, the mechanism by which N and CK interact to regulate rice seminal root growth was clarified. We provide evidence that N deficiency reduces CK content by inhibiting CK biosynthesis and promoting CK degradation, which in turn promotes rice seminal root growth. Specifically, the decrease in CK content results in the down-regulation of OsIAA3 and up-regulation of OsPLT genes, leading to an increase in root meristem cell number and cell proliferation rate, and an up-regulation of the OsXTH and OsEXP genes, which promote cell elongation.

Plant Material and Growth Conditions
Japonica rice Nipponbare (Oryza sativa L.) was used in this study. Rice seeds were sterilized, soaked, and germinated according to the method of Zou et al. [38]. The germinated rice seeds were grown in an artificial climate incubator (HP 1500 GS, Ruihua Instrument & Equipment Co., Ltd., Wuhan, China) with a 12-h light (29 • C)/12-h dark (26 • C) photoperiod.

Chemicals and Treatments
Zeatin (Z) (Sigma-Aldrich Trading Co., Ltd., Shanghai, China), a mixture of trans-Z (tZ) and cis-Z (cZ), was dissolved in dimethyl sulfoxide (DMSO) and diluted to the desired concentration with distilled water. NH 4 NO 3 (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), which was used as the source of N, was dissolved directly in distilled water and diluted to the desired concentration. The solution for the control treatment contained 0.5 mM NH 4 NO 3 (1 mM N, 1 N), the solutions for the N deficiency treatments contained 0.125 mM NH 4 NO 3 (0.25 mM N, 1/4 N), 0.03125 mM NH 4 NO 3 (0.0625 mM N, 1/16 N), or 0 mM NH 4 NO 3 (0 mM N, 0 N), and the solution for the N deficiency + Z treatment contained 0 mM N and 4 nM Z (0 N + Z). All solutions also contained 0. 3  Since the Z solutions also contained DMSO, DMSO was added to the solutions without Z to a final concentration of 0.01% (v/v). The germinated rice seeds were incubated in different solutions for one day, two days, three days, or four days. All solutions were refreshed every two days.

CK Content Analysis
The germinated seeds were incubated in a solution containing 0 N, 1/16 N, 1/4 N, or 1 N (control). After four days of treatment, the seminal roots of the rice seedlings were collected and frozen at −80 • C until use. CKs were extracted, purified, and quantified according to previously described methods [19,39]. Data are reported as means ± standard deviation (SD) of three independent biological replicates.

RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis
The germinated seeds were incubated in a solution containing 0 N, 0 N + Z, or 1 N as a control for one day, two days, or four days; then, the seminal roots of the rice seedlings were collected and frozen at −80 • C until use. Total RNA was extracted according to Liu et al. [40]. First-strand cDNA was synthesized using the Fastking RT Kit (Tiangen Biotechnology Co., Ltd., Beijing, China). The relative expression level was analyzed by qRT-PCR according to the method described previously [40]; the gene-specific primers are listed in Supplementary Table S1. The relative expression level was calculated using the comparative threshold method, using OsACTIN as an internal control. Three biological replicates with three technical replicates were used for the statistical analyses and error range analyses, and data are presented as the means ± SD. The relative expression levels of CK metabolism genes, root meristem size-related genes, and root cell elongation-related genes in rice seminal root are presented in Supplementary Figure S1.

Quantitative Analysis of Root Phenotypes
The seminal roots of the rice seedlings were selected for the quantitative analysis of root phenotypes. Seminal root tips were stained with 4',6-diamino-2-phenylhydrazine solution to measure root meristem size and meristem cell number, and mature cells in rice seminal roots were stained with propidium iodide solution to measure mature cell length. The staining methods have been reported previously [19]. Images were taken on a confocal microscope (Leica SP8, Leica Corporation, Solms, Germany). Root length was measured with Image J software 1.46 (National Institutes of Health, Bethesda, MD, USA). Root meristem size was determined by measuring the length from the quiescent center to the first elongated epidermal cell. Root meristem cell number was expressed as the number of cortex cells from the quiescent center to the first elongated cell. Root growth rate was calculated as described previously [41]. Cell proliferation rate was calculated using the following equation: cell proliferation rate = root growth rate/mature cell length. Data are presented as means ± SD calculated from 10 biological replicates.

Statistical Analysis
Statistical analysis was performed using an independent samples t-test or a one-way analysis of variance followed by Duncan's multiple range test, with at least three biological replicates for each treatment. Values of p < 0.05 were considered statistically significant. All data are presented as means ± SD.

