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.

Auxin is a phytohormone that regulates various aspects of plant growth and development, such as dormancy, root development, shoot elongation and embryo pattern formation.^1–5 Polar auxin transport is critical for the formation and maintenance of the auxin concentration gradient around the meristem, and this different auxin gradient initiates and regulates many aspects of plant growth and development.⁶ In recent decades, increasing evidence has shown that PIN-formed auxin transporters are sufficient for determining the direction of auxin transport.^7–10 There are eight auxin efflux transporters in Arabidopsis with five long-loop PINs, including PIN1-PIN4 and PIN7 proteins, whereas PIN5, PIN6, and PIN8 have short-loop PINs.⁸ Loss of PIN function leads to abnormal auxin accumulation and development in Arabidopsis.^11–18 However, the studies on the function of PINs and their role in monocotyledon crops, except for OsPIN1 in rice¹⁹ and ZmPIN1a and ZmPIN1b in maize,²⁰ remain insufficient. OsPIN1 is involved in adventitious root and tiller emergence and development in rice.¹⁹ In maize, the influence of PIN1-mediated auxin transporters is observed during embryogenesis and endosperm development in monocots, and the expression pattern of three identified ZmPIN1 genes were determined at the transcription level in maize defective endosperm-B18 (de*-B18) mutant and the inbred line B73.^21,22 The expression levels of ZmPIN1a and ZmPIN1b in B73 and barren inflorescence2 (bif2) maize mutants were examined.²³ In our previous study, overexpression of ZmPIN1a increased the number of lateral roots and inhibited their elongation, forming a developed root system with longer seminal roots and denser lateral roots.²⁰

In rice, 12 PIN genes encoding auxin efflux carriers have been identified, and phylogenetic analysis has shown that 11 of these genes are on the same branches as Arabidopsis PIN genes, although one had no close homolog in Arabidopsis.²⁴ In maize, total 14 or 15 PIN coding genes^25,26 were identified in B73 line and group into different groups by different groups. There are 5 and 4 long-loop PINs identified by Forestan et al²⁵ and Yue et al²⁶ respectively, while 7 and 8 short-loop PINs were identified and grouped with the orthologs in Arabidopsis and rice. Beside of these, 2 and 3 maize specific PINs were identified with lower similarity to others. The identified ZmPIN genes have overlapping expression profiles in the root apex, inflorescence in male and female differentiation, and kernel in development.^20,23

In this paper, the putative PIN-formed family of genes in maize was identified by the recent database search, and a phylogenetic analysis of these genes to determine orthologs in Arabidopsis and rice was conducted. The expression patterns in different tissues and at different development stages were detected by real-time RT-PCR in maize inbred line DH4866, which was selected from the progeny of a hybrid of 7922 and 478. This inbred line served as the female parent line of many hybrid lines in Shandong Denghai Seeds Co Ltd (China). The reason why DH4866 was used because it was an important inbred line used in maize breeding with a compacted plant structure and deep root system which meet the demand of high density breeding. And auxin is an important plant hormone supposed to be involved in the architecture difference between different maize lines. To determine which members of the PIN1 genes participate in the abiotic stress response, the promoter motifs and expression changes in response to NAA, low phosphate and PEG treatment in the roots of maize seedlings were analyzed. Compared with ZmPIN1c and ZmPIN1d, ZmPIN1a and ZmPIN1b were more important in the response to abiotic stress in maize. The different effects of ZmPIN1a and ZmPIN1b on root development and growth indicated that maize PIN1 genes function in coordination during maize development and abiotic stress response.

