Grain weight and grain-filling characteristic parameters
Grain weight increased across sampling date (Figure 1). At 5-10 DPA, there was no significant difference among three treatments. The main differences between the three different treatments are concentrated at 15-35DPA. The effects of CN treatment on grain weight were mainly concentrated in the spraying period (15 DPA) and the middle-late filling period (25-35 DPA). The grain weight in these two periods was significantly higher than that of CK. The FU treatment significantly increased the grain weight during the spraying period (15 DPA), but significantly reduced the grain weight in the later stage of grain-filling (30-35 DPA). This result indicates that the grain weight was significantly enhanced by CN treatment and declined by FU treatment.
According to the grain-filling parameters, the grain-filling process of wheat was well simulated using logistic equations. As can be seen from table 2, the results fit the curve with high reliability, with an R2 coefficient of determination values above 0.999. Compared with CK treatment, CN significantly increased the maximum theoretical grain weight by 7.5% and FU treatment significantly reduced the maximum theoretical grain weight by 9.5%. Furthermore, CN treatment significantly prolonged the active grouting period and the effective grouting period, by 12.2% and 7.7%, respectively, compared with CK. There was no significant change in the active grouting period between the FU and CK treatment, but the effective grouting period was significantly shortened by 4.5%. The maximum grouting rate under CK treatment appeared at an earlier time point than CK treatment. And the grain weight at the maximum grouting rate was significantly higher than that of CK treatment. While the opposite tendency was obtained under FU treatment.
Characteristics of grain-filling stage
The whole grain-filling process were subdivided into early, middle and late stages according to the standard logistic curve. The results were shown in table 3. From the perspective of the duration of three periods, the effects of CN treatment on the grouting process were mainly concentrated in the middle and end stages. CN treatment significantly extended the duration of the middle and late stages by 12.1% and 12.1%, respectively. The FU treatment significantly shortened the early duration by 9.6%, and had no significant effect on the duration of the middle and end stages. Compared with CK, the mean grain-filling rate of CN was significantly increased by 9.5% in the early, significantly decreased by 4.2% in the middle and no significant difference in the late. The mean grain-filling rate of FU was significantly lower than that of CK treatment in the middle and late stages, with a decrease of 8.1% and 9.1%, respectively. The increased grain weight of CN treatment in the early, middle and late stages increased significantly by 32.7%, 7.3%, and 7.4% respectively, compared with CK. On the contrary, the increased grain weight of FU was significantly lower than that of CK in the early, middle and late stages, which were reduced by 9.6%, 9.6%, and 9.6 respectively.
Endosperm cell activity
In order to observe the PCD process of wheat endosperm under different treatments, the viability of endosperm cells in the cross-section of grain was detected by Evan's Blue staining (Fig. 2). The selective permeability of dead cell membrane became worse and it is colored blue, however, living cells are not colored. The results showed that the endosperm of CK, CN and FU treatments were not stained at the initial filling stage (before 10 DPA), and the staining sites were mainly concentrated on the pericarp (Fig. 2, A1-C1). The endosperm stained was initially observed at 15 DPA (Fig. 2, A2-C2). With grain filling, the number of stained cells increased gradually, and the whole endosperm was stained at the later stage of grain filling (Fig. 2, A3-C3, A4-C4). There was no obvious difference among three treatments at 10 DPA (Fig. 2, A1-C1). However, from 15 to 25 DPA the number of stained cells was the highest in FU treatment, followed by CK and CN (Fig. 2, A2-C2, A3-C3, A4-C4).
Effects of different treatments on nuclear morphology
DAPI fluorescent dyes have strong specificity and sensitivity to the nucleus and chromosomes in cells and it exhibits blue fluorescence when combined with DNA. DAPI can stain nuclei regardless of whether or not the cells undergo death. Therefore, the morphological changes of the endosperm nuclei can be observed by DAPI staining. The wheat kernels at different developmental stages under various treatments were sectioned and stained. The results are shown in Figure 2. It was observed that some of the nuclei in CK treatment were deformed at 25 DPA, and the FU had more deformed nuclei than the CK. However, most of the nuclei remain small and normally spherical shape under CN, only a few deformed nuclei were detected (Fig. 3, A1-A3). At 30 DPA, almost all nuclear morphologies changed, of which FU was the most varied, followed by CK and CN treatment (Fig. 3, B1-B3). The results indicated that CN was helpful to slow down the process of nuclear morphological changes of endosperm, while the FU accelerated the process.
Ultrastructure observation of endosperm cells under different treatments
In order to further explore the influence of different treatments on the nuclear morphological changes of endosperm, ultrathin sections of wheat endosperm at different development stages were made and observed by transmission electron microscopy (TEM). At 15 DPA, the CK treatment exhibited that the heterochromatin increased in the nucleus, nucleoli disappeared, and the nuclear edge was not smooth (Figure. 4, A1). The nuclei in endosperm cells were normal with evident nucleoli in CN (Figure. 4, A2). And the nucleus morphology of FU was similar to that of CK (Figure. 4, A3). At 20 DPA, the CK treatment showed that the nuclei appeared obvious pits, nucleoli disappeared, nucleoli concentrated, and the morphology was irregular (Figure. 4, B1). Under CN condition, the endosperm cells still had nucleoli, however, the edge of the nucleus was not smooth, with more pits. The nucleus were inlaid between starch granules and exhibited the irregular morphology (Figure. 4, B2). In contrast, no intact nuclei were observed in endosperm cells of FU treatment and the nuclear debris were found between starch granules (Figure. 4, B3).
