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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

The Role of Puroindoline A and B Individually and in Combination on Grain Hardness and Starch Association

Dep. of Plant Sciences and Plant Pathology, Montana State Univ., Bozeman, MT, 59717-3150

^* Corresponding author (mgiroux{at}montana.edu)

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Endosperm texture in wheat (Triticum aestivum L.) is an important criterion affecting end-product quality. Grain hardness is controlled by the Hardness (Ha) locus which contains the puroindoline a (Pina) and puroindoline b (Pinb) genes. Hard wheats possess mutations in Pina or Pinb while soft wheats possess the Pina-D1a and Pinb-D1a alleles. Here, we determined the role of PINA and PINB individually and in combination on grain hardness and association to starch. Pina-D1a or Pinb-D1a overexpressing transgenic lines were crossed to PINA or PINB null hard wheats. The crosses segregated for the transgene and the Ha locus. Random lines from each genotypic class were evaluated in replicated trials over 2 yr. Classes containing only native levels of PINA or PINB were hard textured. Homozygous classes with transgenic addition of PINA to the PINA⁺/PINB null Ha locus or addition of PINB to the PINA⁺/PINB null Ha locus were intermediate in texture with grain hardness values of 43.5 and 45.5, respectively. Soft endosperm texture was only obtained when both PINA and PINB were present with the softest grain observed with addition of PINB to the PINA⁺/PINB null Ha locus. Association of high amounts of either puroindoline to starch required both PINA and PINB. The results indicate that PINA or PINB can act alone leading to intermediate-textured grain or can function together to give a soft grain texture.

Abbreviations: CR, Canadian Red • Ha, Hardness • McN, McNeal • PINA, puroindoline a protein • Pina, puroindoline a gene • PINB, puroindoline b protein • Pinb, puroindoline b gene • SKCS, single kernel characterization system • TX-114, Triton-X114

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
WHEAT is classified and traded as either hard or soft based on endosperm texture. Grain hardness in wheat is one of the most important characteristics affecting milling, baking, and end-use qualities. Hard-textured wheat yields larger flour particles that have more starch damage and absorb more water than soft-textured wheat flour (Symes, 1965, 1969). Soft-textured wheats are used for cakes, cookies, and pastries whereas hard wheats are generally used to make bread (reviewed by Morris and Rose, 1997).

Wheat grain hardness is a simply inherited trait controlled by Ha (Symes, 1965; Baker, 1977) and perhaps one or more minor genes (Anjum and Walker, 1991). The major gene (Ha) has been mapped to the short arm of chromosome 5D (5DS) (Mattern et al., 1973; Law et al., 1978). Soft wheats possess the dominant or wild-type form (Ha) while hard wheats have the recessive or mutated form (ha) (Symes, 1965; Baker, 1977; Law et al., 1978). A hard vs. soft wheat marker protein termed friabilin was reported by Greenwell and Schofield (1986). Friabilin is a 15-kDa protein complex abundantly present on the surface of water-washed starch from soft wheats and scarce on hard wheat starch (Greenwell and Schofield, 1986). The physical interaction of friabilin with wheat starch is poorly understood, however, it has been suggested that friabilin regulates the degree of adhesion between starch granules and the protein matrix (Pomeranz and Williams, 1990; Beecher et al., 2002; Hogg et al., 2004). N-terminal sequencing has shown that friabilin is composed primarily of two proteins, PINA and PINB (Blochet et al., 1993; Gautier et al., 1994; Rahman et al., 1994). The tightly linked genes Pina and Pinb code for the PINA and PINB proteins respectively, which together function as the Ha locus (Sourdille et al., 1996; Giroux and Morris, 1997, 1998). The puroindoline (PIN) active site may relate to their unique tryptophan-rich hydrophobic domain that is thought to be involved in the binding of lipids (Gautier et al., 1994; Marion et al., 1994). Molecular studies have revealed that variation in the coding sequence of Pina or Pinb is associated with the hard grain phenotype (Giroux and Morris, 1997, 1998). All soft wheats surveyed to date possess the Pina-D1a and Pinb-D1a alleles (Giroux and Morris, 1998; Lillemo and Morris, 2000; Morris et al., 2001). To date, numerous Pin gene mutations have been characterized among hard wheats, with a null mutation in Pina (Pina-D1b) or a point mutation in Pinb (Pinb-D1b) being the two most common Ha mutations (Giroux and Morris, 1997, 1998; Morris et al., 2001; Pan et al., 2004; reviewed in Morris, 2002).

