Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain
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- Wiley, P.R., Tosi, P., Evrard, A. et al. Plant Mol Biol (2007) 64: 125. doi:10.1007/s11103-007-9139-x
The purolindolines are small cysteine-rich proteins which are present in the grain of wheat. They have a major impact on the utilisation of the grain as they are the major determinants of grain texture, which affects both milling and baking properties. Bread and durum wheats were transformed with constructs comprising the promoter regions of the Puroindoline a (Pina) and Puroindoline b (Pinb) genes fused to the uidA (GUS) reporter gene. Nine lines showing 3:1 segregation for the transgene and comprising all transgene/species combinations were selected for detailed analysis of transgene expression during grain development. This showed that transgene expression occurred only in the starchy endosperm cells and was not observed in any other seed or vegetative tissues. The location of the puroindoline proteins in these cells was confirmed by tissue printing of developing grain, using a highly specific monoclonal antibody for detection and an antibody to the aleurone-localised 8S globulin as a control. This provides clear evidence that puroindolines are only synthesised and accumulated in the starchy endosperm cells of the wheat grain.
KeywordsGrain textureLocationPuroindolinesTransgenic wheat
Bovine serum albumin
Electrospray ionisation–mass spectrometry
Nitro blue tetrazolium 5-bromo-4-chloro-3-indoyl-phosphate
Phosphate buffered saline
Polymerase chain reaction
Sodium dodecyl sulphate
Puroindolines are small (Mr ≈ 15,000), basic cysteine-rich proteins, which are present in grain of wheat and related cereals (reviewed by Morris 2002). They are related in amino acid sequence and three-dimensional structure to the non-specific lipid transfer proteins, α-amylase inhibitors and other members of the prolamin superfamily (reviewed by Shewry et al. 2004), but differ from all of these proteins in the presence of a “tryptophan motif” which comprises five tryptophan residues in a seven residue sequence in puroindoline (Pin) a and three tryptophan residues in a six residue sequence in Pin b. They are encoded by genes located at the Ha (Hardness) locus on chromosome 5D of hexaploid wheat and their association with grain texture has resulted in considerable interest over the past two decades.
The first evidence that proteins associated with the starch granule surface may play a role in determining grain texture was the report by Greenwell and Schofield (1986) that a protein “band” of Mr ≈ 15,000 was more abundant on the surface of water-washed starch granules of soft wheats than hard wheats. They coined the term “friabilin” for this band to reflect its putative role in determining grain friability and it was subsequently shown to comprise several proteins with the major components being Pins a and b (Blochet et al. 1993; Oda and Schofield 1997).
More recent work, notably by Morris, Giroux and co-workers, has clearly demonstrated that grain hardness in bread wheat can result from two types of mutation in Pin genes, either null mutations in Pina or Pinb or base changes which lead to amino acid substitution in the Pin b protein (Giroux and Morris 1997, 1998; Lillemo and Morris 2000; Giroux et al. 2000; Morris 2002). There is also evidence that Pin proteins are inhibitory to plant pathogenic fungi when tested in vitro (Dubriel et al. 1998; Capparelli et al. 2005) or expressed in transgenic plants (Krishnamurthy et al. 2001; Giroux et al. 2003).
Despite the wealth of information on the sequences of puroindoline proteins and their association with grain texture, a number of fundamental questions remain to be answered on their precise location in the grain and mechanism of action.
Puroindolines appear to be typical products of the secretory pathway, being synthesised with an N-terminal signal peptide, which directs the nascent polypeptide into the lumen of the endoplasmic reticulum (ER). Since they lack recognised ER retention sequences it is assumed that they are transported via the Golgi apparatus to the vacuoles, which are also the site of deposition of the storage proteins. This is supported by the presence of C-terminal peptides, which could be removed in the vacuole by processing proteases (Gautier et al. 1994). The protein bodies fuse to form a continuous protein matrix in the mature wheat grain and this matrix is clearly labelled with anti-Pin-a antibodies (Dubreil et al. 1998). Hence, it must be assumed that their binding to starch granules occurs by diffusion during this late maturation stage and that the proteins are not specifically transported to the amyloplast during earlier stages of development.
Similarly, the tissue location of Pins remains unclear. Their association with the starch granule surface in the mature grain and their relationship to grain texture imply that substantial amounts of Pins are present in the central cells of the starchy endosperm, as starch granules are less abundant in the protein-rich sub-aleurone cells (Kent 1966; Evers 1970) and absent from the aleurone layer (Heard et al. 2001). Previously published immunochemical studies have confirmed that both Pin a and Pin b are indeed located in the starchy endosperm cells (Dubreil et al. 1998).
