Expression analysis of PIN-formed auxin efflux transporter genes in maize

Abstract

The local auxin gradient has a decisive role in auxin signaling, auxin-mediated development and abiotic stress response. PINFORMED (PIN)-formed auxin efflux transporters are very important for determining the direction of auxin transport and maintaining a local auxin concentration gradient. In this study, all candidate PIN genes from the current maize genome sequence database were identified and categorized based on amino acid similarity. The expression pattern of these PINs was analyzed in maize inbred line DH4866, which was selected from the progeny of 7922 and 478, and served as the female parent line of many hybrids in Shandong Denghai Seeds Co Ltd (China). Tissue-specific expression patterns indicated that they may have different roles in different stages of development, especially in the root system. Promoter motif analysis of four maize PIN1 genes and their expression levels in response to NAA, low phosphate levels and PEG treatment indicated that ZmPIN1a and ZmPIN1b may contribute more than ZmPIN1c and ZmPIN1d to root growth regulation and abiotic stress response. Analysis of the ZmPIN1a and ZmPIN1b transgenic lines (in DH4866) indicated that they have different effects on root development and growth, with ZmPIN1a increasing the number of lateral roots and inhibiting their elongation to form a developed root system, while ZmPIN1b increases root biomass by promoting the growth of both lateral and seminal roots. These results indicated that maize PIN1 genes function in coordination during maize development and in response to abiotic stress.

Keywords: -formed auxin efflux transporter; PIN1; expression analysis; maize.

Figure 1.

Phylogenetic tree and expression pattern of PINs in maize. (a) Phylogenetic tree of PINs in maize, rice and Arabidopsis. The red highlights the PIN family genes in maize. The ID numbers of the genes are listed in Table S1. (b) Real-time RT-PCR analysis of maize PIN family genes in different maize tissues and at developmental stages with maize Actin1 (NM_001155179.1) used as an internal control to normalize the relative expression levels.

Figure 2.

Four maize long-loop PIN1 genes have different responses to environmental stress. (a) Promoter motif analysis of the four PIN1 genes in maize. Plant promoters were analyzed by PlantCARE. (b) Real-time RT-PCR analysis of maize PIN1 genes in response to abiotic stress treatment (NAA, low phosphate and PEG). Roots from 3-leaf stage seedlings cultured in different nutrient solutions, as indicated, were used in this study.

Figure 3.

Different contributions to root system development and growth by overexpression of ZmPIN1a and ZmPIN1b and the altered PIN1 protein pattern in maize roots due to overexpression of ZmPIN1a. (a,b) The seedlings and root systems of ZmPIN1a and ZmPIN1b overexpression and WT maize lines under normal nutrient solutions. C Anti-PIN1 immunolocalization on the longitudinal root apex of maize primary roots from different ZmPIN1a transgenic lines and WT.