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Dep. of Plant Sciences and Plant Pathology, Montana State Univ., Bozeman, MT 59717-3150
* Corresponding author (mgiroux{at}montana.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: Ha, Hardness • PINA, protein expressed by the Puroindoline a (Pina) gene • PINB, protein expressed by the Puroindoline b (Pinb) gene • SKCS, single kernel characterization system • QTL, quantitative trait locus
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The majority of grain hardness variation in hexaploid wheat is controlled by a single locus referred to as Hardness (Ha) (Symes, 1965; Symes, 1969; Baker and Dyck, 1975; Baker, 1977). The Ha locus is located on the short arm (Doekes and Belderok, 1976) of chromosome 5D (Mattern et al., 1973) and is simply inherited (Law et al., 1978). Soft wheats possess the dominant or wild-type form (Ha), while hard wheats have the recessive or mutated form (ha) (Law et al., 1978). Located within the Ha locus are the tightly linked genes Pina, Pinb, and Gsp-1a (Sourdille et al., 1996; Giroux and Morris, 1997; Tranquilli et al., 1999; Turnbull et al., 2003). All three genes were previously sequenced in T. monococcum L., a diploid ancestor of T. aestivum, and are contained within a single 105-kb bacterial artificial chromosome (Tranquilli et al., 1999). Soft endosperm texture results when both Puroindolines are present as wild-type alleles (Pina-D1a, Pinb-D1a) (Giroux and Morris, 1998). All hard wheats characterized to date have a mutation in Pina or Pinb relative to the wild-type alleles (Giroux and Morris, 1998; Lillemo and Morris, 2000; Morris et al., 2001; Gazza et al., 2005). The most common mutations in North American hard wheats are a Pina null mutation (Pina-D1b) and a Pinb glycine-to-serine point mutation (Pinb-D1b) (Morris et al., 2001).
Biochemically, wheat grain hardness is most likely determined by the degree of adhesion between starch granules and the protein matrix. This starch–protein interaction is regulated by the protein complex friabilin (Beecher et al., 2002). Friabilin was originally described as a 15-kDa, water-washed starch granule protein associated with wheat grain texture (Greenwell and Schofield, 1986). High levels are associated with starch from soft wheats, while low levels are associated with water washed starch from hard wheats (Greenwell, 1987). Friabilin N-terminal sequencing demonstrated that the puroindoline proteins, PINA and PINB, are the primary components of friabilin (Jolly et al., 1993; Morris et al., 1994). PIN proteins contain a unique tryptophan-rich domain that is thought to be involved in the binding of lipids on the surface of starch granules (Gautier et al., 1994; Marion et al., 1994). Furthermore, the lipid binding properties of PINA and PINB have been shown to considerably influence antifungal properties in rice (Krishnamurthy and Giroux, 2001) and loaf volume in wheat (Dubreil et al., 1997; Igrejas et al., 2001; Hogg et al., 2005).
Even though the causal role of puroindolines on grain hardness has been well documented (Giroux and Morris, 1997; Campbell et al., 1999; Beecher et al., 2002; Hogg et al., 2004), it is not certain which puroindoline plays a more critical role in reducing grain hardness in soft wheats. Increasing the dosage of both Pina and Pinb in hexaploid wheat substitution lines, in which inactive forms of the Puroindolines are replaced with active forms, can reduce grain hardness in soft wheats (See et al., 2004). Hogg et al. (2004) demonstrated that when the hard spring wheat cultivar Hi-Line (Pina-D1a, Pinb-D1b) (Lanning et al., 1992) was transformed with additional Pina-D1a, a harder phenotype resulted than Hi-Line transformed with additional Pinb-D1a at similar expression levels. The same study also demonstrated that the addition of Pinb-D1a increased binding of PINA to starch but the addition of Pina-D1a did not alter PINB binding. However, Hogg et al. (2004) did not fully explore the relative contributions of PINA and PINB to grain softness. Hi-Line natively expresses only functional PINA but a nonfunctional PINB, therefore the true contribution of biolistically added Pina-D1a and/or Pinb-D1a cannot be accurately assessed. Hogg et al. (2004) found that the overexpression of Pinb-D1a in Hi-Line increased binding of PINA to starch as friabilin, but again this does not fully define the relative contributions of PINA and PINB since Hi-Line contains the Pinb-D1b allele. The presence of the variant PINB in Hi-Line prevents detailed analysis of which PIN protein limits grain softness. A more complete analysis of which PIN limits grain softness would consist of the addition of PINA and/or PINB to a soft wheat. It is known that PINA is roughly twice as abundant as PINB in soft wheat varieties (Giroux and Morris, 1998). Therefore, if PINA and PINB bind to starch in an equimolar manner, PINB might be expected to limit grain softness in soft wheats.
