Nitrogen Attenuated Zinc-Mediated Promotion of Rice Tillering Under Low Temperature via Regulating Auxin and Cytokinin Balance

Zhilei Liu Northeast Agricultural University Jinkai Su Northeast Agricultural University Jingrou Meng Northeast Agricultural University Jiamei Song Northeast Agricultural University Haonan Zhang Northeast Agricultural University Pengfei Li Northeast Agricultural University Yankun Sun Northeast Agricultural University Cailian Yu Harbin University of Science and Technology Xianlong Peng (  pxl0508@neau.edu.cn ) Northeast Agricultural University


Introduction
Rice is the most important food crop, feeding almost 1/2 of the human population (Pittol et  Province Shimono et al. 2007). Hence, it is crucial to nd approaches to compensate for the reduction of rice production caused by low temperatures.
Low temperature during the vegetative stage affects the carbon and nitrogen (N) metabolism, inhibits root nutrient uptake, reduces rice tiller growth rate and subsequently decreases the number of tillers Shimono et al. 2012; Shimono et al. 2002). Meanwhile, low temperature weakens the process of dry matter synthesis and transformation, resulting in a prolonged growth period, and nally reduces the rice yield (Shimono et al. 2007). 15°C low temperature stress decreases rice tillering, especially for the temperature-sensitive rice varieties, and 12°C has a strong depression on tillering for even insensitive varieties ; Reddy et al. 2021). Moreover, the depression effect of low temperature on rice tillering is associated with the duration of the low temperature, that is to say the longer the low temperature lasts, the more harmful it is to rice tiller number and growth rate (Liu et Phattarakul et al. 2012). Under both glasshouse and eld conditions, su ciently high N application is effective in enhancing grain Zn concentration in wheat (Cakmak et al. 2010b; Kutman et al. 2010).This is primarily because N stimulates the activities of transporter proteins involved in xylem loading and enhances the production of nitrogenous compounds facilitating Zn transport in plants (Curie et

Rice tillering and plant biomass measurement
The rice tiller numbers were measured before temperature treatment (BTT), ATT, and weekly after recovery to a normal temperature. After sampling, the samples were rinsed with distilled water and dried with lter paper. The number of tillers was measured then plants were placed in an oven at 105°C for 30min, dried at 85°C for 48h and weighed to determine their dry weights. The tiller growth rate was calculated as the tiller number changes per week.

Determination of plant Zn concentration
The measurement of Zn concentration was performed according to Gao

Measurement of hormone concentration
The hormone concentration was determined according to Hou et al. (Hou et al. 2008) with some modi es. Sample processing method and improve and optimize. Weigh 1 g of fresh tiller buds, add 5 mL of precooled 80% methanol, grind into a slurry in an ice bath, seal in plastic wrap and cold soak overnight at 4°C. Then the supernatant was obtained by centrifugation at 8,000 rpm/min for 10 minutes at 4°C. The residue was added to 4 mL of 80% cold methanol and centrifuged for 10 minutes, and the supernatant was combined. The whole ltrate was concentrated under reduced pressure at 40°C to 1/3 of the original volume, and 30 mL petroleum ether was added for extraction and decolorization three times, and the ether phase was discarded. The aqueous phase was extracted 3 times with 20 mL ethyl acetate, the ester phases were combined and evaporated to dryness under reduced pressure at 40°C. Add 2 mL of acetic acid solution with pH 3.5, purify through Sep-Pak C18 cartridge, eluting with methanol, collect and concentrate to dryness under reduced pressure at 40°C. Dissolve with mobile phase and dilute to 2 mL, lter through 0.45 µm microporous membrane, and analyze by HPLC.

