classification and pathways of candidate genes involved in BRA and BRD
Brace roots can not only improve lodging resistance but also have a significant impact on grain yield by absorbing nutrients and water under limited soil moisture conditions[4, 14, 15, 49]. However, the genetic basis of BRA and BRD is still unclear. In this study, the MLM method was used to identify four genes that were significantly related to BRA and BRD (Fig. 6). Functional annotations indicated that these candidate genes were mainly placed in several functional groups, such as DNA binding, hormone signaling pathways, and cell transport.
The plant cell wall is a dynamic structure, and its change is a response to the external environment. Two Leu-rich-like receptor kinases (FEI1 and FEI2) have been identified in Arabidopsis thaliana with a high degree of interaction with plant cell wall synthesis. The FEI system can process the turgor signal transmitted by the SOS5 system and stimulate cellulose synthase to synthesize the cell wall. The gene mutations of FEI1 and FEI2 lead to the break of the signal transmission chains, and plants sense the changes in the external environment and its biological effects on cellulose. Synthesis also plays a role in inhibition[50]. Debarati Basu (2016) compared the growth indicators of FEI mutants in different environments and found that compared with wild-type Arabidopsis thaliana, the hypocotyls of FEI mutants had shorter radicles and a larger diameter[51]. Fluorescent protein staining showed that FEI had a higher expression in root tips. FEI1 was highly expressed in the specific expansion of Arabidopsis root cells. GRMZM2G479243 is highly homologous to the FEI coding gene AT1G31420.2, which is predicted to play a role in the synthesis of brace root cell walls in maize.
The morphogenesis of plant organs depends on the intercellular flow of plant hormones, and directional signal transduction is determined by the polar subcellular localization of the auxin transporter synthesized by the PINFORMED (auxin export carrier PIN) gene[52]. The auxin synthesized from the brace roots is transported to the roots via vascular tubes. After auxin transport reaches the root tip, it is redistributed to the elongation zone of the root system. At this time, IAA enters the cell through the AUX1 carrier in the form of auxin, then enters the cell through the PIN carrier, and finally exports in the form of ions[53]. During this process, auxin is unevenly distributed on both sides of the elongation zone under the influence of gravity, and a geotropic reaction occurs. The phosphorylation kinetics of the PIN protein are affected by protein phosphokinase PP2A and PINOID, and the ROTUNDA3 (RON3) protein, as a regulator of PP2A phosphokinase, can play a role in regulating PP2A activity [54]. RON3 is a unique higher plant-specific gene. The map location technology identified 18 genes, including AT4G24500, that were significantly associated with RON3, which encodes a proline-rich protein and phenotypes the RON3-1 mutant. The identification showed that the number of lateral roots at the seedling stage was significantly reduced, and geotropic growth, which is affected by gravity, was significantly weakened[55]. Furthermore, Zhan (2012) identified AT4G24500 as the SIC gene, which encodes a hydroxyproline-rich protein[56]. The SIC gene is a functional gene necessary for the plant to maintain normal growth and development. The phenotype of SIC mutants was identified. It showed a variety of developmental defects, including reduced plant height, delayed flowering, increased leaf edge serrations, and abnormal roots. In maize, GRMZM2G174736 is a homologous gene of AT4G24500. Therefore, GRMZM2G174736 may play a role in the polar transport of auxin and affect the morphogenesis of maize roots.
Polar transport of the phytohormone auxin is the key to regulating plant growth and development. Polar transport of auxin in Arabidopsis roots requires the action of the MFS transporter, which is zinc-induced promoter-like 1 (ZIFL1). Reverse genetics showed that the ZIFL1.1 transporter regulates various root growth hormone-related processes and may indirectly regulate growth through cytokines used in the process of auxin transport; it may act by regulating the abundance of PIN2 in the plasma membrane of the root tip[57]. Auxin regulates various unrelated processes by directing cell division and expansion, such as embryo, root, and vascular bundle formation, post-embryonic organogenesis, and geotropism[58]. The chemical growth hypothesis better describes the basis for the movement of auxin cells, in which the proton gradient is mainly generated by the plasma membrane H+-ATPases between the neutral cytoplasm and the outside of acidic cells to drive the uptake and efflux of auxin[59]. This hypothesis presumes that there are local auxin influx and efflux carriers in the plasma membrane; thus, the asymmetric positioning of the coupling between adjacent cells provides directionality for cytokine flow[60]. In addition, auxin can also control the growth of Arabidopsis roots by regulating the cellular response to GA[61]. Therefore, GRMZM2G101928, as a homologous gene of AT5G13750.1, may affect the horizontal expansion of maize brace root cells and then affect morphogenesis.
Root growth and development is controlled by root tip meristems, and the size of root tip meristems affects the size of roots. In this study, GWAS was used to identify a candidate gene related to BRD traits and DNA binding, GRMZM2G151934, whose DA1 homologous protein 2 (DA1-related protein 2, DAR2) can regulate the size of root apical meristems. Relevant studies have shown that cytokinin and auxin antagonism can affect cell proliferation and differentiation, thereby regulating the size of roots by affecting the abundance of SHORT HYPOCOTYL2 (SHY2/IAA3). SHY2 affects the distribution of auxin in root meristems by inhibiting the auxin-induced expression of the PIN-FORMED (PIN) auxin transport gene. The PLETHORA (PLT1/2) gene affects the growth of root meristems by promoting stem cells and transporting and expanding cells[62]. In addition, DA1-related protein 2 (DAR2) acts downstream of cytokinin and SHY2 but upstream of PLT1/2 to affect the size of the root meristem[63]. Therefore, GRMZM2G151934, as a homologous gene of AT4G24500.1, may play a role in the growth of brace roots and affect the morphogenesis of maize roots.