The objectives of this study were to investigate which puroindoline limits the degree to which endosperm can be softened, and discover if there is a lower limit to grain softness in wheat. Increasing softness among soft wheats is associated with increased cookie diameter and decreased sucrose retention capacity (Gaines, 2000; Guttieri et al., 2001). Therefore, decreasing grain hardness by overexpressing Pina and/or Pinb may result in improved soft wheat quality. To test which puroindoline limits grain softness in soft wheats, Hi-Line genotypes previously transformed with additional copies of Pina-D1a and/or Pinb-D1a (Hogg et al., 2004) were crossed to the soft wheat ‘Heron’ (Pina-D1a, Pinb-D1a). Resulting progeny segregated for the transgene and the native Pinb alleles residing at Ha. Progeny from each of the four homozygous classes (plus or minus transgene and contain either Pinb-D1a or Pinb-D1b at the Ha locus) were identified and analyzed for grain hardness, kernel weight, and protein content through 2 yr and two environments.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Herbicide Screen
Hi-Line parents from the Heron x Hi-Line crosses were cotransformed with a Bar expression vector, as described previously (Beecher et al., 2002). Bar confers resistance to the herbicides bialaphos (4-[hydroxy(methyl)phosphinoyl]-L-homoalanyl-L-alanyl-L-alanine) and glufosinate [DL-homoalanin-4-yl(methyl)phosphinic acid]. All T0 Hi-Line plants expressing added Pina and/or Pinb also expressed a Bar locus conferring herbicide resistance that cosegregated with the Pin transgene(s) (Hogg et al., 2004). Following Hi-Line x Heron crossing, this co-integration of constructs made it possible to identify transgenic positive/negative Pin progeny through progeny testing utilizing herbicide resistance testing. Approximately 18 F3 seeds, derived from one F2 plant, were germinated in the greenhouse and sprayed with a 0.1% glufosinate solution at the two-leaf stage. Plantlets were scored as resistant or susceptible 7 d after spraying. The F2 parent was classified as a Pin transgene homozygous positive (either Pina, Pinb, or Pina/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.
PCR Analysis
PCR screening was performed to determine the allelic state of the native Ha locus. Progeny arising from the Heron x Hi-Line crosses were classified into two groups: those containing native Pinb-D1a (inherited from Heron) and those containing native Pinb-D1b (inherited from Hi-Line). The primer pair PB5/PB3 was previously designed to amplify Pinb-D1a coding sequences (Gautier et al., 1994), however, this primer pair also amplifies a product from the Pinb-D1a transgene. Therefore, a new primer, CAT3.4, was designed from existing sequence data (Gautier et al., 1994) to be just downstream of the Pinb sequence present in the Pinb-D1a transformation vector (Beecher et al., 2002). CAT3.4 was paired with PB5 to amplify Ha locus-specific Pinb-D1a and Pinb-D1b. The sequences for these primers are as follows:
Leaf tissue was taken from 12 F3 progeny arising from a single homozygous F2 parent. These tissue samples were pooled and genomic DNA was extracted as described previously (Riede and Anderson, 1996). Using this DNA as a template and PB5/CAT3.4 primers, a 469-bp product was amplified. The point-mutation polymorphism between native Pinb-D1a and native Pinb-D1b (Giroux and Morris, 1997) was identified by digesting PCR products with BsrBI (Dubcovsky et al., 1999). The Pinb-D1a PCR product is digested once by BsrBI, yielding 340- and 129-bp products. The Pinb-D1b PCR product is digested twice, yielding 245-, 129-, and 95-bp products.