To validate the cause–effect relationship between puroindolines and grain hardness, Krishnamurthy and Giroux (2001) transformed rice (Oryza sativa L.), which lacks Pin homologs, with the soft type Pina-D1a and Pinb-D1a coding sequences under control of the maize (Zea mays L.) ubiquitin promoter. A decrease in rice kernel hardness was observed as well as reduced particle size and starch damage in whole seed meals after milling. In addition, Beecher et al. (2002) demonstrated that expression of Pina-D1a in the hard wheat variety ‘Hi-Line’ complemented the native Pinb-D1b mutation present in Hi-Line and restored a soft grain phenotype. In a recent study, Martin et al. (2006) demonstrated that complementation of the Pina-D1b allele in hard wheats with the wild-type Pina-D1a sequence also restored a soft phenotype. Further studies of the transgenic lines created in Hi-Line have shown that soft endosperm texture correlates with friabilin abundance and the presence of both PINA and PINB (Hogg et al., 2004). Hogg et al. (2004), however, did not demonstrate the individual role of PINA and PINB on grain hardness or starch binding since all genotypes studied contained a functional PINA. Further, the presence of PINB-D1B in Hi-Line with overexpressed PINA or PINB could have confounded the results of Hogg et al. (2004) preventing identification of the individual role of PINA or PINB on grain hardness and association to starch. In a study by Swan et al. (2006) a subset of the transgenic isolines created by Hogg et al. (2004) were crossed to the soft wheat ‘Heron’. They demonstrated that both PINs limit grain softness in soft wheat but PINB is more limiting to PINA starch association and grain softness in soft wheats than is PINA. This study, however, was limited by the fact that genotypes with added PINA or PINB were in the soft wheat Heron background and hence could not isolate the individual role of Pina or Pinb in grain hardness and association to starch.

To overcome the limitations inherent in the studies of Hogg et al. (2004) and Swan et al. (2006), a subset of the Pin overexpressing transgenic lines of Hogg et al. (2004) were crossed with PINA or PINB null genotypes. This allowed us to determine the action of PINA and PINB on wheat grain hardness in the presence or absence of the other protein. To accomplish this, transgenic overexpressing Pina-D1a or Pinb-D1a lines created in Hi-Line were crossed to hard wheats that were either PINA or PINB null. The progeny segregated for the presence or absence of the transgene and the Ha locus. We identified random progeny from the four homozygous classes per cross which were then evaluated for grain hardness, kernel weight, and protein content over two environments.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Genetic Materials
Two spring wheat cultivars, Canadian Red (CR; hard white) (Clark et al., 1926) and McNeal (McN; hard red) (Lanning et al., 1995) were crossed to a subset of the transgenic lines described in Hogg et al. (2004), which were created using the hard red spring wheat cultivar Hi-Line (Lanning et al., 1992). Four transgenic parents were used. Two were transgenic isolines created using the Pina-D1a coding sequence (HGA3, HGA1) while the other two were created using the Pinb-D1a coding sequence (HGB5, HGB12). Hi-Line has the soft type Pina-D1a and the mutant Pinb-D1b allele which contains a single point mutation in Pinb resulting in a glycine to serine substitution at the 46th residue of the peptide (Giroux and Morris, 1997; Beecher et al., 2002). Canadian Red has the soft type Pina-D1a allele and a null Pinb-D1e allele (Morris et al., 2001). Pinb-D1e contains a single point mutation (TGG to a TGA) leading to a change in residue Trp-39 to a stop codon. McNeal carries a mutant Pina allele (Pina-D1b) which is an apparent deletion of the Pina coding sequence and the wild-type Pinb-D1a allele (Giroux and Morris, 1998).