Determination of quantitative gene expression
Total RNA was extracted from frozen rice plants (approximately 100 mg) using TRIzol reagent and an RNA Puri cation Kit (Invitrogen, Carlsbad, CA, USA), including DNase treatment, according to the manufacturer's protocol. Total RNA was quanti ed using a spectrophotometer following electrophoresis on a 0.8% (w/v) agarose gel to assess the concentration and integrity of each sample. Approximately 1 µg of total RNA was transcribed into cDNA using Superscript III Reverse Transcriptase (Invitrogen, Karlsruhe, Germany). The quality of the cDNA was assessed by qRT-PCR using primers for the Os18S rRNA genes.
qRT-PCR was performed using an Agilent Mx3000 P Analyzer (Agilent Technologies Ltd., Santa Clara, CA, USA) in a 15 µL reaction volume containing 1 µL cDNA, 2 µL primer mix, and 7.5 µL SYBR Green Master Mix (Agilent Technologies Ltd., Santa Clara, CA, USA). The cycle number was adapted for rice root and AMF. For rice genes, 35 cycles were performed, and AMF genes were ampli ed with 50 cycles. qRT-PCR was performed on three independent biological samples and three technical replicates. The IAA key gene expression primers were referred to Xu et al. (Xu et al. 2017) and the CTK key gene expression primers were determined according to Ding et al. (Ding et al. 2014), and all primer sequences are shown in Table   S1. The comparative 2 −ΔΔCT method was used to measure changes in the expression of selected genes relative to untreated controls (Winer et al. 1999).

Statistical analysis
Statistical analysis and correlation analysis were performed using SPSS 25 software. Multiway ANOVA was used to analyze the interaction between temperature, N and Zn, and multiple comparisons were performed by the LSD method. The signi cance level was 5%, and the LSD (0.05) value was obtained by SAS 8.01. The values in the gures and tables are represented as the means ± standard errors.

Rice tiller number
There was a signi cant interaction of T × N × Zn on tiller number at WAR 2-WAR 4 ( Table 1). Low temperature signi cantly suppressed the number of tillers, which were 23.66% (P < 0.05) and 42.03% (P < 0.05) lower under normal and high N conditions, respectively ( Fig. 1). Increasing Zn application increased the rice tiller number by 5.54% under normal N conditions, while rice tiller decreased by 19.43% (P < 0.05) under high N conditions. At WAR 3, low temperature still decreased the rice tiller number by 6.75% (P < 0.05) under normal N conditions, and the rice tiller recovered until WAR 4, but with increased Zn application it recovered to the normal level at WAR 3. Under high N levels, the rice tiller number recovered to normal levels at WAR 3 even without increasing the Zn supply. However, the rice tiller number at WAR 4 was still signi cantly lower than the normal level if the Zn application was reduced at both N levels.
The dry matter accumulation in the shoot and root of rice under different temperature, N and Zn treatments had similar trends with the rice tiller number during these three periods (Fig. S1). At WAR4, shoot dry matter could recover to normal level under normal N conditions, which was still signi cantly lower than normal temperature for high N treatments. Increasing Zn application could help the accumulation of rice dry matter, while rice shoot and root dry matter weight signi cantly decreased when reducing the supply of Zn.Tiller growth rate There were signi cant interactions of T × N × Zn on the tiller growth rate at ATT-WAR 2 and WAR 2-WAR 4 ( Table 1). Low temperature signi cantly decreased the rice tiller growth rate. Under the normal N level, increasing the Zn concentration was bene cial and increased the rice tiller growth rate; however, the rice tiller growth rate was signi cantly decreased with an increase of the Zn concentration under a high N level (Fig. 2).