N Deficiency-Induced Growth of Rice Seminal Roots was Negated by Application of CK
In order to investigate the effect of N deficiency on rice seminal root growth, rice seedlings were cultivated hydroponically under control (1 N) and N deficiency (1/4 N, 1/16 N, and 0 N) conditions. After four days of cultivation, the average lengths of rice seminal roots treated with 1 N, 1/4 N, 1/16 N, and 0 N were 4.6 cm, 6.2 cm, 10.4 cm, and 11.5 cm, respectively ( Figure 1). The 1/4 N, 1/16 N, and 0 N treatments increased the rice seminal root lengths by 35%, 126%, and 150%, respectively, compared with those of the control. This result demonstrated that N deficiency promoted rice seminal root growth, and that the decrease in N was accompanied by an increase in rice seminal root length. Furthermore, the application of Z (a type of CK) at concentrations of 0.25-4 nM significantly inhibited the N deficiency-induced growth of rice seminal roots, and 4 nM Z completely blocked the N deficiency-induced growth ( Figure 1). This result suggested that N deficiency might promote rice seminal root growth by controlling the CK content.

N Deficiency Reduced CK Content in Rice Seminal Roots
To test whether N deficiency induces rice seminal root growth by controlling the content of endogenous CKs, the effects of N deficiency on the contents of endogenous Z, dihydrozeatin (DZ), and isopentenyladenine (iP), which are the predominant CKs in higher plants [42], were analyzed. Since the previous results clearly demonstrated comparable activities of trans-Z (tZ) and cis-Z (cZ) in the inhibition of rice seminal root elongation [43], thus the total content of Z, including tZ and cZ, was analyzed in this study. As shown in Table 1, the content of Z was significantly higher than that of DZ and iP, indicating In this experiment, germinated rice seeds were incubated in different solutions for four days, then photographs were taken, and the lengths of the seminal roots were measured. The data are the means ± SD calculated from 10 biological replicates. Significant differences (p < 0.05) are indicated by different letters (a, b, c, d, e, f). N, nitrogen; Z, zeatin.

N Deficiency Reduced CK Content in Rice Seminal Roots
To test whether N deficiency induces rice seminal root growth by controlling the content of endogenous CKs, the effects of N deficiency on the contents of endogenous Z, dihydrozeatin (DZ), Cells 2020, 9, 916 6 of 17 and isopentenyladenine (iP), which are the predominant CKs in higher plants [42], were analyzed. Since the previous results clearly demonstrated comparable activities of trans-Z (tZ) and cis-Z (cZ) in the inhibition of rice seminal root elongation [43], thus the total content of Z, including tZ and cZ, was analyzed in this study. As shown in Table 1, the content of Z was significantly higher than that of DZ and iP, indicating that Z is the predominant CK in rice seminal roots. Under control (1 N) treatment, the content of Z in the seminal root was relatively high (198.4 ± 11.1 pmol·g −1 FW) ( Table 1). By contrast, under the N deficiency treatments, the content of Z in seminal roots decreased significantly; the contents of Z under the 1/4 N, 1/16 N, and 0 N treatments were only 52%, 36%, and 34% of that of the control, respectively. However, neither DZ nor iP content was affected by N deficiency treatments compared with control (1 N) treatment (Table 1). This result indicated that N deficiency reduced CK content by reducing Z content rather than by controlling DZ or iP content in rice seminal roots.

