Plant growth
For grafted plants, graft combination significantly affected the plant growth, and its interaction with N treatment significantly affected the plant height, shoot and whole dry weight (Table 2). The growth performance of bottle gourd rootstock-grafted (Z/J) plants were better than that of self-grafted (Z/Z) plants (Fig. 1a), correspondingly, the plant height, shoot, root, and whole dry weight, and sound seedling index of Z/J plants were all significantly higher than those of Z/Z plants regardless of the N level (Table 2). Compared with 9 mM N treatment, these growth parameters of Z/Z plants decreased slightly under 4 mM N treatment, while increased in Z/J plants. For example, 4 mM N treatment reduced plant height, root, shoot, and whole dry weight, and sound seedling index of Z/Z plants by 22%, 14%, 27%, 25% and 17%, respectively, whereas increased the plant height, shoot and whole dry weight, and sound seedling index of Z/J plants by 22%, 28%, 23% and 2%, respectively.
Table 2
Effect of graft combination and N treatment on plant growth of the watermelon seedlings
Graft combination | N treatment | Plant height (cm) | Root dry weight (g) | Shoot dry weight (g) | Whole plant dry weight (g) | Sound seedling index |
Z/Z | 9 mM | 26.0 ± 0.36c | 100% | 0.14 ± 0.01b | 100% | 0.71 ± 0.05c | 100% | 0.85 ± 0.06c | 100% | 0.30 ± 0.03b | 100% |
| 4 mM | 20.4 ± 0.35d | 78% | 0.12 ± 0.01b | 86% | 0.52 ± 0.03c | 73% | 0.64 ± 0.04c | 75% | 0.25 ± 0.03b | 83% |
Z/J | 9 mM | 38.8 ± 1.09b | 100% | 0.35 ± 0.02a | 100% | 1.38 ± 0.10b | 100% | 1.73 ± 0.12b | 100% | 0.60 ± 0.03a | 100% |
| 4 mM | 47.2 ± 0.50a | 122% | 0.34 ± 0.03a | 97% | 1.76 ± 0.09a | 128% | 2.12 ± 0.11a | 123% | 0.61 ± 0.04a | 102% |
Analysis of variance | | | | | |
N treatment (N) | * | ns | ns | ns | ns |
Graft combination (G) | *** | *** | *** | *** | *** |
N×G | *** | ns | ** | ** | ns |
Data in the columns are the mean ± standard error (n=5). Different letters indicate significant differences determined by Tukey test at P≤0.05. Results of a two-way ANOVA are indicated, *P༜0.05;** P༜0.01; *** P༜0.001; ns, not significant. Z/Z: self-grafted watermelon seedlings; Z/J: rootstock-grafted watermelon seedlings |
For ungrafted plants, bottle gourd (J) plants showed better growth performance than watermelon (Z) plants under 9 mM and 4 mM N levels (Fig. 2a). Rootstock genotype significantly affected the plant growth (Fig. 3). The root and shoot dry weights, sound seedling index, and R/S ratio of J plants were about 2.0, 1.4, 3.1 and 1.4 times of Z plants under 9 mM N treatment, respectively, and 2.2, 1.6, 3.7, and 1.4 times under 4 mM N treatment, respectively (Fig. 3).
Root morphology
For grafted plants, the root morphology traits were significantly affected by graft combination (Table 3). Between the two graft combinations, the root system of Z/J plants was larger than that of Z/Z plants (Fig. 1b), consistently, the total root length, root surface area, root volume, and root forks of Z/J plants were all significantly higher than those of Z/Z plants regardless of the N level (Table 3). Compared with 9 mM N treatment, these parameters of Z/Z plants decreased slightly under 4 mM N treatment, but increased in Z/J plants. For example, 4 mM N treatment reduced the total root length, root surface area, root volume, and root forks of Z/Z plants by 11%, 15%, 16%, and 22%, respectively, whereas increased the total root length and root forks of Z/J plants by 4% and 11%, respectively.
Table 3
Effect of graft combination and N treatment on root morphology of the watermelon seedlings
Graft combination | N treatment | Total root length (cm) | Root surface area (cm2) | Root volume (cm3) | Root forks |
Z/Z | 9 mM | 491 ± 32.0bc | 100% | 133 ± 6.97b | 100% | 2.79 ± 0.20b | 100% | 3471 ± 396ab | 100% |
| 4 mM | 438 ± 41.2c | 89% | 113 ± 12.0b | 85% | 2.33± 0.32b | 84% | 2699 ± 470b | 78% |
Z/J | 9 mM | 680 ± 53.7ab | 100% | 266 ± 16.7a | 100% | 8.40 ± 0.74a | 100% | 3930 ± 213ab | 100% |
| 4 mM | 704 ± 58.7a | 104% | 267 ± 9.60a | 100% | 8.30 ± 0.86a | 99% | 4371 ± 232a | 111% |
Analysis of variance | | | | |
N treatment (N) | ns | ns | ns | ns |
Graft combination (G) | *** | *** | *** | *** |
N×G | ns | ns | ns | ns |
Data in the columns are the mean ± standard error (n=5). Different letters indicate significant differences determined by Tukey test at P≤0.05. Results of a two-way ANOVA are indicated, *P༜0.05;** P༜0.01; *** P༜0.001; ns, not significant. Z/Z: self-grafted watermelon seedlings; Z/J: rootstock-grafted watermelon seedlings |
For ungrafted plants, N treatment affected the total root length, while rootstock genotype and their interaction significantly affected all the root morphology traits (except that the interaction did not affect the root volume) (Fig. 4). The root system of J plants was significantly stronger than that of Z plants under 9 mM and 4 mM N levels (Fig. 2b). The total root length, root surface area, root volume, and root forks of J plants were about 1.5, 1.7, 2.0, and 2.0 times of Z plants under 9 mM N treatment, respectively, and 1.9, 2.1, 2.4, and 2.5 times under 4 mM N treatment, respectively (Fig. 4).