The plant materials used in this study were from the elite inbred maize line DH4866 and the transgenic homozygous lines derived from independent T₀ plants.²⁰ DH4866, was selected from the progeny of the hybrid of 7922 and 478 by Denghai Seeds Co Ltd (plant variety right: CNA19990064.7). The transgenic homozygous lines obtained by using Agrobacterium-mediated maize shoot-tip genetic transformation used were same as reported by Li et al²⁰ The transgene (ZmPIN1a or ZmPIN1b) was driven by the barley Pht1 promoter²⁷ and the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase gene as a selectable marker gene was driven by CaMV35S promoter²⁰ The gene ID numbers of the genes are listed in Table S1. Sequence analysis was conducted with Clustal W2 and MEGA 5.²⁸ Promoter searches for cis-elements were performed using the PlantCARE website.²⁹ Hydroponic cultures with low phosphate and auxin treatment were analyzed with real-time RT-PCR as described by Li et al.²⁰ and Zhang et al.³⁰ The gene transcript levels were calculated by using the 2^−ΔΔCt method,³¹ and maize Actin1 (NM_001155179.1, Zm00001d010159) was used as an internal control. The entire experiment was repeated three times. The primer sequences used in this study are shown in Supplemental Table 1.

ZmPIN1 immunostaining was performed on roots collected from 3- and 4-day-old plants grown vertically in nutrient solution. Blocking, incubating and washing were conducted as described by Forestan et al.²² Anti-AtPIN1 antibody (aP20 sc-27163, Santa Cruz Biotechnology) diluted 1:250 in 1X PBS with 1% BSA and donkey anti-goat IgG H&L (Alexa Fluor® 568) (ab175474, Abcam) diluted 1:400 in 1X PBS with 1% BSA were used.

PIN family auxin efflux transporters can be categorized into long-loop PINs and short-loop PINs based on the length of the central hydrophilic intracellular loop between their transmembrane domains.⁸ Through recent searches and predictions using the most recent maize database, 11 PIN genes were found and categorized through a phylogenetic analysis into branches with Arabidopsis and rice (Figure 1(a)). In the PIN1 clade, there are four PIN1 genes (ZmPIN1a to ZmPIN1d) in maize and two PIN3-like genes, ZmPIN3a and ZmPIN3b, that each have a long loop similar to homologs in rice, whereas there were five members in the Arabidopsis genes. There were three homologous genes of AtPIN5 in maize located on chromosomes 1, 3, and 3 (Table S1). A homologous gene of AtPIN8 and a homologous gene of OsPIN9 were found in maize: ZmPIN8 and ZmPIN9. The orthologs of AtPIN2, AtPIN4, AtPIN6, and AtPIN7 did not exist in maize, and OsPIN4, OsPIN6 and OsPIN7 did not exist in rice. ZmPIN1b and ZmPIN1c were very similar to OsPIN1a, while the ZmPIN1a gene was homologous to OsPIN1c, and the ZmPIN1d gene was homologous to OsPIN1b and OsPIN1d, as we previously reported.²⁰ As shown in Figure S1, 11 genes were identified in this paper and in the result of Forestan et al. 2012²⁵ and Yue et al. 2015,²⁶ however, two PIN3-like genes, ZmPIN3a and ZmPIN3b were found in this paper.

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.

Tissue-specific expression pattern analysis showed that ZmPIN1b and ZmPIN1a have higher expression levels than other genes, whereas the expression levels of ZmPIN1d, ZmPIN3b, ZmPIN5c, ZmPIN8, and ZmPIN9 were very low (Figure 1(b)). The higher expression levels of these genes in the root, immature tassel, ear and embryo indicate that they participate in morphogenesis of the plant. The expression levels of ZmPIN1b in the shoots and leaves of maize were the highest among these PIN genes, suggesting that ZmPIN1b functions in auxin transport in the shoots. Compared to ZmPIN1b, the expression of ZmPIN1a and ZmPIN1c is lower and ZmPIN1a is higher in the root. In the V5 seedlings, ZmPIN1a is mainly expressed in roots with a 5–10-fold expression level in leaves, especially in the segments of lateral root formation and tips of seminal roots²⁰ ZmPIN3a, ZmPIN3b, and the other short-loop PINs, ZmPIN5a, ZmPIN5b, ZmPIN5c, ZmPIN8 and ZmPIN9, are mainly expressed in the root and in early organogenesis. These suggested that the ZmPIN1a or ZmPIN1b may contribute more in maize root development.

