It had been reported that the bulged surface of fruits could be significantly improved through cultivation measures and exogenous spraying of growth retardant agent . However, the research on mechanism is still unknown. In this study, we explored the mechanism of growth retardant agent (PBZ) relieving bulged surface by means of histomorphology, exogenous hormones and transcriptome analysis.
PBZ retarded the growth and development of cell and vascular bundles
Schematic analysis revealed that the surface of pear fruit and their vascular bundles were unevenly distributed in the bulge part of the fruits in the control group (CK) . But it was evenly distributed in the transverse direction in fruits treated with growth retardant PBZ (PK)(Fig. 1, Additional file Fig. S1). It was previously reported that the developed vascular bundles were densely distributed at the calyx end of the bulged surface of pear fruits. However, these vascular bundles were developed slowly and distributed loosely at the calyx end of fruits in PBZ treated group [4,5]. And phloem cells appeared to become much smaller and tighter treated by PBZ than that of CK group . These findings suggest that PBZ might affect the growth and development of the vascular bundles in the fruits, thus affecting the bulge shape on the surface fruits.
The cells were well division and development in CK group. And the length of cell became long in CK group (Fig. 3a). Furthermore, the control group showed that the cell arrangement was relatively wobbly, the cell sizes and layers were significantly increased in the course of making paraffin wax section. Whereas the cells in the transverse section near the fruit pericarps, treated with growth retardant PBZ, were found compact, small and arranged; and cell layer increased in PK group (Fig. 3b-3d). Researchers showed that palisade and spongy cells became smaller when sprayed with PBZ . Meanwhile,the ratio of palisade to sponge layer thickness was increased[7,31], but cell longitudinal growth was inhibited in PBZ group [30-32]. Our data, together with previously reported studies, suggested that the growth retardant agent (PBZ) could affect cell size,cell layer arrangement and inhibit cell longitudinal growth.
PBZ reduced the content of IAA bulged sruface of fruits
Auxin is one of the important endogenous hormones, which plays an essential role in many processes of plant growth and development, such as promoting cell division and elongation [33-35] and vascular tissue formation and differentiation [36-38]. Our observation of auxin results indicated that IAA content was dropped in fruits treated by PBZ compared with CK group (Fig. 2). Our results were consistent with previous studies showing that IAA content was reduced in plant tissue treated by PBZ [11-13]. In addition, the result further showed that bulged surface fruits caused by strong growing vigor contained with high IAA and was relieved by the way of balancing phytohormone, especially IAA, among tissues.
PBZ degraded the expression of auxin and lignin related genes
GO and KEGG analysis revealed that auxin transport-related genes LAX were down-regulated and auxin responded GH3 genes and auxin efflux carrier (PIN) genes were up-regulated in pear fruits treated by PBZ. In addition, we found that lignin synthesis genes CYP73A2, COMT and cellulose synthesis genes involved in construction of vascular bundle were found down-regulated in the pear fruits treated by PBZ. Meanwhile, transcriptional factor genes such as bHLH and MYB were down-regulated in PBZ treated pear fruits (Fig. 5). Real Time qRT-PCR analysis of these differentially expressed genes were consistent with that of RNA-seq data, which confirmed the reliability of the experimental data. Previous reports showed that auxin affected vascular bundle formation through auxin efflux carrier PIN [16, 39,40]. Auxin efflux carrier genes were highly expressed in mutant vascular bundles [15,40,41]. Similar studies were reported that auxin influx carrier LAX supported vascular bundle development [18,42]. In addition, the active state of IAA was inactivated by GH3 genes [26,43]. Further our results proved that the PBZ treatment might change the auxin pathway genes activity to reduce IAA content, and could affect the distribution of transverse vascular bundles, cell division and cell elongation on the surface fruits, thus regulating the bulged surface in pear fruits.
In addition, it had been reported that, phytohormones, lignin synthesis related genes such as phenylpropane metabolic pathway genes CYP73A2 and COMT [44,21,22], transcriptional factor genes bHLH and MYB, as well as cellulose synthetase genes were involved in vascular tissue, growth, development and metabolism of its secondary cell wall. On the other hand, bHLH genes played an important role in early vascular bundle development [45,46]. MYBs, directly act on VND6 and VND7, which were involved in the formation of vessel and xylem of secondary cell walls [47-49,27,50]. Furthermore, overexpression of MYB52 and MYB54 up-regulates cellulose synthase 8 (CesA8) activity [51,52] and 4-coumarate-CoA ligase (4CL) [53,54] in cellulose and lignin synthesis. Some of the above differentially expressed genes showed that there were six genes which responds to auxin. These results indicated that PBZ treatment might affect vascular bundle development by changing the genes in response to auxin (Fig. 2 and Additional file Table S5).
In a word, this research presented a comprehensive overview of PBZ, which might regulate genes function involved in auxin metabolic pathway. Finally vascular development and cell division were affected by auxin. Thus it was concluded from our data that vascular bundles distribution and cells division might affect the bulged surface of pear fruits. This study will provide theoretical basis for the regulation of bulged surface of pear fruits by growth retardant agent (Fig. 6).