Expression analysis of PIN family genes in Chinese hickory reveals their potential roles during grafting and salt stress

Abstract

Grafting is an effective way to improve Chinese hickory while salt stress has caused great damage to the Chinese hickory industry. Grafting and salt stress have been regarded as the main abiotic stress types for Chinese hickory. However, how Chinese hickory responds to grafting and salt stress is less studied. Auxin has been proved to play an essential role in the stress response through its re-distribution regulation mediated by polar auxin transporters, including PIN-formed (PIN) proteins. In this study, the PIN gene family in Chinese hickory (CcPINs) was identified and structurally characterized for the first time. The expression profiles of the genes in response to grafting and salt stress were determined. A total of 11 CcPINs with the open reading frames (ORFs) of 1,026-1,983 bp were identified. Transient transformation in tobacco leaves demonstrated that CcPIN1a, CcPIN3, and CcPIN4 were localized in the plasma membrane. There were varying phylogenetic relationships between CcPINs and homologous genes in different species, but the closest relationships were with those in Carya illinoinensis and Juglans regia. Conserved N- and C-terminal transmembrane regions as well as sites controlling the functions of CcPINs were detected in CcPINs. Five types of cis-acting elements, including hormone- and stress-responsive elements, were detected on the promoters of CcPINs. CcPINs exhibited different expression profiles in different tissues, indicating their varied roles during growth and development. The 11 CcPINs responded differently to grafting and salt stress treatment. CcPIN1a might be involved in the regulation of the grafting process, while CcPIN1a and CcPIN8a were related to the regulation of salt stress in Chinese hickory. Our results will lay the foundation for understanding the potential regulatory functions of CcPIN genes during grafting and under salt stress treatment in Chinese hickory.

Keywords: Carya cathayensis; PIN; auxin; grafting; salt; transport.

FIGURE 1

The subcellular localization of CcPIN1a, CcPIN3, and CcPIN4. The fluorescence images were captured in a dark field for green and red fluorescence, in a white field for the morphology of the cell, and in combination. GFP, green fluorescent protein fluorescence; RFP, red fluorescent protein fluorescence; Bright, bright field; Merged, GFP/RFP/bright field overlay. Bar = 20 μm.

FIGURE 2

Phylogenetic analysis of PIN proteins from 15 species. (A) The phylogenetic tree. (B) The number and distribution of PINs in different species. Kf, Klebsormidium flaccidium; Pp, Physcomitrella patens; Sm, Selaginella moellendorffii; Cf, Cystopteris fragilis; Pa, Picea abies; At, Arabidopsis thaliana; Pt, Populus trichocarpa; Am, Amborella trichopoda; Jr, Juglans regia; Ci, Carya illinoinensis; Cc, Carya cathayensis; Nc, Nymphaea tetragona; Os, Oryza sativa; Zm, Zea mays; Sb, Sorghum bicolor.

FIGURE 3

Multiple alignments of CcPINs by ClustalW. The possible functional sites are circled with red boxes.

FIGURE 4

Motif and gene structure analysis of CcPINs. (A) Phylogenetic relationships. (B) Motif distribution. (C) Exon-intron structures. (D) Motif information.

FIGURE 5

(A) QQProtein–protein interaction (PPI) network of PIN-formed (PINs) and (B) gene ontology (GO) annotation of proteins in the PPI.

FIGURE 6

Cis-acting elements on the promoters of CcPIN family genes. (A) The distribution of cis-acting elements. (B) The number of cis-acting elements.

FIGURE 7

Tissue-specific expression profiles of CcPIN genes. Total RNA was extracted from roots, stems, leaves, and shoots of 1-year-old Chinese hickory. The relative expression levels of each CcPIN gene in roots were standardized as one. Different letters above the columns denote a significant difference in different tissues (P < 0.05).

FIGURE 8

Relative RNA expression of CcPIN genes during the grafting of Chinese hickory. 0, 3, 7, and 14 d denote 0, 3, 7, and 14 days after grafting, respectively. The rootstocks collected at 0 day were regarded as the control sample. Different letters above the columns denote a significant difference in different treatments (P < 0.05).

FIGURE 9

Relative RNA expression of CcPIN genes under CK (H₂O) and salt stress treatments. 0, 1, 3, and 10 d represent 0, 1, 3, and 10 days after treatment, respectively. The sample from the CK group collected 0 days after treatment was regarded as the control sample. Different letters near the points indicate a significant difference in different treatments (P < 0.05).