Triton X-114 Protein Extraction
Seed from two random transgene positive lines from each crossing event (12 total), one hard wheat control (Hi-Line), and one soft wheat control (Heron) was ground using a Udy Cyclone Mill with a 0.5-mm screen (Seedburo, Chicago, IL). Total puroindoline was extracted from the ground seeds using Triton X-114 (TX114) detergent as described by Giroux et al. (2003). Replicate extracts were resolved on Protean II 10 to 20% Tris-HCl Ready Gels (BioRad, Hercules, CA). Soft-type puroindoline content was quantified using a scale of 1x to 6x, with increments of 1x. The scale was constructed using a Heron puroindoline extract where 1x equaled a 10-µL load (240 µL SDS-PAGE sample buffer per 100 mg ground seeds). Values above 6x could not be resolved, thus lines with levels above 6x were diluted to allow comparison to the same scale.
Friabilin Extraction
Friabilin, or starch-bound puroindoline, was isolated from the surface of starch granules using the same genotypes as for the TX114 protein extraction. Methods were as described by Bettge et al. (1995) with the addition of a ZnSO4 starch purification step (Guraya et al., 2003). As for TX114 extraction, a Udy Cyclone Mill with a 0.5-mm screen was used to grind the seeds into a finely textured meal. The ground seeds (300 mg) was placed into 2-mL tubes, 1 mL of 0.1 M NaCl was added to each tube, and samples were vortexed and incubated 15 min. A dough ball was formed by mixing with a pestle. The starch-containing supernatant was removed and placed into a new, preweighed 2-mL tube. One-half milliliter of 0.1 M NaCl was used to wash the dough ball twice more, placing the starch-containing supernatant wash into the same preweighed tube. The starch suspension was then centrifuged for 3 min at 13 000 g, the supernatant was aspirated off, and the starch pellet resuspended in 1 mL of 75% w/v ZnSO4·7H2O. The tube was spun for 3 min at 13000 g and the supernatant was removed by aspiration. The ZnSO4 wash was repeated once more. The starch pellet was then washed with 1 mL water three times and 1 mL acetone once with centrifugation as above. The starch pellet was allowed to dry completely before the tube was weighed to determine the amount of starch present. The starch pellets were then resuspended in 400 µL of 50% isopropanol in 0.5 M NaCl followed by an incubation at room temperature for 30 min. The suspension was centrifuged for 3 min at 13 000 g, the supernatant was transferred to a new tube, and then 520 µL of acetone was added to the solution. The tubes were then vortexed and incubated overnight at –20°C. The samples were spun for 3 min at 13000 g, the supernatant aspirated, and the pellet was washed once in acetone and then dried. Two hundred forty microliters of SDS-PAGE loading buffer minus any reducing agents (Laemmli, 1970) was added for every 100 mg of starch recovered after the ZnSO4 precipitation. Samples were heated at 70°C for 10 min, and 10 µL (1x) was loaded onto 10–20% Tris-HCl polyacrylamide gels (BioRad, Hercules, CA). Total friabilin was quantified using a scale ranging from 1x to 6x in increments of 1x. This scale was constructed using a Heron friabilin extract, where 1x equals 10 µl. Lines having greater than 6x PINA and/or PINB levels were diluted and compared to the same scale.
