Quality traits. Grain Texture

Contributed by Gabriela Tranquilli (gtranqui@cirn.inta.gov.ar)

References

1. Puroindolines: the molecular basis of wheat grain hardness. Morris CF In: Plant Molecular Biology, 2002, 48: 633-647.

The variation in grain hardness is the single most important trait that determines end-use quality of wheat. Grain texture classification is based primarily on either the resistance of kernels to crushing or the particle size distribution of ground grain or flour. Recently, the molecular genetic basis of grain hardness has become known, and it is the focus of this review. The puroindoline proteins a and b form the molecular basis of wheat grain hardness or texture. When both puroindolines are in their `functional' wild state, grain texture is soft. When either one of the puroindolines is absent or altered by mutation, then the result is hard texture. In the case of durum wheat which lacks puroindolines, the texture is very hard. Puroindolines represent the molecular-genetic basis of the Hardness locus on chromosome 5DS and the soft (Ha) and hard (ha) alleles present in hexaploid bread wheat varieties. To date, seven discrete hardness alleles have been described for wheat. All involve puroindoline a or b and have been designated Pina-D1b and Pinb-D1b through Pinb-D1g. A direct role of a related protein, grain softness protein (as currently defined), in wheat grain texture has yet to be demonstrated.

2. Endosperm texture in wheat. Turnbull KM, Rahman S. In: Journal of Cereal Science, 2002, 36: 327-337.

One of the fundamental means of classifying wheat is through its endosperm texture. It impacts significantly on the milling process affecting among other things flour particle size and milling yield. Hardness in wheat is largely controlled by genetic factors but it can be affected by the environment and factors such as moisture, lipid, and pentosan content. The principal genetic locus controlling endosperm texture in wheat, Ha, is located on the chromosome 5D. At this locus several genes, notably the puroindolines, have been identified. Puroindolines are the major components of the 15 kDa protein band associated with starch granules that is more abundant in soft wheats than in hard. Recently the puroindolines have been shown to enhance grain hardness in rice. In this review we discuss the structure of hard and soft wheat endosperm with particular emphasis on when differences in endosperm texture can be detected in the developing seed. The role of the environment and other factors that may affect the endosperm texture is also examined together with the role of the puroindoline genes at theHa locus. Finally, we compare endosperm hardness in wheat and in barley.

3. A glycine to serine change in puroindoline b is associated with wheat grain hardness and low levels of starch-surface friabilin. Giroux MJ, Morris CF. In: Theoretical and Applied Genetics, 1997, 95: 857:864.

The quantitative level of friabilin 15-kDa protein present on the surface of water-washed starch is highly correlated with wheat grain softness. Friabilin is composed primarily, if not exclusively, of the proteins puroindoline a and b. The transcript levels of these two proteins are similar among hard and soft wheat varieties, and the expression of both is controlled by the short arm of chromosome 5D, also the chromosomal location of the Hardness gene. We report here a glycine to serine sequence change in puroindoline b associated with hard grain texture. This amino acid change results from a single nucleotide mutation and resides in a region thought to be important for the lipid-binding properties of puroindolines. No recombination was observed between the serine puroindoline-b mutation, hard grain texture and low levels of starch surface friabilin among a set of 83 homozygous 5D recombinant lines derived from the soft-textured variety `Chinese Spring' and the substitution line `Chinese Spring' containing the 5D chromosome of the hard-textured variety `Cheyenne'. The sequence change reported here may adversely affect the lipid-binding properties of puroindoline-b and so effect hard grain texture. The results suggest that grain hardness results from puroindoline-b functionality such that the Hardness gene is a direct manifestation of puroindoline structure. We are suggesting the tentative molecular marker loci designations of Pinb-D1a and Pinb-D1b for the glycine and serine puroindoline-b types, respectively. t

4. Wheat grain hardness results from highly conserved mutations in the friabilin components puroindoline a and b. Giroux MJ, Morris CF. In: Proceedings of the National Academy of Sciences, 1998, 95: 6262-6266.

"Soft" and "hard" are the two main market classes of wheat (Triticum aestivum L.) and are distinguished by expression of the Hardness gene. Friabilin, a marker protein for grain softness (Ha), consists of two proteins, puroindoline a and b (pinA and pinB, respectively). We previously demonstrated that a glycine to serine mutation in pinB is linked inseparably to grain hardness. Here, we report that the pinB serine mutation is present in 9 of 13 additional randomly selected hard wheats and in none of 10 soft wheats. The four exceptional hard wheats not containing the serine mutation in pinB express no pinA, the remaining component of the marker protein friabilin. The absence of pinA protein was linked inseparably to grain hardness among 44 near-isogenic lines created between the soft variety Heron and the hard variety Falcon. Both pinA and pinB apparently are required for the expression of grain softness. The absence of pinA protein and transcript and a glycine-to-serine mutation in pinB are two highly conserved mutations associated with grain hardness, and these friabilin genes are the suggested tightly linked components of the Hardness gene. A previously described grain hardness related gene termed "GSP-1" (grain softness protein) is not controlled by chromosome 5D and is apparently not involved in grain hardness. The association of grain hardness with mutations in both pinA or pinB indicates that these two proteins alone may function together to effect grain softness. Elucidation of the molecular basis for grain hardness opens the way to understanding and eventually manipulating this wheat endosperm property.t

5.A leucine to proline mutation in puroindoline b is frequently present in hard wheats from Northern Europe. Lillemo M, Morris CF. In: Theoretical and Applied Genetics, 2000, 100:1100-1107.

Endosperm hardness in wheat (Triticum aestivum L.) is determined by one major genetic factor, the Hardness (Ha) gene on the short arm of chromosome 5D. Grain hardness has previously been reported to result from either a failure to express puroindoline a (Pina-D1b) or a glycine to serine mutation at position 46 in puroindoline b (Pinb-D1b). In this study, which involves a large survey of 343 wheat genotypes of mostly Northern European origin, we report two new mutations in puroindoline b associated with hard endosperm. These were characterized as involving a leucine to proline change at position 60 (Pinb-D1c), and a tryptophan to arginine change at position 44 (Pinb-D1d), respectively. While the former seems to be widely distributed in germplasm around the world, the latter was only found in three winter wheats from Sweden and Netherlands. As discussed in the paper, the three known mutations in puroindoline b can be considered "loss-of-function" mutations (i.e. soft to hard), and structural analysis may serve to predict that their dramatic effect on wheat grain texture is a result of reduced lipid-binding ability. t

6. Prevalence of puroindolines grain hardness genotypes among historically significant North American spring and winter wheats. Morris CF, Lillemo M, