Assessment of gibberellin (GA4+7) mediated changes on grain filling, hormonal behaviour and antioxidants in high-density maize (Zea Mays L.)

Dense plant cultivation is an efficient approach to improve the maize production by maximizing the utilization of energy and nutrient. However, dense plant populations may aggravate the abortion rate of young grains and result in fewer number of kernels per ear. Grain filling rate and duration play a decisive role in maize grain yield. Therefore, increasing plant density, consideration of enhancing the grain filling rate and duration of individual maize plant and regulating crop senescence would be the first priority. In this study, we examined the regulatory effects of GA4+7 under two application methods (shank-smearing and silk-smearing). Shank-soaking with GA4+7 at the rate of 0 (CK1), 10 (T1), 60 (T2), and 120 (T3) mg L-1, while silk-smearing at the rate of 0 (CK2), 10 (S1), 60 (S2), and 120 (S3) mg L-1 were used. The results showed that GA4+7 improved the grain filling rate by increasing the content of auxin, gibberellin and zeatin and abscisic acid in grains compared to control plants. In addition, The auxin, gibberellin and zeatin contents in the grains were positively and significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates; the abscisic acid level was positively correlated with the maximum grain weight and the maximum and mean grain-filling rates. Moreover, GA4+7 increased the activities of superoxide dismutases, catalases, peroxidases, and reduced the malondialdehyde content in leaves compared with untreated plants. At the rate of 60 mg L-1, GA4+7 showed the greatest effect for shank-smearing and silk-smearing (T2 and S2), followed by 10 mg L-1 (T1) for shank-smearing and 120 mg L-1 (S3) for silk-smearing. Our results suggest that application of 60 mg L-1 GA4+7 for smearing application could efficiently be used for changed the level of hormones in grains and antioxidant enzymes in ear leaf, would be useful for enhancing grain filling rate and delaying leaves senescence, and resulting in an increasing of maize grain yield.

The contents of indole-3-acetic acid (IAA) and abscisic acid (ABA) were higher in superior grains than in inferior [23], 55 and the increased ABA and reduced IAA shortened grain-filling period in the inferior grains [24]. Liu, et al. [25] and Ali, 56 et al. [26] have reported a positive and significant correlation of ABA, IAA, and Z + ZR contents with the maximum grain 57 weight and grain-filling rates. In addition, a negative and significant correlation of ETH concentration with grain weight 58 and grain-filling rates was reported by Liu, et al. [25]. Additionally, the higher gibberellins (GAs) contents were present at 59 early stages of grain filling [27]. These studies showed that cereals grain filling is markedly affected by alterations in 60 hormones level in grains.

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In recent decades, plant growth regulators (PGRs) including gibberellins (GAs) are gaining interest among agriculture 62 scientists are broadly used in agronomic crops. GAs comprise a large family of hormones that are ubiquitous in higher 63 plants and have long been known as endogenous plant growth regulators, promoting several aspects of plant growth and 64 developmental processes, such as cell division, stem elongation, seed germination, dormancy, leaf expansion, flower and fruit development [28,29]. They were first discovered in 1930s by Teijiro Yabuta and Yusuke Sumiki and named after the 66 pathogenic fungus called Gibberella fujikuroi culture [30]. Since then 136 different types of gibberellins structures have 67 been identified and isolated from different fungi and plants sources [31,32]. Despite a large number of GAs, relatively a 68 few gibberellins such as GA1, GA3, GA4, GA7 are believed to have intrinsic biological activity in higher plants [29] 69 (chemical structure of GAs with high bioactivity shown in Scheme 1). GA3 is one of the most widely used plant growth 70 hormones. With the deepening of research, other hormones of GAs, especially GA4 and GA7 attract more and more 71 attention due to their special effects on plants. Some researchers suggested that GA 4+7 could successfully induce the fruit 72 set and increased fruit size in cucumber [33], pear [34], apple [35]. In addition, compared to pollinated fruit, GA 4+7 -treated 73 fruits accumulated more quantities of sucrose and fewer organic acids [34]. Therefore, the application of GA 4+7 proved to 74 be an utmost agricultural remedy in horticultural crops, which would otherwise dramatically increase yield and income.

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However, the application of GA 4+7 in cereal crops is limited and the effect of exogenous GA 4+7 on the regulation of maize 76 grain filling and its mechanism remains unclear.

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These previous literature have indicate that the potential of GA 4+7 to control grain filling of maize is limited, and the 87 possible effects on hormone change and antioxidants have not been investigated in details. Therefore, the objectives of the 88 present study were to instigate the effects of shank-smearing and silk-smearing with different concentrations of GA 4+7 on 89 grain filling rate, hormonal changes and its relation with grain filling and possible changes in antioxidants. We provided 90 the first detailed description of the GA 4+7 application in improving grain filling rate of maize and antioxidants, with the aim 91 of achieving higher grain yield in high-density maize. The hybrid maize seeds (Zhengdan, 958, a local hybrid) were provided by China National Seeds Group Co., LTD.

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week after silking stage (R1). The experiment was laid out in a randomized complete block design (RCBD) using three row-row distance of 17 and 60 cm, respectively. All plots were supplied with 225 kg N ha −1 , and 120 kg P 2 O 5 ha −1 . All P 6 119 fertilizers and 60% of the N fertilizer were applied at pre-sowing. The remaining 40% of urea (N 46%) was applied as a 120 top dressing at the twelfth leaf stage (V12). During the entire growth period, disease, pest and weed control in each 121 treatment were well controlled. Irrigation was applied when it was necessary.  The grain-filling data were fitted using the Richards growth equation [39]: The grain-filling rate (G) was calculated as the derivative of Eq (1):

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(2) Where W is the grain weight (mg), A is the final grain weight (mg), t is the time after anthesis (d), and B, k and N are 135 coefficients determined by regression analysis.

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The quantification of IAA, ZR, GA 3 and ABA was done using 50 µL sample (standard sample or the test sample) and 150 50 µL of the diluted antibody were added into each well of the elisa plate, and the plate was placed in the wet box at 37 °C 151 for 0.5 h. The plate was then washed 4 times with phosphate-buffered saline (PBS) containing 0.1%(v/v) Tween-20, and

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Then add 100 µL of the diluted IgG-HRP was added to each well, and the plate was put in the wet box at 37 °C for 0.5 h.

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Afterwards, the plate was washed 4 times again, and then it's the color reaction. To each well will be added 100 µL chromogenic reaction was also carried out in the wet box, and the reaction was terminated by adding 50 µL 2 mol L -1

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H 2 SO 4 . The absorbance of the solution was read at 490 nm by a spectrophotometer.

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The logit curve was used to calculate the ELISA results. Hormone concentration was expressed as ng g -1 fresh weight.

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The breakdown of H 2 O 2 was followed by measuring the absorbance change at 240 nm.      The IAA and ZR contents in maize grains exhibited a similar pattern during the grain filling process. The IAA and

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ZR contents increased linearly during the initial grain-filling stage and attained the maximum peak curves at 28 DAS for 239 all treatments (Fig. 3 and 4)   with GA 4+7 at the rate of 0, 10, 60, and 120 mg L -1 , respectively. CK2, S1, S2 and S3 represent silk-smearing with GA 4+7 255 at the rate of 0, 10, 60, 120 mg L -1 , respectively. Different letters within each growth stage are significantly different (P < 256 0.05). The vertical bars represent the ± the standard error of the mean (n = 3).

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The GA 3 contents in the grains exhibited a gradually decreasing trend during grain filling, except for S3 treatment 259 (Fig. 5). S3 treatment decreased the level of GA 3 in grains from 14 to 21 DAS and rose abruptly to a peak of 28 DAS, and 260 then decreased steadily after the respective peak. The trend is different from other treatments, and it is necessary to deepen 261 the research to explore the mechanism of its raise. Compared with control treatments, T2 and S2 treatments significantly 262 increased the GA 3 contents in grains and followed by T1 and S3. The average GA 3 contents under T1, T2, and T3 treatments 263 during the whole grain filling stage were increased by 9.1%, 26.4%, 5.1% compared with CK1, while that of S1, S2, and 264 S3 treatments were greater by 11.6%, 25.9%, and 22.3% compared with CK2. with GA 4+7 at the rate of 0, 10, 60, and 120 mg L -1 , respectively. CK2, S1, S2 and S3 represent silk-smearing with GA 4+7 267 at the rate of 0, 10, 60, 120 mg L -1 , respectively. Different letters within each growth stage are significantly different (P < 268 0.05). The vertical bars represent the ± the standard error of the mean (n = 3). whereas that of S2 and S3 increased by 7.9 ng g FW -1 , 14.9 ng g FW -1 , respectively, i.e., 13.6% and 7.9% increases 277 compared with CK2. However, T3 and S1 had no significant effect relatively on increasing ABA contents compared with 278 control treatment (CK).  The SOD activity showed a downward trend from 15 to 45 DAS under all treatments (Fig. 7). The SOD activity was 286 significantly improved in GA 4+7 -treated plants at a varied level, Compared to control. 10 mg L -1 (T1 and S1) and 60 mg L -1

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vertical bars represent the ± the standard error of the mean (n = 3). The POD activity in ear leaf displayed a gradually decreasing pattern of change from 15 to 45 DAS. And at the same 300 stage, with the increase of GA 4+7 concentrations, POD activity decreased gradually (Fig. 8). T1 treatment had the best 301 effect on increasing the POD activity, followed by T2. There was no significant difference between T1 and T2. The POD 302 activity revealed a significant increase of 9.0%, 45.6%, 10.8% and 15.8% in T1-treated plants, compared to CK1 at 15, 25,

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35 and 45 DAS, while that of T2 was 6.1%, 37.4%, 14.2%, and 15% higher than CK1. Whereas at the early grain filling 304 stage (from 15 to 25 DAS), the POD activity of T3 is higher than that of CK1, then the POD activity of T3 decreased 305 rapidly, and at 45 DAS, the POD activity of T3 was significantly lower than that in CK1. The treatments of silk-smearing 306 with GA 4+7 significantly increased the POD activity at all sampling stages compared with CK2, except for T3 treatment at 307 15 DAS. At 15 and 25 DAS, S1 had the best effects on increasing the POD activity, followed by S2. And from 35 to 45 308 DAS, the POD activities of T1, T2, and T3 were not significantly different from each other. S1, S2, and S3 increased the 309 average POD activities of the whole grain filling stages by 17.8%, 18.0% and 15.3%. with GA 4+7 at the rate of 0, 10, 60, and 120 mg L -1 , respectively. CK2, S1, S2 and S3 represent silk-smearing with GA 4+7 312 at the rate of 0, 10, 60, 120 mg L -1 . Different letters within each growth stage are significantly different (P < 0.05). The

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vertical bars represent the ± the standard error of the mean (n = 3).  (Fig. 9). With GA 4+7 shank-smearing applications, T1 treatment showed the best effect of CAT activity at all 317 sampling stages, followed by T2. And at 25 DAS, the CAT activity of T2 was significantly less than that of T1, but there 318 was no significant difference in CAT activity between T1 and T2 in other sampling periods. The CAT activity in T3 was 319 significantly higher than that of CK1 and significantly less than that of T2. All concentrations of GA 4+7 silk-smearing 320 treatments significantly increased CAT activity compared with CK2. The CAT activity of S2 was significantly higher than 321 S1, S3, and CK2. The average of CAT activity of all sampling stages in T1, T2, and T3 treatments was increased by 58.4%,
324 Fig. 9. The effects of GA 4+7 smearing application on CAT activity in maize. CK1, T1, T2 and T3 represent shank-325 smearing with GA 4+7 at the rate of 0, 10, 60, and 120 mg L -1 , respectively. CK2, S1, S2 and S3 represent silk-smearing 326 with GA 4+7 at the rate of 0, 10, 60, 120 mg L -1 . Different letters within each growth stage are significantly different (P < 327 0.05). The vertical bars represent the ± the standard error of the mean (n = 3). The MDA content increased gradually from 15 to 45 DAS in all the treatments, with the increase in GA 4+7 treatments 330 significantly lower than that of the untreated control (Fig.10). All the silking-smearing treatments (S1, S2, and S3) had

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G max : maximum grain-filling rates; G mean : mean grain-filling rates.

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Furthermore, ABA and GAs also play important roles in regulated grain filling. Yang,et al. [27] suggested that a 381 higher ABA concentration in grains enhanced the remobilization of prestored carbon to the grains and accelerated the grain 382 filling rate. And the previous studies showed that ABA content in grains was significantly and positively correlated with 383 the maximum and mean grain filling rates and maximum grain weight [25][26]55]. In our present study, the ABA content 384 in grains were increased by exogenous GA 4+7 , and there was positively correlated between ABA and grain filling rate 385 (Table.4). However, the correlation between GAs and grain filling rate and grain weight were not in consistent. Liu, et al.

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[25] and Liu, et al. [55] suggested that the content of GAs in the grains was not significantly correlated with the maximum  photosynthetic pigments content and subsequent in the delayed aging.

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With the knowledge of that developing seeds contain large amounts of hormones, which possess the ability of inducing 406 directional movement of nutrients within plants. The leaves senesce appears when a "sink" (young developing leaves or 407 seeds) needs the nutrients which were moved from the rest of the plant. Seth and Wareing [60] suggested that hormone-408 directed transport plays an important role in directing the movement of nutrients towards developing seeds. These indicated 409 that the application of exogenous GA 4+7 changed the level of hormones in seeds, thus affecting the antioxidant enzymes in 410 leaves and the senescence of maize. However, there are little research about exogenous GA 4+7 on reactive oxygen 411 metabolism in maize, and the specific mechanism needs further study.

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Application of GA 4+7 under high density leads to an increase in the level of IAA, ZR, GA 3, and ABA. This increase 414 is attributed to improving the grain filling rate, grain weight and the number of kernels ear -1 were also increased, as well as 415 increased the yield. In addition, GA 4+7 applications improved the activities of antioxidant enzymes in leaves senescence.

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The grain filling period could be prolonged by delaying the senescence of maize so that the grain filling was sufficient and 417 eventually increased the yield. Our results showed the positive effect of GA 4+7 on maize grain filling rate and antioxidant 418 enzymes, and could effectively be used for crop improvement, especially for cereal crops. At the rate of 60 mg L -1 , GA 4+7

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showed the greatest effect for shank-smearing and silk-smearing (T2 and S2), followed by 10 mg L -1 (T1) for shank-420 smearing and 120 mg L -1 (S3) for silk-smearing. The results from the present study illustrate that GA 4+7 application could 421 efficiently be used for altering the level of hormones in grains and antioxidant enzymes in ear leaf, would be useful for 422 enhancing grain filling rate and delaying leaves senescence and increasing grain yield of maize. 18