However, the same study (Dubriel et al. 1998) showed that they were also present in the aleurone and a more recent report showed that they are concentrated in the aleurone (Capparelli et al. 2005). Analysis of Pin promoters in transgenic rice plants also showed differences in the expression patterns conferred by the Pin a and Pin b promoters (Digeon et al. 1999; Evrard et al. 2007). Thus, whereas the Pinb gene promoter drove expression of the GUS reporter gene in the starchy endosperm, aleurone and seed epidermis of transgenic rice plants, the Pina gene promoter also drove expression in the embryo and, to a lower extent, in non-seed tissues. Furthermore, expression of the Pina promoter was induced by wounding or infection with a fungal pathogen (Digeon et al. 1999; Evrard et al. 2007). Finally, supporting evidence for expression of Pins in the aleurone comes from studies of the expression of the related hordoindoline proteins in developing barley grains, using in situ hybridisation to locate the corresponding mRNA (Lee et al. 2006). However, a similar in situ study of developing wheat demonstrated that Pin b was expressed in the starchy endosperm and pericarp but not the aleurone cells in a 9-day-old developing grain of wheat (Drea et al. 2005, see also online database at http://www.bioinf.scri.sari.ac.uk/cgi-bin/insitu/home).
We report here two lines of investigation which also clearly demonstrate that Pin genes are expressed in the starchy endosperm cells but not the aleurone layer of wheat: the expression of Pin promoter::uidA reporter constructs (encoding the β-glucuronidase (GUS) enzyme) in developing grain of transgenic bread and durum wheats and immunolocalisation in bread wheat with a highly specific monoclonal antibody.
Materials and methods
The Pinb::uidA construct was as described by Digeon et al. (1999), containing 1062 bp of sequence from the Pinb gene promoter of cv. Andain. For the Pina::uidA construct 1214 bp of sequence from the Pina gene promoter of bread wheat (Triticum aestivium L.) cv. Chinese Spring (NCBI accession number CS131558) were amplified with forward 5′-GTCGACTGATCTGCATGACTGTGTGC-3′ and reverse 5′-CCCGGGGTTGTCAGTGTGTTTTGG-3′ primers and cloned into SalI/XmaI restricted pSPORT1-GUS upstream of the bacterial uidA gene.
The pAHC20 selectable marker construct (Christensen and Quail 1996), which contains the bar gene encoding phosphinothricin acetyltransferase (PAT), was used to confer resistance to the herbicide phosphinothricin (PPT).
Immature scutella of bread wheat cv. Cadenza and durum wheat (Triticum durum Desf.) cv. Ofanto were used as targets for transformation by particle bombardment using the protocol of Sparks and Jones (2004). The following modifications were made: bombardments were carried out at a pressure of 900 psi; induction medium for Ofanto contained 3% (w/v) maltose instead of 9% (w/v) sucrose; first-round selection was conducted on regeneration medium containing 2 mg/l PPT, subsequent rounds of selection used 3 mg/l PPT and 4 mg/l PPT for Ofanto and Cadenza, respectively; all media contained 0.1% (v/v) Plant Preservative Mixture (Plant Cell Technology Inc., Washington DC, USA). The presence of the transgenes in putative transgenic plants was confirmed by PCR.
Histochemical GUS assay
Transverse sections of 300 μm and 600 μm thickness of developing wheat caryopses from 8 days after anthesis were cut in 0.1 M sodium phosphate buffer using a Leica VT1000S vibrating blade microtome (Leica Microsystems UK Ltd., Milton Keynes, UK). Expression of the uidA reporter gene was analysed by incubating the sections in X-Gluc (5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid) buffer containing 1 mM X-Gluc, 100 mM sodium phosphate buffer, pH 7, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide and 0.1% (v/v) Triton X-100 for 2 h at 37°C. Staining was visually assessed using a Zeiss Axiophot stereomicroscope (Carl Zeiss Ltd., Welwyn Garden City, UK) for the 300 μm sections or a Leica MZ8 stereomicroscope (Leica Microsystems UK Ltd.) for the 600 μm sections. Sections could not be cut from caryopses before 8 days after anthesis due to the milky nature of the tissue but analyses of whole grain or hand cut segments showed no GUS activity.
Cryostat tissue printing
The method was based on Conley and Hanson (1997). Cadenza seeds were collected at 28 dpa and submerged in a small dish of cryostat embedding medium (Jung Tissue Freezing Medium, Leica) on cryostat holders that were then quickly frozen in liquid nitrogen. Frozen specimens were then transferred to a cryostat microtome (Leica CM 1850) where sectioning was carried out at a temperature of −17°C. Serial sections 20 μm thick were arranged on the knife, to be collected by gentle pressure on nitrocellulose coated slides (Oncyte Film-slides, Grace Bio-Labs Inc.).