The expression of the Pina and Pinb transgenes is under control of the GluDy10 seed-specific high molecular weight glutenin promoter (Blechl and Anderson, 1996). These transgenic plants also express the Bar gene (De Block et al., 1987) that confers resistance to bialaphos {4-[hydroxy(methyl)phosphinoyl]-L-homoalanyl-L-alanyl-L-alanine, Meiji Seika Kaisha Ltd., Tokyo, Japan} and glufosinate ammonium {ammonium 4-[hydroxy(methyl)phosphinoyl]-DL-homoalaninate, AgrEvo, Wilmington, DE). In each transgenic line used, the Pin transgene cosegregated with Bar (Hogg et al., 2004). Transgenic lines in this study were selected based on total PIN expression levels. HGB12 and HGA3 had slightly higher total PIN expression levels than HGB5 and HGA1, respectively (Hogg et al., 2004).

Four hundred single F₂ plants from each of the four CR crosses (CR/HGA3, CR/HGA1, CR/HGB5, and CR/HGB12) and the three McN crosses (McN/HGA3, McN/HGB5, and McN/HGB12) were planted in the greenhouse at Montana State University-Bozeman Plant Growth Center. These plants were segregating for the native Ha locus from CR, McN, or Hi-Line and for the added transgene (Pina or Pinb). Single heads (F₃ seeds) from F₂ plants were harvested and screened for herbicide resistance. Twelve F₂–derived F₃ seeds from each head in the crosses were tested in the greenhouse for the transgene as described below.

Herbicide Screening
To identify F₂–derived F₃ seed pools homozygous for the presence or absence of the transgene, 18 F₂–derived F₃ seeds per line were planted in the greenhouse. Plants were sprayed with 0.1% glufosinate ammonium (AgrEvo) at the two-leaf stage. The plants were scored as being resistant or susceptible after 7 d. Resistant plants stayed green while susceptible plants were killed. The F₂ parent was classified as a Pin transgene homozygous positive (Pina or Pinb) or a Pin transgene homozygous negative genotype if >11 consecutive F₃ progeny were glufosinate resistant or glufosinate susceptible, respectively. F₃ progeny with mixed herbicide results, most often segregrating 3:1 resistant/susceptible, were considered to have a heterozygous-Pin F₂ parent. These lines were excluded from subsequent experiments.

Field Trials
Two seeds of each homozygous F₂ plant were grown at the Arthur H. Post Field Research Farm near Bozeman, MT, in the summer of 2004 under rain-fed conditions. The plants were then genotyped for the Ha locus. F₂–derived F₃ single plants homozygous for the Ha locus were harvested as individual plants. The number of plants homozygous for the segregating transgene and the native Ha locus within a cross varied from 14 to 42. In the summer of 2005, F₃–derived lines confirmed to be homozygous for the segregating transgene and native Ha locus plus parental controls were planted in a randomized block design with two blocks. The entries comprised of 441 F₃–derived F₄ lines and six parental controls. Each plot was a 3-m row seeded with 4 g with row spacing of 30 cm. The same trial was grown with and without irrigation in separate adjacent experiments at the Arthur H. Post Field Research laboratory near Bozeman, MT. Irrigated plots received 7.6 cm of water 1 wk before and 1 wk after anthesis. At maturity, plots were cut with a binder (Mitsubishi Agricultural Machinery Co., Ltd, Tokyo, Japan), threshed with a Vogel bundle thrasher (Bill's Welding, Pullman, WA), cleaned, and weighed. Grain hardness and grain protein were then determined on a subsample of seeds from each plot.