N Deficiency Inhibited CK Biosynthesis and Promoted CK Degradation by Affecting the Transcription Levels of CK Metabolic Genes in Rice Seminal Roots
CK homeostasis is mainly controlled by CK biosynthesis and degradation [44,45]. In rice, six OsIPTs (OsIPT1, OsIPT3, OsIPT4, OsIPT5, OsIPT7, and OsIPT8) and 11 OsCKXs (OsCKX1-11) are the key genes modulating CK biosynthesis and degradation, respectively [46,47]. Thus, to investigate the mechanism by which N deficiency regulates CK content, the effects of N deficiency on the transcription levels of these six OsIPTs and 11 OsCKXs in rice seminal roots were investigated. On the one hand, N deficiency could reduce CK content by decreasing CK biosynthesis via the down-regulation of OsIPT genes. As shown in Figure 2a, after one day of treatment, of the six OsIPT genes tested, there were significant differences in the transcription levels between control and N deficiency treatments for four genes; the transcription levels of OsIPT3 and OsIPT4 were significantly down-regulated in response to all the N deficiency treatments, and the transcription levels of OsIPT7 and OsIPT8 were significantly down-regulated in response to the 1/16 N and 0 N treatments. After two days of treatment, the transcription levels of OsIPT3, OsIPT4, and OsIPT8 were significantly down-regulated by the 1/16 N and 0 N treatments (Figure 2b). After four days of treatment, the transcription levels of all six OsIPT genes were significantly down-regulated by treatment with ≤1/4 N (Figure 2c). These results suggest that N deficiency might reduce CK content by inhibiting CK biosynthesis via the down-regulation of OsIPT genes. On the other hand, N deficiency could reduce CK content by promoting CK degradation via the up-regulation of OsCKX genes. As shown in Figure 2d-f, OsCKX10 and OsCKX11 did not respond to N deficiency treatments, but the other eight OsCKX genes were up-regulated to different extents by different N deficiency treatments. Among the nine N deficiency-responsive OsCKX genes, OsCKX4 responded most strongly to N deficiency; the transcription level of OsCKX4 was up-regulated by more than 2-fold after one and two days of N deficiency treatment.

N Deficiency-Induced Decrease in CK Content Enhanced Root Meristem Cell Proliferation Rate and Promoted Root Cell Elongation
We investigated the effects of control (1 N), N deficiency (0 N), and N deficiency + Z (0 N + Z) treatments on seminal root meristem cell proliferation rate and mature cell length to clarify the mechanism by which the N deficiency-induced decrease in CK content increases seminal root length. The results demonstrated that the meristem sizes and the cell numbers of the seminal roots in the N deficiency treatment group increased significantly compared with those in the control group, but this effect was completely negated by the application of Z (Figure 3a-c). Moreover, the lengths of mature cells in the mature zone of rice seminal roots were also significantly increased by N deficiency treatment; the average root cell length in the mature zone was increased by 45.5% under N deficiency treatment (Figure 3d,e). However, this increase in cell length was completely negated by the addition of Z (Figure 3d,e). Further, the results demonstrate that N deficiency strongly enhanced the root growth rate and root meristem cell proliferation rate, but these effects were

N Deficiency-Induced Decrease in CK Content Enhanced Root Meristem Cell Proliferation Rate and Promoted Root Cell Elongation
We investigated the effects of control (1 N), N deficiency (0 N), and N deficiency + Z (0 N + Z) treatments on seminal root meristem cell proliferation rate and mature cell length to clarify the mechanism by which the N deficiency-induced decrease in CK content increases seminal root length. The results demonstrated that the meristem sizes and the cell numbers of the seminal roots in the N deficiency treatment group increased significantly compared with those in the control group, but this effect was completely negated by the application of Z (Figure 3a-c). Moreover, the lengths of mature cells in the mature zone of rice seminal roots were also significantly increased by N deficiency treatment; the average root cell length in the mature zone was increased by 45.5% under N deficiency treatment (Figure 3d,e). However, this increase in cell length was completely negated by the addition of Z (Figure 3d,e). Further, the results demonstrate that N deficiency strongly enhanced the root growth rate and root meristem cell proliferation rate, but these effects were negated by the addition of Z (Figure 3f,g). In addition, compared with control treatment, 0 N and 0 N + Z treatments had no influence on the transcription level of root meristem cell division-related genes, such as OsCycB1;1 and OsCDKB2;1 (Figure 3h,i). These results suggest that the N deficiency-induced decrease in CK content enhanced the root meristem cell proliferation rate and promoted root cell elongation, and that the enhanced cell proliferation rate was contributed by the increased meristem cell number rather than by the changed meristem cell division activity. negated by the addition of Z (Figure 3f,g). In addition, compared with control treatment, 0 N and 0 N + Z treatments had no influence on the transcription level of root meristem cell division-related genes, such as OsCycB1;1 and OsCDKB2;1 (Figure 3h,i). These results suggest that the N deficiency-induced decrease in CK content enhanced the root meristem cell proliferation rate and promoted root cell elongation, and that the enhanced cell proliferation rate was contributed by the increased meristem cell number rather than by the changed meristem cell division activity.