NO3− content and NO3− uptake
For grafted plants, the total NO3− content was significantly affected by graft combination and N treatment (Fig. 5a), and the whole plant NO3− uptake was significantly affected by graft combination and its interaction with N treatment (Fig. 5b). The total NO3− content and NO3− uptake of Z/J plants were all significantly higher than those of Z/Z plants, especially in the roots. Compared with 9 mM N treatment, 4 mM N treatment reduced the total NO3− content and NO3− uptake of Z/Z plants by 17% and 40%, while the total NO3− content of Z/J plants decreased by 9% and NO3− uptake increased by 14%. Furthermore, the NO3− content and NO3− uptake in Z/J plant roots decreased, but these values in leaves increased under 4 mM N treatment.
For ungrafted plants, both total NO3− content and NO3− uptake were significantly affected by N treatment and rootstock genotype (Fig. 6). Compared with 9 mM N treatment, 4 mM N treatment reduced the NO3− content in Z plant roots and leaves by 32% and 25%, and NO3− uptake by 38% and 42%, respectively. However, 4 mM N treatment had no significant effect on NO3− content and NO3− uptake in J plant roots, while NO3− uptake in leaves decreased by 32% (Fig. 6). Furthermore, Z plants showed significantly higher NO3− uptake than that of Z plants regardless of the N level (Fig. 6b). For example, the whole plant NO3− uptake of J plants was about 1.4 times and 2.4 times higher than that of Z plants under 9 mM and 4 mM N treatment, respectively.
Gene expression of nitrate transporters and N metabolizing enzymes
Relative expression of nitrate transporters and N metabolizing enzymes genes was conducted in self-grafted and rootstock-grafted watermelon plants. According to the Cucurbitaceae Genome database, we identified two nitrate transport genes in watermelon and bottle gourd roots, respectively, and four N metabolizing enzymes genes in watermelon leaves (Table 1). The relative expression of these genes was significantly affected by N treatment, graft combination and their interaction (Fig. 7). The mRNA levels of NRT1.5 and NRT2.1 in the roots, and NR, NiR, GS1 and GS2 genes in the leaves of Z/J plants were up-regulated significantly under 4 mM N treatment compared to the 9 mM N treatment. However, these genes in Z/Z plants did not change significantly under 4 mM N treatment, except for the up-regulated expression of NiR and GS2 genes. Moreover, the gene expression of nitrate transporters and N metabolizing enzymes in rootstock-grafted watermelon seedlings was significantly higher than that in self-grafted plants. For example, under 4 mM N treatment, the relative expression of NRT1.5, NRT2.1, NiR, GS1 and GS2 genes in Z/J plants were 27.3, 2.9, 2.6, 1.8 and 2.5 times higher than that in Z/Z plants, respectively.
NA, NUpE, NUtE and NUE
The NA, NUpE, NUtE and NUE were all significantly affected by N treatment, graft combination and their interaction (except that the N treatment did not affect NUtE) (Figs. 8 and 9). Obviously, the NA in roots, stems and leaves of Z/J plants was remarkablely higher than that of Z/Z plants, and increased significantly under 4 mM N treatment, especially in leaves (Fig. 8). In addition, compared with 9 mM N treatment, the NUpE, NUtE and NUE of Z/J plants were increased significantly under 4 mM N treatment (Fig. 9). These NUE traits were all significantly higher than those of Z/Z plants, especially at 4 mM N level. For example, the total NA, NUpE, NUtE and NUE of Z/J plants under 4 mM N treatment were 3.8, 1.2, 3.3, and 3.8 times higher than that of Z/Z plants, respectively.
leaf photosynthesis
Graft combination significantly affected Pn, Tr and chlorophyll content, and its interaction with N treatment significantly affected the Pn and chlorophyll content (Fig. 10). The Pn, Tr and chlorophyll content of Z/J plants were all higher than those of Z/Z plants regardless of the N level. Compared with 9 mM N treatment, these parameters of Z/Z plants decreased slightly under 4 mM N treatment but increased in Z/J plants. For example, 4 mM N treatment reduced the Pn, Tr and chlorophyll content of Z/Z plants by 60%, 11%, and 16%, respectively, whereas increased by 35%, 15% and 27% in Z/J plants, respectively.
Correlation analysis
Pearson’s correlation analysis between the NUE and other parameters of the self-grafted and rootstock-grafted watermelon seedlings under 9 mM and 4 mM NO3−-N conditions was conducted (Fig. 11). The NUE was found to be positively correlated with the plant height, shoot dry weight, whole plant dry weight, NA, NUtE, Pn, Tr, and chlorophyll content; and all correlations between these parameters above were positive. NUtE was also positively correlated with the root dry weight, sound seedling index, total root length, root surface area, root volume, and whole plant NO3− uptake; meantime, all these parameters were positively correlated with each other, and they were also positively correlated with the plant height, shoot dry weight, whole plant dry weight, NA and Tr (except that RV has no correlation with PH). In addition, the whole plant NO3− uptake was also positively correlated with Pn and chlorophyll content. The NUpE was positively correlated with the relative expression of NRT1.5, NRT2.1 and GS1 genes, and the relative expression of NRT1.5 and NRT2.1 genes, as well as the NiR, GS1 and GS2 genes were all positively correlated with each other.