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Structure and expression of barley starch phosphorylase genes

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Abstract

The function of starch phosphorylase has long been debated on the regulation of starch metabolism during the growth and development of plants. In this study, we isolated starch phosphorylase genes (Pho1 and Pho2) from barley, characterized their gene and protein structures, predicated their promoter’s cis-elements and analyzed expression patterns. Multiple alignments of these genes showed that (1) both Pho1 and Pho2 genes possess 15 exons and 14 introns in all but three of the species analyzed, Aegilops tauschii (for Pho1 which contains 16 exons and 15 introns), potato (for Pho1b which contains 14 exons and 13 introns), and Triticum uraru (for Pho2 which contains 15 exons and 14 introns); (2) the exon–intron junctions of Pho1 and Pho2 flanking the ligand-binding sites are more conservative than the other regions. Analysis of protein sequences revealed that Pho1 and Pho2 were highly homologous except for two regions, the N terminal domain and the L78 insertion region. The results of real-time quantitative PCR (RT-qPCR) indicated that Pho2 is mainly expressed in germinating seeds, and the expression of Pho1 is similar to that of starch synthesis genes during seed development in barley. Microarray-based analysis indicated that the accumulation of Pho1 or Pho2 transcripts exhibited uniform pattern both in various tissues and various stages of seed development among species of barley, rice, and Arabidopsis. Pho1 of barley was significantly down-regulated under cold and drought treatments, and up-regulated under stem rust infection. Pho2 exhibited similar expression to Pho1 in barley. However, significant difference in expression was not detected for either Pho1 or Pho2 under any of the investigated abiotic stresses. In Arabidopsis, significant down-regulation was detected for Pho1 (PHS1) under abscisic acid (ABA) and for Pho2 (PHS2) under cold, salt, and ABA. Our results provide valuable information to genetically manipulate phosphorylase genes and to further elucidate their regulatory mechanism in the starch biosynthetic pathway.

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Abbreviations

Aa:

Acid residues

ABA:

Abscisic acid

AGP:

Adenosine 5′ diphosphate glucose pyrophosphorylase

BE:

Branch enzyme

CTAB:

Hexadecyltrimethylammonium bromide

DAF:

Days after flowering

dNTP:

Deoxynucleotide triphosphat

GAPDH:

Glycerinaldehyde-phosphate dehydrogenase

L78:

About 80-amino acid long insert region

NCBI:

National Center for Biotechnology Information

ORF:

Open reading frame

Pho:

Phosphorylase

Pho1 (PHS1):

Plastidial phosphorylase

Pho2 (PHS2):

Cytosolic phosphorylase

PLP:

Pyridoxal phosphate

RT-qPCR:

Real-time quantitative polymerase chain reaction

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (31000167 and 31230053) and the China Transgenic Research Program (2011ZX08002-001,004 and 005). We appreciate the referees for critical reading of the manuscript.

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Authors and Affiliations

  1. Triticeae Research Institute, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China

    Jian Ma, Qian-Tao Jiang, Xiao-Wei Zhang, Xiu-Jin Lan, Zhi-En Pu, Yu-Ming Wei & You-Liang Zheng

  2. CSIRO Plant Industry, 306 Carmody Road, St Lucia, QLD, 4067, Australia

    Jian Ma & Chunji Liu

  3. Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, T1J 4B1, Canada

    Zhen-Xiang Lu

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  1. Jian Ma
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Correspondence to Chunji Liu or You-Liang Zheng.

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Jian Ma and Qian-Tao Jiang authors contributed equally to this paper.

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Supplementary material 1 (DOC 152 kb)

425_2013_1953_MOESM2_ESM.tif

Supplementary Fig. S1 Structure and isolation of Pho1 and Pho2 genes. a Aegilops tauschii Pho1 gene structure and PCR primers for amplicaton of barley Pho1. b Aegilops tauschii Pho2 gene structure and PCR primers locations for barley Pho2. c PCR banding pattern of barley Pho1 and Pho2 genes. Boxes and thick lines present exons and introns, respectively. The positions of primers (see Table 1) are indicated by arrows. Triangles illustrate the approximate positons of the start of (ATG) and the stop (TGA) codons. 1, Pho1-F2/R2; 2, Pho1-F3/R3; 3, Pho1-F4/R4; 4, Pho1-F5/R5; 5, Pho1-F6/R6; 6, Pho2-F1v/R1v; 7, Pho2-F2/R2; 8, Pho2-F3/R3; 9, Pho2-F4/R4; 10, Pho2-F5/R5; 11, Pho2-F1v2/R3; 12, Pho2-F4/R5. M. DNA 1kb plus ladder (TIFF 3328 kb)

425_2013_1953_MOESM3_ESM.tif

Supplementary Fig. S2 Multiple alignments of Pho1 and Pho2 amino acid sequences among different species using CLC Main Workbench 6.7.2. Hv, barley; Ta, Triticum aestivum; At, Arabidopsis thaliana; St, Solanum tuberosum; Cr, Chlamydomonas reinhardtii; Ca, Caldilinea aerophila; Hs, Homo sapiens (muscle) (TIFF 192 kb)

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Ma, J., Jiang, QT., Zhang, XW. et al. Structure and expression of barley starch phosphorylase genes. Planta 238, 1081–1093 (2013). https://doi.org/10.1007/s00425-013-1953-6

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