Grain Hardness and Grain Protein Measurement
The Single Kernel Characterization System (SKCS 4100, Perten Instruments, Springfield, IL.) was used to determine grain hardness. One hundred seeds from each plot in the 2005 replicated trial and 50 seeds from 2004 single plants were analyzed. Grain protein content was determined on whole grain samples using near-infrared transmission using an Infratec 1225 Grain Analyzer (Foss North America Inc., Eden Prairie, MN).

DNA Isolation and Ha Locus PCR Analysis
To identify the allelic state of the Ha locus, leaf tissues were taken from F₃ progeny lines arising from a single F₂ parent homozygous positive or negative for the transgene. Young leaf tissues were pooled from >10 individual F₃ plants from a single F₂ parent and genomic DNA extracted according to Riede and Anderson (1996). To determine the puroindoline genes resident at Ha in F₃ progeny lines, the polymorphisms inherent between the parental Ha locus genotypes were exploited (Giroux and Morris, 1997, 1998; Morris et al., 2001). Pinb coding sequence was amplified by PCR from genomic DNA using the primers described by Massa et al. (2004) to generate a 469-bp product containing 447 bp of Pinb coding sequence. The temperature regime used consisted of a 4 min initial denaturation step at 94°C, followed by 40 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 90 s, and a final extension at 72°C for 5 min. To distinguish Pinb-D1e from CR and Pinb-D1b from Hi-Line in CR/HG crosses, amplified Pinb PCR products were digested with BstN1 at 60°C for 1 h and then separated on 2.5% Metaphor agarose 1x TBE gels (Cambrex Bio-Science Inc., Rockland, ME). BstN1 cuts the Pinb-D1b PCR product once yielding 235- and 234-bp fragments while no BstN1 sites are present in the Pinb-D1e PCR product. To differentiate Pinb-D1a from McN and Pinb-D1b from Hi-Line in McN/HG crosses, Pinb PCR products were digested with BsrB1 at 37°C for 1 h followed by separation using 2.5% Metaphor agarose gels. BsrB1 cuts the Pinb-D1a PCR product once yielding 340- and 129-bp products while the Pinb-D1b PCR product is cut twice yielding 245-, 129-, and 95-bp products (Swan et al., 2006).

TX-114 Protein Extraction and Analysis
Fractionation of total puroindoline and separation via SDS-PAGE was done via Triton X-114 phase partitioning as previously described by Giroux et al. (2003). Whole meal UDY ground flour (UDY Co., Fort Collins, CO) from each of three random lines of CR/HGA3, CR/HGB12, McN/HGA3, and McN/HGB12 was used for TX-114 protein extraction. Puroindoline abundance was quantified visually using a scale of 1x to 8x. Heron (PI 290910; soft white wheat) was used to construct the scale where 1x = 6 µL load (240 µL SDS-PAGE sample buffer/100 mg whole seed meal). Means for each group were computed from three randomly selected individual genotypes per group.