During ATT-WAR 2, under normal N levels, increasing the Zn application increased the rice tiller growth rate, while the rice tiller growth rate decreased by 29.85% (P < 0.05) if the Zn concentration was decreased. Increasing the N supply reduced the rice tiller growth rate after the low temperature treatment, and increasing the Zn application further inhibited the rice tiller growth rate, with the rice tiller growth rate decreasing by 44.44% (P < 0.05), while decreasing the Zn supply increased the rice tiller growth rate by 14.29%.
During WAR 2-WAR 4, the rice tiller growth rate after the low temperature treatments was higher than that after the normal temperature treatments, and the rice tiller growth rate increased by 39.34% (P < 0.05) and 135.71% (P < 0.05) for the normal and high N levels, respectively. Increasing the Zn supply level increased the tiller growth rate by 29.41% (P < 0.05) and 10.91% (P < 0.05) under normal and high N levels, respectively.
Shoot and root Zn concentration T × N × Zn had signi cant interaction effects on the shoot zinc concentration at ATT and WAR 4, and on the root Zn concentration at all stages (Table 1). Low temperature stress increased rice shoot and root Zn concentration by 13.12% (P < 0.05) and 34.59% (P < 0.05), respectively at normal N level; and by 62.83% (P < 0.05) and 70.14% (P < 0.05), respectively at high N level (Fig. 3). Under low temperature stress, increasing Zn application had little effect on rice shoot Zn concentration at normal N condition, but signi cantly elevated root and shoot Zn concentrations at high N level. At WAR 2 and WAR 4, the shoot and root Zn concentrations of rice were lower than ATT, and high N treatments showed higher Zn concentration than that at normal N level. Additionally, increasing Zn application signi cantly increased shoot and root Zn concentration both at normal and high N conditions.
In order to identify the relationship between shoot Zn concentration and rice tiller numbers at different temperature and N conditions, the correlation of Zn concentration and tiller was analyzed. At normal N level, there was signi cant positive correlations between shoot Zn concentration and tiller number both under normal and low temperature conditions (Fig. 4). At high N level, signi cant negative correlation at ATT and positive correlation at WAR2 and WAR4 was observed under normal temperature conditions. However, there was negative correlations between rice tillers and shoot Zn concentration at ATT and WAR2 under low temperature conditions. And quadratic curve relationship was observed at WAR4, the Zn concentration with the highest rice tiller number was 32.62 mg kg − 1 (Fig. 4f). N absorption T × N × Zn had a signi cant interactive effect on N concentration at ATT and WAR 2 ( Table 1). Increasing the N levels signi cantly increased the shoot N uptake not only under normal but also under low temperature conditions (Fig. 5a). The N concentration of rice treated with high N levels decreased by 8.26% (P < 0.05) under low temperature treatment and increased by 7.63% (P < 0.05) under normal N levels. Under low temperature, increased Zn application increased N uptake by 4.39% (P < 0.05) at the normal N level but decreased N uptake by 5.94% (P < 0.05) at the high N level. At WAR 2 and WAR 4, there was little effect of N and Zn on the rice shoot N concentration.
T × N × Zn all had signi cant interactive effects on N accumulation in all stages (Table 1). Low temperature signi cantly decreased the rice N accumulation, but there was no signi cant difference between N levels. Under low temperature conditions, increasing the application of Zn increased the N accumulation by 16.61% (P < 0.05) at normal N levels. However, increasing the Zn supply under a high N level decreased the N accumulation by 20.90% (P < 0.05), which increased by 20.55% (P < 0.05) with decreased Zn application (Fig. 5b).
A negative effect of low temperature on N accumulation remained at WAR 2, which was more severe between N accumulation and tiller increment during ATT-WAR 2 and WAR 2-WAR 4 (Fig. 6), and there was also a signi cant positive correlation between the Zn application and the N accumulation increment under normal N levels (Fig. 7However, there was a signi cant positive correlation between the Zn application level and the N accumulation increment only during WAR 2-WAR 4 at high N level (Fig. 7).