F4 Seed Analysis
F2–derived F3 lines determined to be homozygous for the presence or absence of the transgene and homozygous for the Pinb-D1a (from Heron) or Pinb-D1b (from Hi-Line) allele at the Ha locus were evaluated in a replicated field trial in 2004. One hundred ninety-eight lines plus the Hi-Line and Heron parental controls were evaluated in a randomized block design with two replications. All plots were single 3-m-long rows spaced 30 cm apart with 2 g of seed planted per 3-m row. The experiment was grown under irrigated and rainfed conditions at the Arthur H. Post Field Research Farm, Bozeman, MT. Irrigated plots received an additional 6.4 cm of water 1 wk before flowering and 6.4 cm of water 1 wk after flowering. Each plot was harvested and threshed to obtain F4 seed for analysis. Grain hardness and kernel weight were determined using the Perten Single Kernel Characterization System (SKCS) 4100 (Perten Instruments, Springfield, IL) on samples of 100 seeds per plot. Grain protein content was determined by near-infrared transmittance for whole grain using the Tecator Infratec 1225 Grain Analyzer (Foss North America, Silver Spring, MD).
Statistical Analysis
The six transgenic events (representing six crosses) with four homozygous classes for each cross (presence or absence of transgene and Pina-D1a/Pinb-D1a from Heron or Pina-D1a/Pinb-D1b from Hi-Line) gave 24 total genotype classes with varying numbers of random lines within the 24 classes. Data obtained from F2–derived F3 lines were analyzed utilizing a mixed effects analysis of variance model for randomized block combined over environments using PROC MIXED in SAS (SAS Institute, 2000). Entries variation was partitioned into sources due to genotype classes and progeny lines within genotype classes. Environments and genotype classes were considered fixed, while blocks within environments, progeny lines within genotype classes and the interaction with environments were considered random effects. A similar model was applied to data from spaced F2 plants. Specific comparisons among genotype class means were made using ESTIMATE statements in SAS. Data from the independent observations from the TX114 and friabilin gels were analyzed via analysis of variance and a least significant difference was computed.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Grain Analysis
Soft wheats typically have mean SKCS grain hardness values between 20 and 50, while hard wheats are typically between 51 and 90. Heron and Hi-Line had mean SKCS grain hardness values of 26.9 and 79.8, respectively, in 2003 and 30.3 and 80.8, respectively, in 2004. Parental transgenic lines with added Pinb-D1a and with Pina-D1a and Pinb-D1a were all softer than Heron. The two parental transgenic lines with added Pina-D1a were intermediate in grain texture between Heron and Hi-Line (Table 1).
Since the six transgenic parents were transgenic isolines for the added transgene(s) the six cross means where progeny are homozygous negative for the transgene and carry the same Pinb allele should be the same within the limits of sampling variation. Any differences among the six cross means for each Pinb allele may be attributed to genetic alterations that occurred during tissue culture regeneration. The only difference detected when comparing events for the same transgene was between HGA1 and HGA3 for both grain protein and kernel weight with the Pinb-D1a allele, and we observed no differences between transgenes when averaged over the two events (Table 2 and 3). There was a hardness difference of about 48 SKCS hardness units observed between progeny lines that were homozygous negative for the transgene and homozygous for either Pinb-D1a (hardness of 31.0) or Pinb-D1b (hardness of 77.9).