Friabilin Protein Extraction and Analysis
The method for friabilin extraction and analysis used combined the friabilin method of Bettge et al. (1995) with the addition of a ZnSO₄ starch purification step (Guraya et al., 2003). One hundred milligrams of UDY milled whole wheat flour was steeped for 30 min in a 2-mL microfuge tube containing 0.5 mL of 0.1 M NaCl. The solution was vortexed and transferred to 2-mL preweighed microfuge tubes containing 1 mL of 80% (w/v) ZnSO₄ and centrifuged at 13000 x g for 3 min. The ZnSO₄ solution and the supernatant consisting of gluten and bran were then decanted and the starch pellet resuspended two more times in 80% (w/v) 1 mL ZnSO₄ by vortexing, followed by recentrifugation, and decanting. The starch was then washed with 1 mL water by vortexing followed by a brief centrifugation, and the supernatant removed followed by two more identical water washes. To the tubes containing water-washed starch, 1 mL acetone was added and the tubes were vortexed, centrifuged briefly, and decanted. The pellet was allowed to completely dry before being weighed to allow equal loading of samples. To the dried starch, 200 µL of 50% isopropanol and 0.5 M NaCl were added and the samples were vortexed. The samples were then incubated for 1 h at room temperature. After incubation the samples were centrifuged for 3 min at 13000 x g and the supernatant was transferred to a new 2-mL tube. Five hundred twenty microliters of cold acetone was added to the supernatant, vortexed, and then incubated overnight at –20°C. The samples were removed from –20°C, centrifuged for 3 min at 13000 x g, and the supernatant aspirated off. The pellet was washed once with 500 µL of acetone and allowed to dry. After the pellet was completely dry, the correct amount of SDS sample buffer (240 µL buffer 100 mg^–1 starch) was added. The samples were heated for 10 min at 70°C with occasional vortexing and then fractionated on 10 to 20% Tris-HCl, 160 by 160 by 1.5 mm, 20-well polyacrylamide gels (Bio-Rad, Hercules, CA). Gels were then stained using Coomassie brilliant blue R-250 (Fischer Scientific, Hampton, NJ). The amount of puroindoline associated with starch was quantified using a Heron (soft wheat) scale ranging from 1x to 5x where 1x = 10 µL. Means for each group were computed from three randomly selected individual genotypes per group.

Statistical Analysis
Four homozygous classes with varying numbers of random lines within each class were identified for each of the seven crosses. The two transgenes (Pina and Pinb), two null parents (CR and McN), and four homozygous classes within each cross gave 16 genotype classes with two independent events within each transgene except the HGA1 x McN cross was missing. Data obtained from F₃–derived F₄ lines were analyzed utilizing a mixed effects analysis of variance model for randomized block combined over environments using PROC MIXED in SAS (SAS Institute Inc., Cary, NC). Entries variation was partitioned into sources due to genotype classes, transgenic event within genotype classes, and progeny lines within genotype class by event combination and their interactions with environment. All effects were considered fixed, except blocks within environments, progeny lines within genotype class by event combination, and the interactions with environment were considered random. A similar model was applied to data from spaced F₃ plants. Specific comparisons among genotype class means were made using ESTIMATE statements in SAS.

	RESULTS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Screening for Ha Locus
Seven homozygous classes were created by crossing transgenic isolines in Hi-Line (Pina-D1a/Pinb-D1b) background that overexpressed Pina-D1a or Pinb-D1a coding sequences with CR (Pina-D1a/Pinb-D1e) and McN (Pina-D1b/Pinb-D1a) (Table 1). The homozygous classes segregated for the altered native Pin allele from Hi-Line (Pinb-D1b), CR (Pinb-D1e), or McN (Pina-D1b) and the presence or absence of the transgene. The native Pin genotype of all lines was determined by cleaved amplified polymorphism marker analysis. Figure 1 shows the cleaved amplified polymorphism marker test illustrating the segregation of Pinb-D1e from CR and Pinb-D1b from Hi-Line in CR crosses. Allele variation between Pinb-D1a present in McN and Pinb-D1b allele present in Hi-Line among McN crosses was determined as described by Swan et al. (2006). Based on herbicide screening and PCR analysis, four genotypic groups from each of the seven crosses were identified with 14 to 42 lines per class.

View larger version (87K): [in this window] [in a new window]   Fig. 1. Cleaved amplified polymorphism sequence (CAPS) test used to distinguish between the Pinb-D1e (Canadian Red [CR]) and Pinb-D1b (Hi-Line) alleles at Ha locus in segregating populations among CR crosses. BstNI does not digest the Pinb-D1e PCR product (469 bp) from CR. Pinb-D1b PCR product from Hi-Line is cut once yielding 234- and 235-bp products. Lanes 1 and 2 represent progeny lines possessing the Pinb-D1e allele while lanes 5 and 6 represent progeny lines carrying the Pinb-D1b allele. Lanes 3 and 4 are heterozygous for the Pinb-D1e and Pinb-D1b allele. M denotes a 100-bp ladder marker (Promega, Madison, WI) that goes up to 900 bp.