MDA concentration and antioxidase activities
There was a signi cant interactive effect of T × N × Zn on MDA concentration and SOD, CAT and POD activities during the ATT period and on POD activity at WAR 2 (

Tiller bud IAA and CTK concentration
There was an interaction of T × N× Zn on tiller bud IAA concentration (Table 4). Low temperature treatment increased the tiller bud IAA concentration under normal N levels by 29.63% (P < 0.05) compared with normal temperature treatment, while CTK/IAA ratio decreased by 17.42% (P < 0.05); the IAA and CTK concentrations under high N levels increased by 105.52% (P < 0.05) and 94.05% (P < 0.05), respectively, but CTK/IAA ratio was not signi cantly different from that under normal temperature conditions (Table 3). Although increasing the Zn supply had no signi cant effect on the tiller bud CTK and IAA concentrations at normal N levels, the CTK/IAA ratio increased by 7.03%. Increasing the Zn application at a high N level increased the IAA concentration by 30.54% (P < 0.05) but had no effect on the CTK concentration, and the CTK/IAA ratio decreased by 27.13% (P < 0.05).  ATT represents after temperature treatment, and WAR represents weeks after recovery to normal temperature. * and **present signi cance at P < 0.05 and P < 0.01, respectively, and ns means no signi cance.
There was an interaction of T × N × Zn on tiller bud IAA and CTK concentration and CTK/IAA ratio at WAR 2, and IAA concentration and CTK/IAA ratio at WAR 4 (Table 4). At WAR 2, the tiller bud IAA concentration after the low temperature treatment decreased signi cantly, but there was little change in the CTK concentration ( Genes expression related to hormone metabolism T × N × Zn had an interaction effect on the expression levels of the key IAA and CTK metabolism genes, except OsIPT2. At WAR 2, T × N × Zn had an interaction effect on OsIPT2 expression (Table 4). Low temperature inhibited the expression of OsYUCCA1 but triggered the expression of OsYUCCA2, OsYUCCA4, OsIPT1 and OsIPT2 (Fig. 8a). Increased Zn application signi cantly promoted the expression levels of OsPIN1b, OsYUCCA2 and OsYUCCA4 under normal N conditions, but the expression levels of the three genes were signi cantly reduced under high N concentrations. In addition, increasing the Zn supply signi cantly inhibited the expression of OsYUCCA1 at both N levels and promoted the expression of OsIPT1 and OsIPT2. At WAR 2, increased Zn application signi cantly promoted the expression of OsYUCCA2, OsPIN1b, and OsIPT2 at normal N levels but decreased the expression of OsYUCCA1, OsYUCCA4, and OsIPT1 (Fig. 8b). Increasing the Zn concentration inhibited the expression of OsYUCCA1, OsYUCCA4, and OsIPT1 at high N levels but promoted the expression of OsPIN1b, OsYUCCA2 and OsIPT2. The results of this experiment also demonstrated that 7 days exposure to a low temperature (12℃) signi cantly increased the MDA concentration, declined root activities (data not shown), decreased the N absorption (Fig. 5), inhibited tillering growth (Fig. 2), and nally decreased the number of rice tillers number (Figs. 1 and 9) and dry matter accumulation (Fig. S1). Meanwhile, the antioxidant enzyme activity (SOD, POD, CAT) was signi cantly increased under low temperature stress ( Table 2).

