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Alfred2014 PDF
Alfred2014 PDF
Faculty of Science, Engineering and Technology, Swinburne University of Technology, PO Box 218, Melbourne, Victoria 3122, Australia
Department of Environment and Primary Industries, Horsham, Victoria 3400, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 March 2014
Received in revised form
29 May 2014
Accepted 5 June 2014
Available online 5 July 2014
The puroindoline genes are causatively associated with wheat grain hardness, a commercially signicant
property. The proteins puroindoline (PIN) A and B are both required in their wild-type (WT) to impart
soft grain texture, and absence of/mutations in either/both PIN(s) results in hard wheat. However, there
is no biochemical clarity yet that explains this interdependence. This work critically analyses the roles of
the tryptophan-rich domain (TRD), the little-known hydrophobic domain (HD), and certain other residues, in the physical associations of PINs. Site-directed mutagenesis-PCR was used to delete the TRD or
HD and introduce an Arg39Gly substitution in PINA. The PINB-D1c mutant (Leu60Pro) was also investigated. The yeast two-hybrid system was used to assess the proteineprotein interactions (PPI) of proteins. The TRD deletion or Arg39Gly substitution in PINA did not adversely affect its PPI, while deletion of
HD resulted in a signicant reduction. No effect on PPI was observed for Leu60Pro PINB. The results of
this expression system strongly suggest that the HD is essential (but not sufcient) in higher-order associations of PINs. We propose a two-event model that explains the co-operative action of the PINs and
why mutations outside the TRD may alter grain texture.
2014 Elsevier Ltd. All rights reserved.
Keywords:
Grain texture
Proteineprotein interaction
Puroindolines
Tryptophan
1. Introduction
The two puroindoline genes of common wheat have been causatively associated with grain texture, a commercially highly signicant property. The putative full-length PINA and PINB proteins,
encoded by the wild-type (WT) alleles Pina-D1a and Pinb-D1a, are
148 amino acids long helicoid proteins that exhibit a unique
tryptophan-rich domain (TRD) comprised of 5 Trp residues in PINA
but only 3 in PINB (Gautier et al., 1994). The PINA and PINB are lipidbinding proteins and both must be present together in their wildtype forms to impart soft grain texture, and the deletions of, or
diverse mutations in, either or both Pin gene(s) result in hard
common wheats (reviewed in Bhave and Morris, 2008a; NadolskaOrczyk et al., 2009; Pauly et al., 2013). The functionality of PINs has
been associated with their lipid-binding properties, the TRD being
Abbreviations: Gsp-1, grain softness protein-1 gene; GSP, grain softness protein;
HD, hydrophobic domain; Pin, puroindoline gene; PIN, puroindoline protein; TRD,
tryptophan rich domain.
* Corresponding author. Faculty of Science, Engineering and Technology, Swinburne University of Technology, PO Box 218, John Street, Hawthorn, Victoria 3122,
Australia. Tel.: 61 3 9214 5759; fax: 61 3 9819 0834.
E-mail address: mbhave@swin.edu.au (M. Bhave).
http://dx.doi.org/10.1016/j.jcs.2014.06.001
0733-5210/ 2014 Elsevier Ltd. All rights reserved.
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Fig. 1. Design of primers for siteedirected mutagenesis in the putative PINA protein. A. Section of PINA containing the TRD (in grey), showing the alignment of the forward
(PINAR39G-F) and reverse (PINAR39G-R) primers. The point mutation to be introduced is underlined. B. Section of PINA containing the TRD, showing the alignment of the forward
(PINADTRD-F) and reverse (PINADTRD-R) primers. PINADTRD-F has 9 nucleotides directly upstream of the 50 end of the TRD, 9 nucleotides directly downstream of the 30 end of the
TRD, and a further 11 nucleotide 30 region (grey). Likewise, PINADTRD-R has 9 nucleotides directly upstream of the 50 end of the TRD, 9 nucleotides directly downstream of the 30
end of the TRD, and a further 11 nucleotide 30 region (grey). C. Section of PINA containing the HD, showing the alignment of the forward (PINADHD-F) and reverse (PINADHD-R)
primers. PINADHD-F has 9 nucleotides directly upstream of the 5 end of the HD, 9 nucleotides directly downstream of the 30 end of the HD, and a further 11 nucleotide 30 region
(grey). Likewise, PINADHD-R has 9 nucleotides directly upstream of the 50 end of the HD, 9 nucleotides directly downstream of the 30 end of the HD, and a further 11 nucleotide 30
region (grey).
Fig. 2. Deduced tertiary structure of PINA using SWISSeMODEL. The sequence of the
putative mature PINA (Gautier et al., 1994) (Genbank ABD72477) was used as template
for homology modelling of its tertiary structure using SWISS-MODEL (http://
swissmodel.expasy.org/). The TRD (Phe34 to Gly46) is located as an extended loop
between a-helix 1 and 2 and the putative hydrophobic domain (HD) (Ile75 to Phe85) is
located as a smaller loop between a-helix 3 and 4.
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PINA-bait (Fig. S7A, spots 32e36), and stronger growth on YSDWLAH3AT than PINA-baits (Fig. S7B, spots 8, 9, 10). With
PINArTRD as prey, strong growth was noted with PINA, GSP-1 and
PINAR39G (Fig. S7A, spots 12, 24, 30), weak with PINB (spot 18), and
none on YSD-WLAH3AT. Interestingly, one control, the
PINADTRD/ev, exhibited strong growth (Fig. S7A, spot 21) and
enzyme assay results (10.9 MU) (Table S2), suggesting an interaction of the mutant fusion protein with an unknown protein encoded by pGADT7. This value was included in the correction average
applied to all relevant samples. No false-positive growth was seen
for ev/PINADTRD or any other controls. The enzyme activity values
conrmed the stronger induction by PINADTRD baits paired with
PINA (16.25 MU; P 0.005), PINB (20.43 MU; P 0.003), GSP-1
(8.18 MU; P 0.001) and self (12.46 MU; P 0.001), compared to
PINA/PINA and PINA/PINB (Fig. 3, Table S2). As prey, the value for
PINB/PINADTRD (1.82 MU) was low, similar to PINB/PINA (1.59 MU;
P 0.125), and PINA/PINADTRD (4.84 MU) was comparable to PINA/
PINA (5.13 MU; P 0.072). Thus PINADTRD as bait seemed to have
retained the strong activity of WT PINA baits, and stronger reactions with PINB-prey than with itself. Due to its weak interactions, GSP-1 was not used as an interacting partner in further
work.
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Fig. 5. begalactosidase reporter activity of the interactions of PINBeD1c with wildetype PINA and PINB. The b-galactosidase activity, presented in MU, is the mean of two
independent experiments, each reaction in an experiment being conducted in triplicate. The values shown have been corrected for the relevant empty vector control
activities.
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Fig. 6. Suggested model for the proteineprotein and proteinelipid interactions of PINs. A. WT-PINA and WT-PINB interact with each other and stabilise the interactions of each
other with lipids such as remnants of the amyloplast membrane at the surface of the starch granules. B. The Leu60Pro substitution in PINB alters the way WT-PINA and PINBLeu60Pro interact, impeding the TRD of both proteins from lipid interactions.
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