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The effects of the addition of Pina-D1a and Pinb-D1a to a soft wheat has not previously been studied. The mean SKCS hardness for progeny homozygous for the added transgene and the Pinb-D1a allele were less than their corresponding transgene negative control group (P < 0.05 data not shown) indicating that the addition of either or both Pina-D1a and Pinb-D1a gave softer grain even in the presence of wild-type soft Pin alleles. Addition of both Pina-D1a and Pinb-D1a had greatest effect on grain texture (hardness of 13.2), followed by Pinb-D1a (hardness of 14.6) and Pina-D1a (hardness of 19.8) having least effect on grain texture (Table 2). The three means (HGA, HGB, and HGAB) averaged over the two events were each different from each other (Table 3 and Fig. 2 ). The two HGA events were different as were the two HGAB events for SKCS hardness. Genotype means were similar between events for the same transgene for kernel weight and grain protein except HGB3 gave lower grain protein than HGAB18. The same SKCS differences were observed in 2003 from single plants as were observed in 2004.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Giroux and Morris (1998) suggested two major ideas regarding the control of grain hardness: (i) starch friabilin is the factor controlling grain softness, and (ii) puroindolines are the causal genes for grain hardness in wheat. In support of these ideas, Campbell et al. (1999) found that the segregation of the Ha locus, which contains the tightly linked Pina and Pinb genes, in a hard x soft wheat cross accounted for approximately 60% of the variation in wheat grain hardness. Further evidence came with the report that transgenic expression of Pina-D1a and Pinb-D1a in rice seeds reduced grain hardness (Krishnamurthy and Giroux, 2001). Rice does not contain Pina, Pinb, or homologs of puroindolines, thus the ability of puroindolines to modify grain texture became evident. To establish a direct cause-and-effect relationship between puroindolines and grain texture in wheat, Beecher et al. (2002) showed complementation of the Pinb-D1b mutation in the hard wheat Hi-Line with the soft-type Pinb-D1a sequence resulted in a soft phenotype, solidifying the importance of the two wild-type puroindolines in soft wheat texture. Results obtained from the current study further support the above hypothesis that puroindolines are the casual agents in grain hardness. The addition of soft-type Pina-D1a, Pinb-D1a, or both Pina-D1a and Pinb-D1a resulted in increased puroindoline levels (Table 4) and reduced grain hardness when compared to the native parents and transgene-negative controls (Table 2). Further, the addition of Pinb-D1a resulted in a softer phenotype than the addition of Pina-D1a. The greater "softening effect" of Pinb-D1a relative to Pina-D1a was seen in progeny lines that had either of the native Pinb alleles. We had previously observed this to be true in studies of Pina-D1a/Pinb-D1a overexpressing transgenics of Hogg et al. (2004, 2005) (Table 1). In Hogg et al. (2004) the addition of Pinb-D1a to Hi-Line (Pina-D1a, Pinb-D1b) resulted in much softer grain and increased friabilin PINA and PINB more than the addition of Pina-D1a. However, the results of Hogg et al. (2004) can only indicate that both PINs are required for grain softness and do not address which or both PINs limit grain softness in soft wheats. Grain hardness data obtained from single plants in 2003 confirms results obtained from the replicated trial averaged over irrigated and rainfed environments in 2004. This demonstrates that puroindoline effects on grain hardness show minimal interaction with environments. It is clear that the addition of the transgene is the major difference between genotype class means. However, variation in grain hardness was observed within genotype classes (Fig. 2). This variation could be attributable to differences in kernel weight, grain protein or other unmeasured traits. Results obtained from this study indicate that additional, transgenic puroindolines increase the degree to which endosperm is softened when both wild-type Pin alleles (Pina-D1a and Pinb-D1a) are present at the soft-type Ha locus.
It is believed that PINA and PINB bind starch granule surface lipids in soft wheats and not hard wheats (Greenblatt et al., 1995), which is consistent with the findings that much higher levels of PINA and PINB are bound to soft wheat starch than hard wheat starch. This suggests that puroindolines found at the surface of starch granules affect grain hardness. Furthermore, differences in the amount of either puroindoline protein should influence grain texture if PINA and PINB act independently from one another. The levels of both PINs were increased by See et al. (2004) who substituted chromosome 5Am from T. monococcum for 5A and 5S from Aegilops searsii Feldman Kislev ex K. Hammer for 5B of bread wheat. They found that the additional copies of the puroindoline genes carried on the substituted chromosomes increased friabilin levels and resulted in softer grain. Hogg et al. (2004) used transgenic