Discussion
As important catalytic factors and essential components of many enzymes and proteins, Zn is an essential micronutrient for rice growth and development (Rehman et al. 2012 Although short duration or low strength low temperature exposure had little effect on the rice tiller number, a long duration or strong strength low exposure temperature irreversible damage on rice tillering, and the rice tiller number could not recover to normal level even for low temperature insensitive cultivars ). Consequently, it is important to improve rice low temperature resistance and nd appropriate approaches to reduce low temperature damage to rice tillers and promote the recovery of tillers after exposure to low temperature. In this study, low temperature reduced rice tiller numbers by 23.66% but increasing Zn application increased the tiller number by 5.54% (Fig. 1). Rice tiller numbers recovered to normal levels at WAR 4 without increasing Zn supply but could recover one week earlier if the Zn application was increased. It also can be seen from the tiller growth rate that Zn supply signi cantly increased the tiller growth rate and promoted tiller growth (Fig. 2), demonstrating that Zn could increase the number of rice tillers after exposure to low temperatures and promote tiller growth and recovery.
Zn enhanced rice tiller recovery by improving nutrient uptake and hormones balance Tillering growth is inseparable from the supply of N and Zn nutrients , and our previous study identi ed that the increase of tiller numbers after exposure to low temperature was signi cantly and positively correlated with the N accumulation increment ). In cold regions, applying Zn after regreening can also facilitate earlier tiller germination and increase effective panicles number (Zhang et al. 2013). In the current research, increasing Zn application could improve Zn and N absorption at normal N condition, when suffering low temperature stress (Figs. 3 and 5). Signi cant positive correlation between tiller number and shoot Zn concentration were observed not only under normal temperature but low temperature stress (Fig. 4), and there was a signi cant positive correlation between N accumulation and tiller number increment after increasing Zn supply at ATT-WAR 2 and WAR 2-WAR 4 ( Fig. 6). Meanwhile, there was a signi cant positive correlation between the Zn application and the N accumulation increment in both periods as well ( Fig. 7a and b), indicating that maintaining su cient Zn and N nutrition was crucial important for rice tillering and promoting nutrient absorption might be one of the possible mechanisms of Zn improving rice tiller growth and recovery after low temperature stress ( Fig. 9).
Rice tiller growth mainly consists of two processes: tiller bud germination and elongation (Li et  Upadhyay and Panda 2010). In this experiment, the IAA and CTK concentrations and CTK/IAA ratio of rice tiller buds decreased signi cantly with a reduced Zn application (Table 3), and increasing Zn supply increased rice Zn concentration (Fig. 3), promoted the expression of key genes for IAA and CTK synthesis and IAA transport in rice (Figs. 8 and 9), therefore rice still had a higher CTK/IAA ratio under low temperature stress (Table 3). After recovery to normal temperature, increasing Zn application reduced the IAA concentration in the tiller buds, but had less effect on CTK, so a higher CTK/IAA ratio was still presented in the tiller buds (Table 3). This was mainly due to the promotion of OsPIN1b expression under increasing Zn application conditions (Fig. 8b), indicating that during the recovery of rice from low temperature stress, Zn promoted IAA transport to other parts, reduced IAA accumulation in tiller buds, increased CTK/IAA ratio, broke the dormancy of the tiller buds, and promoted tiller growth (Fig. 9).
Nitrogen affected the contribution of Zn on rice tiller growth under low temperature Zn uptake and utilization by plants are both affected by the N application level (Nie et al. 2019b). N could promote the synthesis of nitrogenous compounds that are bene cial for Zn transport and improve Zn uptake by the plant root system (Erenoglu et al. 2011;Ji et al. 2021). Our experiment obtained a similar result that increasing the N supply could signi cantly increase the Zn concentration of rice (Fig. 3), and the contribution of Zn to rice resistance to low temperature exposure was also affected by the concentration of N. Compared with normal N levels, high N application increased MDA concentrations under low temperature stress (Table 2), reduced the rice tiller growth rate (Fig. 2), and signi cantly reduced the tiller numbers (Fig. 1). Increasing Zn application caused higher MDA accumulation (Table 2), lower dry matter production (Fig. S1), signi cantly reduced the tiller growth rate and tiller numbers at high N level (Fig. 1and 2). Although increasing Zn application increased shoot and root Zn concentration under high N condition, the increase of Zn concentration might due to the decrease of dry matter production, and the results of the correlation analysis showed that there was a signi cant negative correlation between the shoot Zn concentration and the number of tillers when suffering low temperature stress (Fig. 4d). These results suggested that increasing Zn supply at high N level might aggravate the damage of low temperature. Consequently, the ability of Zn help rice resisting to low temperatures was associated with the level of N application, but the internal mechanism is still unclear and needs further study.
The results of plant hormone showed that the IAA concentration of tiller buds treated with high N concentration increased signi cantly under low temperature stress, an increase in Zn application signi cantly increased the IAA concentration and then signi cantly decreased the CTK/IAA ratio ( Table 3).