Grain Analysis Among Pinb-D1e or Pinb-D1b Genotypic Classes with Added Pina-D1a or Pinb-D1a
To determine the role of PINA alone on grain hardness in the absence of PINB, transgenic lines with added PINA in the CR (Pinb-D1e) Ha locus or Hi-Line (Pinb-D1b) Ha locus backgrounds were evaluated. In the presence of both native Pinb alleles (Pinb-D1e or Pinb-D1b) and an invariant Pina-D1a, addition of Pina gave intermediate grain texture (SKCS mean value of 43.5 and 42.5 respectively), while the addition of Pinb gave soft grain (SKCS mean value of 15.9 and 17.3 respectively) (Table 2 and Fig. 2 ). Grain hardness, kernel weight, and grain protein did not differ among transgenic events with the same transgene in the presence of either Pinb-D1e or Pinb-D1b allele (Table 3). The grain texture was not different when comparing native Pinb-D1e versus Pinb-D1b with added Pina or with added Pinb (Table 4). The same relative hardness differences on addition of Pina or Pinb was observed in 2004 single plants as were observed in 2005 replicated trials.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Scatter plots of F3–derived F4 progeny vs. F3 single plant grain hardness for segregating progeny from crosses of: (A) McNeal (McN) (Pina-D1b/Pinb-D1a) x Hi-Line Ha locus homozygous lines with added Pina or Pinb and the McN Ha locus. Pina-D1b denotes genotypes possessing the Pina-D1b/Pinb-D1a alleles and lacking a transgene, Pina-D1b (PINA+) denotes genotypes possessing the McN Pina-D1b/Pinb-D1a alleles with added Pina transgene and Pina-D1b (PINB+) denotes genotypes possessing the Pina-D1b/Pinb-D1a alleles with added Pinb transgene; (B) Canadian Red (CR) (Pina-D1a/Pinb-D1e) x Hi-Line Ha locus homozygous lines with added Pina or Pinb and the CR Ha locus. Pina-D1b denotes genotypes possessing the Pina-D1b/Pinb-D1a alleles and lacking the transgene, Pina-D1b (PINA+) denotes genotypes possessing the Pina-D1b/Pinb-D1a alleles with added Pina transgene, and Pina-D1b (PINB+) denotes genotypes possessing the Pina-D1b/Pinb-D1a alleles with added Pinb transgene.

Grain Analysis in the Absence of Added Pina or Pinb Transgene
Comparisons between added Pina and added Pinb homozygous classes where the progeny are homozygous negative for the transgene represent average contribution from transgenic lines contributing Pina versus those contributing Pinb. The significant differences for these comparisons were confined to protein content in the presence of the Pinb-D1b native Pin allele inherited from the transgenic parent (Table 3) when crossed to the CR parent and both grain hardness and kernel weight in the presence of the Pinb-D1b allele inherited from the transgenic parent when crossed to the McN parent. However, these differences for grain hardness and kernel weight were not confirmed from the 2004 single plant trial (Table 3). Differences among events for the same transgene occurred only for HGB5 versus HGB12 for grain hardness and kernel weight in the presence of the Pinb-D1b allele from the transgenic parent and for grain hardness in the presence of the Pina-D1b allele from McN in crosses to McN. These same differences were confirmed in the 2004 single plant trial.