isolines overexpressing puroindolines to conclude that decreased levels of grain hardness are associated with both functional PINA and PINB, and not total puroindoline content. Results obtained from this experiment support the above findings and demonstrate the effects of increasing PINA, PINB or both PINs in a soft wheat variety. Nontransformed soft wheat (Heron) had a TX114 soluble PINA to PINB ratio of 2:1 and friabilin PINA and PINB levels in a 1:1 ratio (Fig. 3A,B). HGA lines expressing PINA and homozygous for the native Pinb-D1a allele showed increases in TX114 PINA and friabilin PINA levels with no effect on PINB friabilin levels (Table 4). The addition of PINA alone had the smallest effect on the reduction of grain hardness relative to PINB or PINA/PINB additions. Pinb-D1a type HGB lines transformed only with additional Pinb, exhibited increased friabilin PINA and PINB levels. HGB lines were significantly softer then Heron, negative controls, HGAB3, and both HGA lines. HGB lines and HGA lines yielded similar TX114 and friabilin total puroindoline levels but significantly different PINA to PINB friabilin ratios and mean grain hardness values. Results support the hypothesis that total puroindoline content does not dictate grain softness. Rather, the proportion of functional PINA to PINB plays a more critical role. The overexpression of PINA and PINB separately in the Pina-D1a, Pinb-D1a background suggests that low PINB levels limit the binding of PINA and that PINB is the limiting factor in the reduction of grain hardness. The data in Fig. 3 and Table 4 support the hypothesis that the HGA group is harder than the HGB group, despite having higher total PIN levels. Data obtained from the HGAB lines, transformed with Pina and Pinb, further demonstrate this point. HGAB3 and HGAB18 were different in grain hardness (Table 3, Fig. 2) and, not surprisingly, in puroindoline levels (Table 4, Fig. 3). HGAB3 had less TX114 soluble and starch bound PINA than the HGA lines and its PINB level was less than the HGB lines resulting in a friabilin PINA/PINB ratio of 2:1. With the low friabilin PINB coupled with the hypothesis that PINB is the limiting factor in grain hardness reduction, the mean grain hardness of HGAB3 at 21.2 was explained. HGAB18 displayed the greatest amount of TX114 and friabilin total puroindoline with PINA levels similar to the HGA lines and PINB amount greater then the HGB lines. Most interestingly, HGAB18 had a 7:6 ratio between friabilin PINA and PINB. This suggests that there was sufficient PINA available to interact with the PINB present, leading to the softest mean grain hardness at 7.3 (Table 2, Fig. 3). The ratio of friabilin PINA to PINB coupled with the hardness data also lead to the conclusion that soft wheat varieties are limited in the degree of softness by both low PINB levels and low total PINA and PINB. Increased softness in soft wheats could be achieved by increasing the level of PINB or by increasing both PINA and PINB, in correct ratio.
In conclusion, this study has brought to light several interesting observations regarding the interaction of the two puroindolines. Reduction in grain hardness is not correlated with total puroindoline content but with both functional PINA and PINB. The data also indicates that the reduction in grain hardness in soft wheats is limited by the expression of PINB. Lines expressing additional TX114 and friabilin PINB showed reduced grain hardness when compared to lines expressing additional PINA. Additionally, overexpression of friabilin PINB in soft wheat increased the amount of starch granule–bound PINB and PINA, while overexpression of friabilin PINA only increased the amount of PINA. We were also given no indication that grain hardness has reached a minimum value in wheat. Lines overexpressing puroindolines in a ratio close to 1:1 continued to decrease in grain hardness. The information presented here indicates that increasing puroindoline abundance in soft wheats could give grain texture softer than conventional soft wheat genotypes. Such genotypes may prove useful in improving break flour yields and soft wheat quality.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication December 16, 2005.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  L. Feiz, B. S. Beecher, J. M. Martin, and M. J. Giroux In Planta Mutagenesis Determines the Functional Regions of the Wheat Puroindoline Proteins Genetics, November 1, 2009; 183(3): 853 - 860. [Abstract] [Full Text] [PDF]
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 J. M. Martin, F. D. Meyer, C. F. Morris, and M. J. Giroux Pilot Scale Milling Characteristics of Transgenic Isolines of a Hard Wheat Over-Expressing Puroindolines Crop Sci., March 1, 2007; 47(2): 497 - 504. [Abstract] [Full Text] [PDF]
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H. W. Wanjugi, A. C. Hogg, J. M. Martin, and M. J. Giroux The Role of Puroindoline A and B Individually and in Combination on Grain Hardness and Starch Association Crop Sci., January 22, 2007; 47(1): 67 - 76. [Abstract] [Full Text] [PDF]
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