Promoter analysis indicates that four maize PIN1 genes may be involved in different biological processes and stress responses. Many light-responsive elements exist in the promoter regions of polar auxin transporter genes, indicating that they are involved in photomorphogenesis, which corresponds to the biological function of auxin. To identify which member contributes to the abiotic stress response and is thus useful for transgenic breeding, the number and position of response elements to drought stress, low phosphate stress and auxin were analyzed (Figure 2(a)). In the −500 bp (from ATG) region of the ZmPIN1a promoter, four ABREs, one low phosphate response element and one auxin response element were found. Two ABREs, one low phosphate response element and one auxin response element were found in the −500 bp region of the ZmPIN1d promoter. These results suggest that these elements are involved in resistance to drought and low phosphate and may be the main node between drought and low phosphate stress and auxin polar transport.

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.

As shown in Figure 2(b), four ZmPIN1 genes have different expression patterns in maize roots in response to NAA, low phosphate, and osmotic stress. Compared with ZmPIN1c and ZmPIN1d, for example, ZmPIN1a and ZmPIN1b were upregulated by NAA and low-phosphate stress and downregulated by osmotic stress. ZmPIN1a was induced 2.65-fold by NAA and 2.95-fold by low phosphate and was downregulated 0.36-fold by PEG treatment. A similar response was observed for ZmPIN1b, with a 1.94-fold induction by NAA, 2.32-fold induction by low phosphate, and 0.35-fold downregulation by PEG treatment. ZmPIN1c and ZmPIN1d were slightly affected in the root when subjected to abiotic stress treatment, and their expression were low especially ZmPIN1d. As with the tissue-specific expression levels, they may have different roles in the growth and abiotic stress response of different tissues.

ZmPIN1a and ZmPIN1b were selected for examination of their roles in maize plant growth and in response to abiotic stress by using transgenes driven by a root-specific expression promoter (barely Pht1;1 promoter).²⁰ ZmPIN1a and ZmPIN1b have different roles in maize root development, with ZmPIN1a increasing the number of lateral roots and inhibiting their elongation, thus forming a developed root system, while ZmPIN1b shows increased root biomass by promoting the growth of both lateral and seminal roots (Figure 3(a,b)). Why does the auxin concentration in the root tips of overexpressed ZmPIN1a plants substantially increase? We investigated the cellular localization of gene products and carried out PIN1 immunostaining using an anti-AtPIN1 antibody. We also determined the IAA transport capacity of the ZmPIN1a sense lines, antisense lines and WT.²⁰ PIN1 immunostaining of maize root showed that the signal intensity in the pericycle region of the primary root and the lateral root primordium is stronger in the overexpressed lines than it is in the WT and antisense lines (Figure 3(c)). In the meristematic zone, the meristem region showed an intensification of PIN proteins in the overexpressed lines, and the polar region was indistinguishable.

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.

The local auxin gradient formed and maintained mainly by polar auxin transport is sufficient to determine the direction of an organ in which the PIN auxin efflux transporters have a decisive role in the auxin gradient and signaling.⁶ In this study, all possible PIN genes from the recent maize genome sequence database were identified and categorized based on amino acid similarity. Tissue-specific expression patterns indicate that they may have unique roles in different developmental stages; this may be particularly true of roots with high expression levels. Promoter motif analysis and expression in response to NAA, low phosphate and PEG treatment indicate that ZmPIN1a and ZmPIN1b may contribute more than ZmPIN1c and ZmPIN1d to the maize root response to abiotic stress. Analysis of the ZmPIN1a and ZmPIN1b transgenic lines indicates that they have different effects on root development and growth.

Funding Statement

This work was supported by the National Natural Science Foundation of China [31571674]; National Major Projects for Genetically Modified Organisms Breeding in China [2016ZX08003004-003].

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.