Whole mature seeds were imbibed by placing them onto moistened filter paper in Petri dishes sealed with Parafilm for 40 h at 4°C. Excess moisture was removed with tissue paper and the seeds cut in half across the centre with a double-sided razor blade using a sliding motion to obtain a pure cut. The cut surfaces were then pressed for approximately 10 s onto nitrocellulose-coated slides.
Nitrocellulose coated slides with tissue prints were soaked in washing buffer (PBS, pH 7, 0.3% (v/v) Tween 20) twice for 10 min each, with shaking, to release the bulk of the attached tissues. The slides were then soaked for 40 min in blocking buffer (5% (w/v) Marvel skimmed milk in washing buffer). At this point any tissue remnants adhering to the membranes could be wiped off with a paper tissue.
Slides were rinsed in washing buffer for 1 min and then incubated twice for 60 min and 30 min in primary antibody solution prepared in 0.5% (w/v) bovine serum albumin (BSA) in washing buffer. Dilutions of 1:2500 were used for both 8S globulin polyclonal antibody (Yupsanis et al. 1990) and Durotest monoclonal antibody (R-Biopharm Rhône Ltd.). Slides were rinsed with washing buffer four times for 8 min each and then incubated for 2 h in the secondary antibodies. A dilution of 1:6250 was used for both the anti-rabbit alkaline phosphatase conjugate and the anti-mouse alkaline phosphatase conjugate antibodies.
Blots were then rinsed for 10 min in washing buffer, twice for 10 min in washing buffer plus 0.05% (w/v) SDS and then once again in washing buffer. They were then rinsed briefly in distilled water and incubated in alkaline phosphatase buffer (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 5 mM MgCl2) for 10 min before addition of alkaline phosphatase substrates. Substrates were applied to tissue prints until the coloured reaction product could be seen distinctly, usually 3–4 min for the 8S globulin antibody and 8–10 min for the Durotest antibody. The tissue prints were also stained with Ponceau S (Sigma Diagnostics P7767) for total protein after immunolabelling with the Durotest antibody. Tissue prints were examined in a Leica MZ8 stereomicroscope.
Electrophoresis and western blotting
Total protein extracts from wholemeal flours of cvs. Cadenza, Ofanto, Chablis, Riband and transgenic lines of Ofanto expressing Pina and Pinb were prepared by adding 1 ml of extraction buffer (2 M thiourea, 7 M urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer pH 6–11) to 30 mg of flour. Samples were left at room temperature for 90 min before the addition of an equal volume of 2× Laemmli buffer to each sample. Eight μl of each extract were loaded on a pre-cast Nu-PAGE 4–12% (w/v) Bis-Tris gradient gel (Invitrogen) and run at 200 V. Separated proteins were blotted on nitrocellulose membranes using a semidry apparatus (Bio-Rad) following the manufacturer’s instructions. Membranes were probed with the Durotest antibody (R-Biopharm Rhône) using a peroxidase conjugated secondary antibody (Sigma) and the Super Signal West Pico Chemiluminescent substrate (Pierce) following the manufacturer’s protocols.
Two dimensional gel electrophoresis and mass spectrometry
Protein fractions enriched in puroindolines were extracted as described in Branlard et al. (2003). The dry pellet was dissolved in rehydration buffer (2 M thiourea, 7 M urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer pH 6–11) and incubated at room temperature for 2 h. An aliquot was taken (equivalent to ∼150 μg protein) and made up to 250 μl with rehydration buffer. Isoelectric focussing (IEF) was performed on 13 cm Immobiline DryStrips pH 6–11. Rehydration of the DryStrips with the protein extract was performed for 12 h, as part of the IEF programme on an IPGphor (GE Healthcare). Isoelectric focussing parameters were as follows: 500 V 1 h; 1000 V 1 h; 8000 V 4.25 h or until 35,500 volt hours were attained. After IEF, DryStrips were either frozen at −80°C or incubated in equilibration buffer (50 mM Tris/HCl pH 8.8, 6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS plus a few grains of bromophenol blue) for 15 min. (N.B. DTT was omitted throughout, as the Durotest antibody does not recognise the reduced protein (Greenwell 1992)). After incubation, DryStrips were applied to the top of 15% Tris/glycine Laemmli gels. Following running of the second dimension, gels were either blotted onto Hybond C+ (Amersham), as described above, and probed with the Durotest antibody or fixed and silver stained. The silver stain method used (Yan et al. 2000) was compatible with subsequent mass spectrometry.