Decrease in CK Content Increased Root Meristem Cell Number by Affecting the Transcription Levels of Root Meristem Size-Related Genes
It has been reported that root meristem size is positively regulated by PLT and negatively regulated by IAA3 [25,27]. There are 10 OsPLT genes in rice; among them, only six (OsPLT1-6) are expressed in roots [35]. Therefore, to clarify whether the N deficiency-induced decrease in CK increased meristem cell number by affecting the transcription of meristem size-related genes in rice seminal roots, the effects of control (1 N), N deficiency (0 N), and N deficiency + Z (0 N + Z) treatments on the transcription levels of OsPLT1-6 and OsIAA3 were investigated. After one day of N deficiency treatment, the transcription levels of OsPLT1-6 were significantly increased, while the transcription level of OsIAA3 was significantly decreased, but these effects were antagonized by the application of Z (Figure 4a). After two days of treatment, the transcription levels of most of the root meristem size-related genes changed in response to N deficiency, but these transcriptional changes were smaller in the presence of Z (Figure 4b). After four days of treatment, the transcription levels of the four meristem size-related genes under N deficiency were still slightly but significantly different from those under the control condition, and the transcriptional regulation of these genes was also antagonized by the application of Z (Figure 4c). These results suggest that the N deficiency-induced decrease in CK content led to an increased root meristem cell number through the down-regulation of OsIAA3 and the up-regulation of root-expressed OsPLTs.

Decrease in CK Content Increased Root Cell Length by Up-Regulating Transcription of Root Cell Elongation-Related Genes
It has been reported that seven OsXTHs and eight OsEXPs are specifically expressed in rice roots [31,32], and that these root-specific genes play important roles in regulating root cell elongation in rice seedlings [31,32]. To clarify whether the N deficiency-induced decrease in CK increased the cell length in rice seminal roots by affecting the transcription levels of cell elongation-related genes, the effects of the control (1 N), N deficiency (0 N), and N deficiency + Z (0 N + Z) treatments on the transcription levels of the root-specific OsXTHs (OsXTH1, OsXTH2, OsXTH4, OsXTH13, OsXTH15, OsXTH16, and OsXTH25) and OsEXPs (OsEXP3, OsEXP11, OsEXP12, OsEXP13, OsEXP15, OsEXP17, OsEXPB2, and OsEXPB8) were investigated. After one day of N deficiency treatment, two-thirds of the root-specific OsXTHs and OsEXPs were up-regulated (Figure 5a). After two days of treatment, all root-specific OsXTHs and OsEXPs except for OsXTH16, OsXTH25, and OsEXP11 were up-regulated by the N deficiency treatment (Figure 5b). After four days of treatment, most of the root-specific OsXTH and OsEXP genes were still up-regulated under the N deficiency treatment (Figure 5c). At every time point, the effects on transcription were reduced in the presence of Z (Figure 5a-c). Taken together, all root-specific OsXTHs and OsEXPs except for OsXTH25 and OsEXP11 were up-regulated to different extents by N deficiency, and the up-regulation of these genes was inhibited by Z treatment. These results indicate that the N deficiency-induced decrease in CK content increased root cell length by up-regulating the transcription levels of cell elongation-related genes, including six OsXTHs and seven OsEXPs, in rice seminal roots. N deficiency, but these transcriptional changes were smaller in the presence of Z (Figure 4b). After four days of treatment, the transcription levels of the four meristem size-related genes under N deficiency were still slightly but significantly different from those under the control condition, and the transcriptional regulation of these genes was also antagonized by the application of Z (Figure 4c). These results suggest that the N deficiency-induced decrease in CK content led to an increased root meristem cell number through the down-regulation of OsIAA3 and the up-regulation of root-expressed OsPLTs.

Increasing Root Length is a Strategy for Responding to N Deficiency
Rice seminal roots are the first roots to emerge from seeds after germination and are responsible for water uptake and nutrient absorption during seedling establishment [37]. Here, we provide evidence that N deficiency treatments strongly promote seminal root growth of rice seedlings under a relatively short duration (four days) of N deficiency. Moreover, even under a long duration (30 days or 60 days), rice seminal root growth is still promoted by N deficiency treatments (Supplementary Figure S2). This result is consistent with previous reports that N deficiency treatment promotes root growth in other plants, including wheat [48] and Arabidopsis [49,50]. Root length is an essential parameter allowing crops to increase N use efficiency, as more and deeper roots may improve the uptake of N from deeper soil layers and reduce N losses to the environment [9]. Taken together, these findings suggest that increasing root length is a strategy used by plants to