Effects of different treatment on the number of endosperm nuclei
The PCD process was accompanied by the nuclear disintegration. In order to observe the influence of different treatments on the number of endosperm nuclei, we counted the endosperm nuclei at different sampling stages. The period of rapid proliferation of endosperm cell nuclei was 5 between 10 DPA. From 20 to 30 DPA, it was the period of rapid decline of the number of endosperm nuclei. There was no significant difference in the number of endosperm cells among three treatments at 5, 30 and 35 DPA, respectively. The number of endosperm cells were greater in CN and CK than in FU (Figure 5, note the difference was significant between CK and FU). At 25 DPA, the number of endosperm cells in CN was significantly higher than that in CK and FU (Figure 5, note the difference was not significant between CK and FU). This result suggested that the number of endosperm nuclei could be significantly increased by spraying with cobalt nitrate, while it could be significantly decreased by spraying with fluridone.
DNA content and DNA hydrolase activity in wheat kernels
The degradation of endosperm nucleus could be confirmed by measuring the change of total DNA content in grain. The DNA content in the grain varied depending on sampling time and different treatment (Figure 6A). The variation trend of DNA content in three treatments was similar, showing a single peak. The DNA content under CK and FU treatments was gradually increased and reached its peak at 20 DPA. However, the peak time of CN treatment was later, at 25 DPA. It should be noted that at the early stage of grain filling (5 DPA), whereas there was no significant difference among three treatments. From 25 to 35 DPA, the CN treatment had the greatest DNA content among the treatments.
Degradation of genomic DNA, often accompanied by an increase in DNA hydrolase activity. As shown in Figure 6B, the activity of DNA hydrolase showed the similar pattern among different treatments. The DNA hydrolase activity in CK and FU treatments gradually increased from 5 to 15 DPA, and increased suddenly to the peak at 20 DPA and then decreased. The DNA hydrolase activity was greater in FU than in CK and CN. However, the CN reached the maximum at 25 DPA and then decreased gradually, maintaining the greatest DNA hydrolase activity among three treatments between 25 to 35 DPA. Combined with the above anatomical results, the period of 15 to 20 DPA is the rapid growth period of PCD and also the key period of grain filling. Meanwhile, the nuclease activity of CK and FU reaches its peak rapidly with the rate of change greater than CN, while CN reaches its peak at 25 DPA. These results suggest that the CN treatment can slow down genomic DNA degradation during the critical grouting period (15-20d). Spray application of fluridone treatment resulted in an increase in nuclease activity and accelerated degradation of genomic DNA.
ACC and ABA content in wheat kernels
1-Aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, its variation trend can reflect the fluctuation of ethylene content. As shown in Figure 7A, the ACC content in the grain varied depending on sampling date and different treatment. From5 to 10 DPA, the ACC content was declined in CN, on the contrary, the content of ACC was rapidly increased in CK and FU treatment. The FU treatment had the highest ACC content and CN had the lowest ACC content among three treatments from 10 to 30 DPA (Figure 7B).
As shown in Figure 7B, the ABA content in grains of the three treatments had a consistent trend in different periods, showing a single peak. The CK and CN reached the maximum value at 25 DPA, while the FU reached the maximum value at 20 DPA. There was no significant difference in ABA content between CK and CN, but under FU treatment it was significantly lower than CK and CN treatments from 10 to 35 DPA. The results showed that spraying cobalt nitrate had no significant effect on ABA content in wheat grains, however, spraying fluridone could significantly reduce ABA content in wheat grains.
Genes involved in ethylene receptors and PCD
The relative expression pattern of four genes (ers1, ers2, etr1, etr2) encoding ethylene receptors, and one gene (dad1) involved in PCD were shown Fig.8. The expression patterns of the four ethylene receptor genes were similar, and all had lower expression levels in the mid-filling(15-25DPA)which was the rapid period of PCD. The relative expression of the four receptor genes was significantly higher in CN group than in the CK group at all times, except for 5 DPA. In contrast, the opposite phenomenon was observed in FU group. The expression of the dad1 increases gradually as the grain filling, reaching a maximum at 35 DPA. Compared with the CK treatment, the relative expression of dad1 was significantly increased in the CN treatment. The opposite results were observed in the FU group, except at 5, 30, and 35 DPA. The results showed that spraying cobalt nitrate significantly increased the relative expression of the ethylene receptor genes and the PCD repressor gene, and spraying fluridone significantly decreased their expression.
Relationship between DNA content, ABA content and grouting rate in wheat grains under different treatments.
Correlation analysis showed that there was a significant positive correlation between grain filling rate to DNA content and ABA content under the three treatments, respectively (Table.4). The correlation coefficient between grain filling rate and DNA content under CN treatment was 0.937. Furthermore, there was a significant positive correlation between ABA content and DNA content in the three treatments, and the correlation coefficients were all greater than 0.8.