TX-114 Extractable Puroindoline Levels
To determine the total puroindoline levels present in Pinb-D1e and Pina-D1b genotypes with either added Pina or Pinb, TX-114 protein extracts from three randomly selected lines per genotypic class were fractionated using SDS-PAGE and visualized by direct staining. Amounts of PINA and PINB for the soft wheat Heron were used as references. The CR parent had no PINB, but its PINA level was relatively equal to Heron. The McN parent had no PINA, and PINB was not detected with load concentrations used. Adding Pina increased PINA protein to more than six times that in Heron in both lines with the Pinb-D1e and the Pina-D1b alleles. Adding Pina to Pinb-D1e genotypes gave no PINB protein, while PINB in the Pina-D1b genotypes increased to amounts equal to the Heron control. Adding Pinb to either Pinb-D1e or Pina-D1b genotypes increased PINB protein about three times that in the Heron control (Fig. 3 , Table 5). PINA protein did not increase over that in the respective parents with addition of Pinb. Total PIN protein levels, which were obtained as the sum of the two relative amounts, was higher with the addition of Pina than for addition of Pinb (Table 5).

View larger version (117K): [in this window] [in a new window]   Fig. 3. SDS-PAGE gel of total TX-114 extractable proteins levels from PIN null and transgenic overexpressing lines. Heron (Pina-D1a/Pinb-D1b) is a soft wheat control while Canadian Red, PINB null (Pina-D1a/Pinb-D1e) and McNeal, PINA null (Pina-D1b/Pinb-D1a) are the hard wheat controls. TX-114 extractable puroindoline protein levels were performed on three randomly selected lines per group as shown. The position of PINA and PINB is marked.

View larger version (94K): [in this window] [in a new window]   Fig. 4. SDS-PAGE gel of starch-associated puroindolines (PINA and PINB). Total puroindolines were extracted from water-washed starch granules of control and soft and intermediate-textured PIN overexpressing lines. Heron (Pina-D1a/Pinb-D1a) is a soft wheat control while Canadian Red, PINB null (Pina-D1a/Pinb-D1e) and McNeal, PINA null (Pina-D1b/Pinb-D1a) are the hard wheat controls. Starch-associated proteins (PINA or PINB) were extracted from three randomly selected lines per group as noted. The position of PINA and PINB is marked.

	DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

In support of this theory, Hogg et al. (2004) characterized transgenic lines with added Pina, Pinb, or both in the hard wheat Hi-Line. They observed soft grain texture and increased puroindoline and friabilin abundance with addition of PINB, or both PINA and PINB to Hi-Line. Further, Swan et al. (2006) crossed Hi-Line transgenic lines expressing Pina, Pinb, or both Pina and Pinb to the soft wheat Heron and found that progeny lines with the soft Ha locus of Heron and increased PINB had softer endosperm texture than those with increased PINA. They concluded that PINB is the limiting factor in friabilin formation and reduction in grain hardness. The results of Hogg et al. (2004) and Swan et al. (2006) indicated that both PINA and PINB proteins are required for friabilin formation and for soft grain phenotype. Our current results lend support to the theory that both PINA and PINB are required for a wild-type soft grain endosperm phenotype in wheat. Expression of Pinb transgene in Pinb-D1e and Pina in Pina-D1b resulted in increased TX-114 soluble puroindoline levels, increased friabilin levels, and a soft phenotype (Table 2 and Fig. 3). Although addition of Pina to Pina-D1b decreased grain hardness leading to a soft texture, the softest phenotype resulted from the addition of Pinb to Pinb-D1e (Table 2, Fig. 2). These results concurred with Swan et al. (2006) that addition of Pinb leads to softer grain than the addition of Pina, and that Pinb is more limiting to grain softness than Pina. Further, addition of the Pinb transgene to Pinb-D1b also resulted in a soft phenotype (Table 2). Our study together with Beecher et al. (2002), Hogg et al. (2004), and Martin et al. (2006) demonstrates that a soft phenotype can be restored by complementing either the mutated Pinb (null), Pinb glycine-serine (Pinb-D1b), or Pina (null) with the corresponding functional Pin allele. It has been proposed that the absence of PINA in Pina-D1b and absence of PINB in Pinb-D1e hard wheats limits the interaction of PINB and PINA respectively with starch granules leading to hard endosperm phenotype (Giroux and Morris, 1997, 1998; Capparelli et al., 2003; Gazza et al., 2005). It has also been hypothesized that PINA and PINB interact to form friabilin and together affect grain texture (Hogg et al., 2004). Here, our main objective was to determine the individual role of each puroindoline protein in the absence of the other on grain hardness. Results obtained in this study show that addition of PINA in absence of PINB and addition of PINB in absence of PINA gave intermediate textured grain. A wild-type soft phenotype was obtained only in the presence of both PINA and PINB (Fig. 2). High amounts of either PIN individually led to an intermediate texture (Table 5, Fig. 3). This supports the hypothesis that total puroindoline content does not dictate grain softness, rather the presence of both functional PINA and PINB.