Following silver staining the protein spots in an equivalent position to those identified by the Durotest antibody were excised and destained using Farmer’s reducing agent (20% (w/v) sodium thiosulphate; 1% (w/v) potassium ferricyanide mixed 1:1:1 with water).
In-gel digests were then carried out as described by Jensen et al. (1999) using trypsin. Following overnight incubation peptides were isolated as in Jensen et al. (1999) and dried down by speed-vac. Peptides were concentrated and desalted using C18 Zip-Tips (Millipore) using the method of Kristensen et al. (2000). Peptides were then loaded into nanoflowtips (Waters) using gel loader tips (Eppendorf) and analysed by mass spectrometry.
Electrospray ionisation mass spectrometry (ESI-MS) was performed on a quadrupole time-of-flight (Q-Tof) I mass spectrometer (Micromass, Manchester, UK) equipped with a z-spray ion source. Instrument operation, data acquisition and analysis were performed using MassLynx/Biolynx 4.0 software. The sample cone voltage and collision energy were optimised for each sample. The Microchannel plate (MCP) detector voltage was set at 2800 V. Scanning was performed from m/z 100–3500.
Expression of promoter::uidA constructs
Durum wheat lacks the Pina and Pinb genes due to deletions within the Ha (Hardness) loci on the A and B genomes (Chantret et al. 2005). Hence it provides an ideal background to explore the expression patterns of Pin transgenes. A series of experiments was therefore carried out to transform both durum wheat cv. Ofanto and the hard bread wheat cv. Cadenza with constructs containing the uidA reporter gene (encoding the β-glucuronidase enzyme, or GUS) under control of the Pina or Pinb gene promoters. These constructs were called Pina::uidA and Pinb::uidA, respectively, and the uidA reporter gene allows promoter activity to be located in tissue sections using a simple assay in which the GUS enzyme catalyses the conversion of X-Gluc to a colourless intermediate which undergoes oxidative dimerisation to give an insoluble indigo-coloured product, 5,5′-dibromo-4,4′-dichloro-indigo (diX-indigo).
Segregation ratios and expression patterns of T0 transgenic lines expressing the GUS reporter gene
Segregation in T1
No. of lines
Ofanto Pin a
Ofanto Pin b
Cadenza Pin a
Cadenza Pin b
Cadenza Pin a
Ofanto Pin a
Cadenza Pin b
Ofanto Pin a
Ofanto Pin b
Nine lines which showed 3:1 segregation ratios and comprised all background/transgene combinations were selected for detailed analysis (Table 2), using histochemical analysis of homozygous or hemizygous T3 seed with null segregant sibling lines as controls.
Plants were grown in containment glasshouses, spaced to avoid cross-pollination, and developing heads tagged at anthesis. Developing grain were then harvested from the central part of the head at times between one and 55 days after anthesis.
Characteristics of transgenic lines and seeds selected for expression analysis
The variation in expression levels and the differences in the rates of seed development in the two species (bread and durum wheats) made it difficult to compare the precise times of initiation of GUS expression in the various lines. However, in general, initiation occurred at about 10–12 days after anthesis, corresponding broadly to the initiation of starch and gluten protein synthesis, with no obvious differences between the promoter constructs or species. No expression was detected before 9 days in any of the lines.
Three lines out of the nine did show a slightly different expression pattern in that significantly higher expression was observed in the starchy endosperm cells adjacent to the modified aleurone layer in the groove of the grain. These were CadA2 (Pina::uidA) in Cadenza, CadB4 (Pinb::uidA) in Cadenza and OfA6 (Pina::uidA) in Ofanto (illustrated in Fig. 2E, F). Thus there was no clear association between this variant pattern and either the species or promoter construct.
Finally, there was no evidence of expression as a result of wounding or damage of the tissues.
Immunochemical localisation of Pins
Characterisation of the monoclonal antibody
The Durotest monoclonal antibody was raised to a “friabilin” fraction from bread wheat (Greenwell et al. 1992). It is highly specific for puroindolines and hence is marketed in a test kit format for detecting the adulteration of pasta wheat semolina with bread wheat flour. Although the epitope recognised by the Durotest antibody has not been mapped, it is probably “conformational” as the antibody does not react with Pins after reduction of disulphide bonds or after denaturation during fixation for immunocytochemical analysis of tissue sections (Greenwell (1992) and authors’ unpublished results).