Increasing Root Length is a Strategy for Responding to N Deficiency
Rice seminal roots are the first roots to emerge from seeds after germination and are responsible for water uptake and nutrient absorption during seedling establishment [37]. Here, we provide evidence that N deficiency treatments strongly promote seminal root growth of rice seedlings under a relatively short duration (four days) of N deficiency. Moreover, even under a long duration (30 days or 60 days), rice seminal root growth is still promoted by N deficiency treatments (Supplementary Figure S2). This result is consistent with previous reports that N deficiency treatment promotes root growth in other plants, including wheat [48] and Arabidopsis [49,50]. Root length is an essential parameter allowing crops to increase N use efficiency, as more and deeper roots may improve the uptake of N from deeper soil layers and reduce N losses to the environment [9]. Taken together, these findings suggest that increasing root length is a strategy used by plants to respond to N deficiency. However, because N is an essential element, a long duration of severe N deficiency such as 0 N treatment inhibits adventitious root production and reduces the whole root system volume (Supplementary Figure S2).

N Deficiency Promotes Rice Seminal Root Growth by Reducing CK Content
We found that the CK contents in rice seminal roots in the N deficiency treatment groups were lower than those of the control, and that a decrease in the concentration of N was accompanied by a decrease in the content of CKs as well as a longer seminal root ( Figure 1, Table 1). Moreover, the induction of rice seminal root growth was significantly negated by CK application (Figure 1). Previous work has shown that the application of high or moderate concentrations of N induces CK accumulation in the roots of dicotyledonous plants such as Arabidopsis [51] and monocotyledonous crops, including rice [52], wheat [48], maize [53], and barley [54]; this accumulated CK inhibits root growth [19,55]. Usually, CK content in the roots of plants incubated with high or moderate concentrations of N is supraoptimal for rapid root growth, and thus mild decreases in CK content promote root growth [20,21]. All these findings lead to the conclusion that N deficiency promotes rice seminal root growth by reducing CK content. Moreover, our results indicated that N deficiency reduces CK content by reducing Z content rather than by controlling DZ or iP content in rice seminal roots (Table 1).

N Deficiency Reduces CK Content by Inhibiting CK Biosynthesis and Promoting CK Degradation
To investigate the regulatory mechanism by which N deficiency affects CK metabolism, the effects of N deficiency on the expression of six OsIPTs and 11 OsCKXs were analyzed in this study. We found that N deficiency inhibited CK biosynthesis and promoted CK degradation through down-regulation of the OsIPT genes and up-regulation of OsCKX genes (Figure 2a-f), respectively, which in turn led to reduced CK content. Our result is supported by studies in Arabidopsis and tobacco that found that expressing CKX genes specifically in the roots results in reduced CK content through the enhancement of root-specific degradation of CK [56]. Moreover, our result is also supported by experiments in rice showing that an increase in endogenous CK content achieved through the knockdown of OsCKX4 or overexpression of OsIPT inhibits the growth of rice seminal roots [47,57]. In addition, our findings are also consistent with a study in rice in which OsIPT4 was found to be responsible for the increased content of CK in roots in response to nitrite (a type of N fertilizer) treatment [23]. Taken together, these results suggest that N deficiency reduces CK content by inhibiting CK biosynthesis and promoting CK degradation.

N Deficiency-Induced Decrease in CK Content Promotes Rice Seminal Root Growth by Enhancing Meristem Cell Proliferation Rate through Increased Meristem Cell Number
The root meristem cell proliferation rate is positively correlated with meristem cell number and meristem cell division activity [26]. We found that the N deficiency-induced decrease in CK content increases the root meristem cell number by up-regulating OsPLT1-6 and down-regulating OsIAA3 (Figuers 3c,4), which in turn promotes rice seminal root growth by enhancing the root meristem cell proliferation rate (Figure 3g). However, the N deficiency-induced decrease in CK content could not affect meristem cell division activity in rice seminal root (Figure 3h,i). This result is consistent with a previous report that CK had no influence on meristem cell division activity in Arabidopsis primary root [24]. All the evidence suggests that the N deficiency-induced decrease in CK content increases root meristem cell number by up-regulating OsPLT1-6 and down-regulating OsIAA3, which in turn promotes rice seminal root growth by enhancing the root meristem cell proliferation rate through an increased meristem cell number. Our result is supported by previous studies showing that a mild decrease in endogenous CK content promotes root growth [20,21], while CK treatment strongly inhibits root growth [19]. Our result is also consistent with the finding in rice that the transcription of all OsPLT1-6 genes declined to the lowest level after 24 h of CK treatment [35], and that the transcription of the OsIAA3 gene increased significantly within four days of CK treatment [19].