Friabilin is abundant on water-washed starch granules of soft wheat and little or none is found on water-washed starch granules from hard wheat (Greenwell and Schofield, 1986). Here, we report that PINA and PINB weakly associate with starch granules in absence of the other protein leading to an intermediate-textured endosperm (Table 3, Fig. 3). However, friabilin was found in abundance only when both PINA and PINB were present. It is evident that the amount and association of friabilin components on the surface of starch granules affect endosperm texture. Overexpression of PINA in Pinb (null) and overexpression of PINB in Pina (null) resulted in starch granule associated puroindolines and intermediate texture with slightly more PINB associating to starch granule surface than PINA. Capparelli et al. (2003) and Gazza et al. (2005) suggested that that in the absence of PINA, less PINB associates with starch granule surface. In this study, we observed that in the absence of PINA, overexpressed PINB associates with starch granule surface more efficiently than PINA. Previously, Greenblatt et al. (1995) postulated that the association of puroindoline to starch granules involves either polar bound phospholipids or glycolipids. Regardless of the specific mechanism of interaction of starch granules with PINA or PINB leading to intermediate endosperm texture, our study shows that either PINA or PINB can bind independently with the starch granule surface to produce intermediate-textured grain, or interact together to give wild-type soft-textured grain.

The effect of overexpressed puroindoline on a wide range of milling and baking traits was demonstrated by Hogg et al. (2005). In addition, the effect on dough properties and bread quality on puroindoline free flours reconstituted with puroindoline has been demonstrated (Dubreil et al., 1997). Martin et al. (2001) also observed an association of puroindoline sequence variation with several milling and baking traits in an recombinant inbred population population segregating for Pina-D1b and Pinb-D1b alleles. However, to clearly understand the roles of puroindolines on milling and baking traits, a baking and milling study of the unique genotypes created in this study varying randomly in PINA and PINB abundance would further illustrate the individual role each puroindoline protein plays on baking and milling qualities. These unique populations would also be useful in studying the antifungal effects conferred by an individual PIN protein in the absence of the other in wheat.

	CONCLUSIONS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
In conclusion, this study has shown that PINA and PINB can act independently in the absence of the other protein leading to an intermediate endosperm texture or together to produce wild-type soft-textured grain. It is also evident that overexpressed PINA or PINB in absence of the other protein associates with starch granules leading to an intermediate phenotype, however, wild-type soft phenotype leading to full friabilin abundance and function is observed when both functional PINA and PINB are present. The biochemical basis for the association of PINA or PINB to starch granules in the absence of the other leading to an intermediate grain texture is unknown. However, our results support the hypothesis that Pina and Pinb are the causative factors in grain hardness and that wild-type Pina and Pinb genes are required for a wild-type soft texture.

	ACKNOWLEDGMENTS

 
We thank Fletcher Meyer, Jim Berg, and Susan Lanning for their agronomic expertise.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
This research was supported by USDA-ARS National Research Initiative Competitive Grants Program grant 2004-01141 and by the Montana Agricultural Experiment Station.

Received for publication May 10, 2006.

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