In this study, we used the TC19 maize mutant, which had been screened after Co60-γ-ray irradiation and had been self-pollinated for multiple generations on the background of B73. Compared with Chang 7 − 2, the grain length, grain width, grain thickness, and 100-kernel weight of TC19 were significantly increased, whereas the ear length and grain weight were reduced. Kernel number per grain, 100-kernel weight, and ear number are components of maize yield. The phenomenon of improved grain weight with reduced yield has been observed previously [14]. Some quantitative trait loci (QTLs) have been found to affect the balance between the kernel and ear [15, 16]. Thus, the relationship between kernel, ear per plant, and field conditions should be carefully considered in plant breeding.
The grain type and grain weight of maize seeds not only are controlled by genetic factors, but also are affected by environmental factors, such as temperature, moisture, disease, and insect pests. To explore the difference between the grains of Chang 7 − 2 and TC19, we analyzed the grain length, grain width, grain thickness, and 100-kernel weight under multiple different environmental conditions. The results showed that environment had a great effect on the grain size of maize. However, the grain size and weight of TC19 under each environmental condition were always higher than in Chang 7 − 2, indicating that grain development mainly is genetically controlled. This is consistent with previous studies [17].
In this study, grain width was the main contributor to the difference in grain size between Chang 7 − 2 and TC19. The grain width increased fastest in TC19 at 14–28 DAP, at which stage it exceeded Chang 7 − 2, indicating that 14–28 DAP is an important period for grain enlargement. This stage is the grain-filling stage [18], and thus the grain-filling stage is critical for grain width.
Plant hormones are one of the most important factors affecting the growth and development of seeds [19]. Many genes related to plant hormones have been confirmed to play key roles in regulating plant kernel development [20]. In recent years, it has been discovered that cytokinin and brassinolide play a vital role in regulating seed size, and in addition, auxin, ABA, and gibberellin have regulatory effects on seed development to a certain extent [21]. In this study, we detected changes in IAA, GA3, CTK, and BR during grain development in Chang 7 − 2 and TC19. In all periods, the concentrations of IAA and BR in TC19 were significantly higher than in Chang 7 − 2. The content of GA3 was significantly higher in TC19 than in Chang 7 − 2 in the first three stages, but not significantly so in the latter two periods. The content of CTK differed significantly only at 21 DAP, being significantly higher in TC19 than in Chang 7 − 2, and the difference between the two maize varieties was not significant at the other periods. We propose that auxin and brassinolide may have contributed significantly to the increased size of the TC19 grains.
Through transcriptomics analysis of the grains of Chang 7 − 2 and TC19 at 14 days, 21 days, and 28 days after pollination, we found that the signal transduction pathway of plant hormones may have a notable influence on the grain size. Further screening of the DEGs revealed that five genes exhibited high expression levels, including ARF3, IAA15, AO2, DWF4, and XTH. Among them, ARF3, IAA15, and AO2 are related to the IAA biosynthesis or signal transduction pathway, and DWF4 and XTH are related to the BR biosynthesis or signal transduction pathway [22]. IAA15 is a member of the AUX/IAA gene family [23], and ARF3 is a member of the ARF family [24]. The Aux/IAA protein, as a type of transcription inhibitor, has been proven to play an extremely important role in the auxin signal transduction pathway. The auxin response requires the degradation of the Aux/IAA inhibitor, which causes the release of the ARF transcription factor that interacts with it and regulates the target gene. In this experiment, IAA15 was highly expressed in Chang 7 − 2, whereas ARF3 was lowly expressed in Chang 7 − 2, indicating that IAA signal transduction in Chang 7 − 2 was inhibited compared with TC19. AO2 encodes 3-indole acetaldehyde oxidase, which is a key enzyme in the indolepyruvate pathway; the latter being the main pathway through which higher plants synthesize IAA. In this study, the expression of the AO2 gene in TC19 was higher than in Chang 7 − 2 in the three periods, indicating that TC19 grains have higher levels of IAA, which was consistent with the endogenous hormone measurement results. DWF4 encodes sterol C-22α hydroxylase. Sterol C-22α hydroxylase acts as the rate-limiting link in the process of BR biosynthesis. A high expression of DWF4 increases the BR content in grains. In this study, the expression level of DWF4 in TC19 was always higher than that in Chang 7 − 2, which was consistent with the higher BR content in TC19. XTH encodes xyloglucan endotransglycosidase/hydrolase, which is a cell wall relaxase and a key enzyme in plant cell wall remodeling. Studies have shown that XTHs play roles in cell volume growth, and their expression is induced by brassinolide. The higher expression level of XTH is consistent with the higher BR content in TC19. DWF4 may have an indirect regulatory effect on the XTH gene—an aspect that could be further studied in the future (Fig. 10).
In addition to the genes related to auxin and brassinolide, some genes related to other hormones were differentially expressed between the two maize lines, but these genes may be involved in the process of grain development because of their low expression levels. The molecular regulatory mechanism of plant seed size is complex, and many genes and metabolites are involved in this process. The regulation of seed size by hormones forms only a part of this process. To study the regulatory mechanisms of seed size, the relationships and interactions between different substances or different genes need to be explored.