Two dimensional analysis of fractions from Cadenza (hard) and Riband (soft) (Fig. 5C, D) showed binding of the antibody to one or two spots, respectively (Fig. 5E, F). Q-Tof mass spectroscopy confirmed the identity of the single immunoreactive spot in Cadenza as Pin a. No peptide sequences corresponding to Pin b were identified in the tryptic digests which suggests that Cadenza is null for Pin b. This conclusion is supported by capillary electrophoresis, which failed to show appreciable amounts of Pin b in extracts of mature grain (authors’ unpublished results with Dr. D. Bhandari, CCFRA, UK). In contrast, spot 2 from Riband gave sequences diagnostic only for Pin a while spot 1 gave sequences diagnostic for Pin a and Pin b. The diagnostic sequences used for these identifications were IQGSIQGLDGGIFGFQR and DVAGGGGGAQ for Pin a and GGFLGIWR for Pin b.
These results therefore confirm that the Durotest antibody is specific for the Pin a and Pin b proteins.
Because the Durotest monoclonal antibody does not recognise denatured Pins it could not be used for conventional immunolocalisation using fixed sections. A tissue printing method was therefore used with an antibody to the aleurone-located 8S storage globulin (Kriz 1999) as a control. This antibody was raised against the homologous 8S globulin of oats but is also highly specific for 8S globulins from wheat, barley and rye (Burgess and Shewry 1986; Yupsanis et al. 1990).
We generated 35 independent transgenic lines of bread and durum wheat, which were PCR positive for the uidA gene, of which 23 showed expression of the GUS enzyme. In 21 of these expression was restricted to the endosperm while two lines showed expression in the seed coat only. The latter also showed non-Mendelian segregation ratios and it was assumed that their expression pattern resulted from rearrangement/insertion site.
Nine of the lines were studied in detail by microscopic examination of sections of developing grains and other tissues following staining in X-Gluc buffer. In all cases, expression was only observed in the starchy endosperm cells, but with less expression in the sub-aleurone cells which are rich in protein but contain less starch. It should also be noted that three of the nine lines also showed higher expression in the starchy endosperm cells immediately adjacent to the modified aleurone cells in the groove. The aleurone and sub-aleurone cells in this region function as transfer cells for nutrients and signals from the plant and the expression of Pins could relate to a role in defence against infection during development. This is consistent with studies of maize in which the basal endosperm transfer cells express a series of four genes (betl1 to betl4), which encode small S-rich proteins with anti-fungal properties (Thompson et al. 2001; Serna et al. 2001). Furthermore, Pins have also been shown to confer resistance to fungal infection when expressed transgenically in rice, apple and wheat (Krishamurthy et al. 2001; Gerhardt et al. 2002; Giroux et al. 2003; Faize et al. 2004).
We have also used a highly specific monoclonal antibody to confirm that Pins are located in starchy endosperm cells but not in aleurone cells by tissue printing using an antibody to the aleurone-located 8S globulin as a control. However, this method did not confirm the difference in distribution between the central and outer layers of the starchy endosperm shown by the promoter-GUS assays. This difference may relate to the fact that the immunochemical analysis measures the total accumulated Pin proteins while the GUS expression assay measures the transient activity of the enzyme encoded by the uidA gene. Hence, the latter is more accurate for measuring temporal and spatial differences in gene expression.
Although our data on the absence of Pin gene expression from aleurone cells are convincing they conflict with several other reports in the literature.
The difference between our observations with Pin-GUS constructs with those reported for the same Pinb::uidA (Digeon et al. 1999) construct and for related Pina::uidA and Pinb::uidA (Evrard et al. 2007) constructs in rice may be related to loss of specificity of the promoter in other cereals. Although the Pin promoters have not been evaluated in any species other than wheat and rice, we have previously reported evidence that another seed-specific promoter of wheat, the Glu-1D-1 promoter encoding the high molecular weight subunit of glutenin 1Dx5, is expressed in pollen when used to drive transgene expression in barley (Zhang et al. 2001). The 1Dx5 transgene is also inefficiently transmitted through the pollen in transgenic maize and one explanation could be that the gene is expressed in the pollen resulting in mortality (Sangtong et al. 2002). Hence we do not consider that it is possible to draw valid conclusions about gene expression patterns based on transgene expression in other species, even between related cereals such as barley, wheat, maize and rice.
However, we have no explanation for the different results reported by Dubreil et al. (1998) and Capparelli et al. (2005) who used polyclonal antibodies to immunolocalise rather than the monoclonal antibody described here.
Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. We are grateful to Cristina Sanches-Gritsch and Raffaella Carzaniga (Rothamsted Research) for discussions and advice on microscopy and to Dr. Marie-Françoise Gautier (INRA, Montpellier) for providing the Pinb::uidA reporter construct and for further advice and discussion.