The gene expression data also showed that an increased Zn application under high N level suppressed rice OsPIN1b expression and reduced the transport of IAA from tiller bud to other parts (Fig. 8a), thus excessive Zn application under high N level led to a higher CTK/IAA ratio. Possible reasons for the above results were that signi cant higher Zn concentration under high N condition lead to excessive IAA accumulation in tiller bud ( Fig. 3 and Table 3). When recovered to normal temperature for 2 weeks, rice shoot and root Zn concentrations were lower than ATT, while rice plants of increasing Zn application under high N condition still had higher Zn concentration, which was negatively affected tiller number (Fig. 4e). Furthermore, increasing Zn application promoted the expression of the key genes for CTK synthesis and IAA transport, resulting in signi cant decrease in tiller bud IAA concentration and signi cant increase in CTK/IAA ratio (Table 3 and Fig. 8b). Previous studies have shown that excessively high IAA or low CTK/IAA ratio could cause tiller buds to enter dormancy (Haver et al. 2002). According to the Zn concentration, hormonal changes and tiller recovery, although the tiller number decreased signi cantly under low temperature stress with the Zn application increasing under high N level, the low temperature did not cause tiller bud death and instead they entered dormancy due to the inhibition of tiller bud germination and elongation caused by high IAA concentration. After the low temperature stress was relieved, the shoot Zn concentration and IAA concentration in the tiller buds were reduced, and CTK/IAA ratio increased along with the enhancement of IAA transport. Thus, the dormancy of the tiller buds was broke, tillering growth was spurred at WAR 3 and then quickly recovered to normal levels. However, there was no signi cant difference of tiller numbers between normal and high Zn levels (Fig. 1). Consequently, the rice tiller number could recover to normal level with a moderate amount of Zn application at high N level. Nevertheless, this result was only veri ed from the hormonal perspective, and the internal mechanisms need to be further explored from morphological and molecular perspectives.
Although high N levels attenuated the contribution of Zn in improving rice low temperature resistance, rice growth after recovery to normal temperature still required an additional nutrient supply ). An increased N supply increases the chlorophyll concentration, enhances the rice photosynthetic rate and accelerates carbon and N metabolism, and then enhances the leaf area index, biomass, and tiller number, therefore alleviating the low temperature damage that occurred during the vegetative growth stage ( The same conclusion was obtained in this study: rice grown under a high N supply recovered faster than rice grown under low N supply (Fig. 1). Meanwhile, a synergistic effect between N and Zn emerged after recovery to normal temperature. Increasing Zn application promoted the increase of N accumulation (Fig. 5b) and higher N level also improved Zn absorption (Fig. 3) and transport in rice (Fig. 8), enabling the contribution of Zn in enhancing rice recovery from low temperature. However, the tiller number and shoot Zn concentration presented a quadratic curve relationship at WAR4, meaning that excessive Zn was not still bene t for tiller growth under high N condition and the appreciate shoot Zn concentration was 32.62 mg kg − 1 in this experiment (Fig. 4f). Consequently, Zn could regulate tiller bud germination, promote rice growth recovery under high N levels after recovery to normal temperature by affecting IAA and CTK balance and providing su cient nutrients for tillering growth through the synergistic effect of N and Zn.
In conclusion, low temperature during the vegetative growth stage damaged the rice antioxidant system, suppressed nutrient uptake and decreased the number of tillers. Zn could enhance rice low temperature resistance and promote tillering recovery from low temperature stress; while the promotion of Zn was related to the environmental N level. Increasing Zn application under suitable N level could diminish the damage of low temperature stress and promote tiller recovery rapidly after low temperature via increasing nutrient absorption and regulating IAA and CTK balance. Increasing Zn application under high N level aggravated the low temperature damage and further reduced tiller number. The inhibition on rice tillering by Zn under high N was mainly due to higher shoot Zn concentration and IAA accumulation, and lower CTK/IAA ratio in tiller buds, which is the main reason lead to tiller dormancy. When the low temperature was relieved, appropriate Zn application under high N level promoted IAA transport from tiller buds, tiller dormancy was broken, grew spurts and then rapidly recovered to normal level.  The relationship between shoot Zn concentration and tiller numbers after temperature treatment (ATT) (a and b), 2 weeks after recovery (WAR 2) (c and d) and WAR 4 (e and f) at different N levels, n=9. N1 and N2  Zn (0.30 μM ZnSO4·7H2O) level, respectively. 22°C represents the normal temperature, and 12°C represents the low temperature. ATT represents after temperature treatment, and WAR represents weeks after recovery to normal temperature. Data represent means ± standard errors. from different independent treatments. LSD (0.05) is the least signi cant difference between treatments at P<0.05.

Figure 6
The relationship between the increment of N accumulation and tiller 2 weeks after recovery (WAR 2) (a) and WAR 4 (b). n=36, * and **present signi cance at P<0.05 and P<0.01, respectively, and ns means no signi cance.

Figure 7
The relationship between Zn concentration and the N accumulation increment 2 weeks after recovery   Schematic diagram of Zn increasing rice tillering under low temperature stress. Low temperature causes the decline of root activity, inhibits the nutrient uptake, cause the generation of ROS, disturbs hormones balance and then reduces the number of tillers. Increasing or appropriate Zn application under low temperature can enhance root activity, increase nutrient absorption, promote the synthesis of IAA and cytokine CTK, promote the transport of IAA from tiller bud to other parts, then maintain tiller bud hormones balance, accelerate tillers growth. Meanwhile, Zn can also alleviate the damage of ROS under low temperature stress. ROS, reactive oxygen species; IAA, auxin; CTK, cytokinin; YUCCA, IPT and PIN1, key genes for IAA and CTK synthesis and transport; black arrow, the damage of low temperature to rice; red arrow, the effect of Zn under low temperature stress; green circle, hormones status in tiller bud.

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