Decrease in CK Content Promotes Rice Seminal Root Growth by Promoting Root Cell Elongation
Root-specific OsXTHs and OsEXPs have been implicated in the control of rice root cell elongation [19,34]. Here, we provide evidence that all root-specific OsXTHs and OsEXPs except for OsXTH25 and OsEXP11 were up-regulated by N deficiency, but that this up-regulation was blocked by the application of Z (Figure 5a-c). This finding is consistent with our previous finding that the application of exogenous kinetin (a synthetic CK) down-regulated the transcription of OsXTHs and OsEXPs, such as OsXTH1 and OsEXP3, which in turn inhibited rice seminal root growth [19]. These pieces of evidence suggest that the N deficiency-induced decrease in CK content promotes rice seminal root growth by promoting root cell elongation through the up-regulation of root-specific OsXTHs and OsEXPs.

Conclusions
In summary, our results demonstrate that N deficiency reduces CK content by inhibiting CK biosynthesis and promoting CK degradation through the down-regulation of CK biosynthesis genes and up-regulation of CK degradation genes, respectively ( Figure 6). Moreover, our results indicate that the N deficiency-induced decrease in CK content promotes rice seminal root growth not only by enhancing the root meristem cell proliferation rate through an increased root meristem cell number, but'also by promoting root cell elongation through the up-regulation of root-specific OsXTHs and OsEXPs ( Figure 6). In addition, our results suggest that the N deficiency-induced decrease in CK content increases the root meristem cell number by up-regulating root-expressed OsPLTs and down-regulating OsIAA3 ( Figure 6). Taken together, our data suggest that an N deficiency-induced decrease in CK content promotes rice seminal root growth by promoting root meristem cell proliferation and root cell elongation. Further study is required to characterize the molecular mechanisms involved in the N-regulated transcription of OsIPTs and OsCKXs. Another important issue requiring further in-depth study is the role played by CK signaling in regulating the transcription of OsIAA3, OsPLTs, OsXTHs, and OsEXPs. Cells 2020, 9, x FOR PEER REVIEW 13 of 17

Decrease in CK Content Promotes Rice Seminal Root Growth by Promoting Root Cell Elongation
Root-specific OsXTHs and OsEXPs have been implicated in the control of rice root cell elongation [19,34]. Here, we provide evidence that all root-specific OsXTHs and OsEXPs except for OsXTH25 and OsEXP11 were up-regulated by N deficiency, but that this up-regulation was blocked by the application of Z (Figure 5a-c). This finding is consistent with our previous finding that the application of exogenous kinetin (a synthetic CK) down-regulated the transcription of OsXTHs and OsEXPs, such as OsXTH1 and OsEXP3, which in turn inhibited rice seminal root growth [19]. These pieces of evidence suggest that the N deficiency-induced decrease in CK content promotes rice seminal root growth by promoting root cell elongation through the up-regulation of root-specific OsXTHs and OsEXPs.

Conclusions
In summary, our results demonstrate that N deficiency reduces CK content by inhibiting CK biosynthesis and promoting CK degradation through the down-regulation of CK biosynthesis genes and up-regulation of CK degradation genes, respectively ( Figure 6). Moreover, our results indicate that the N deficiency-induced decrease in CK content promotes rice seminal root growth not only by enhancing the root meristem cell proliferation rate through an increased root meristem cell number, but also by promoting root cell elongation through the up-regulation of root-specific OsXTHs and OsEXPs ( Figure 6). In addition, our results suggest that the N deficiency-induced decrease in CK content increases the root meristem cell number by up-regulating root-expressed OsPLTs and down-regulating OsIAA3 ( Figure 6). Taken together, our data suggest that an N deficiency-induced decrease in CK content promotes rice seminal root growth by promoting root meristem cell proliferation and root cell elongation. Further study is required to characterize the molecular mechanisms involved in the N-regulated transcription of OsIPTs and OsCKXs. Another important issue requiring further in-depth study is the role played by CK signaling in regulating the transcription of OsIAA3, OsPLTs, OsXTHs, and OsEXPs.  Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/4/916/s1, Figure S1: The relative expression levels of genes investigated in this study, Figure S2: Effects of long-term treatment with N deficiency on rice seminal root growth, Table S1: List of primers used in this research.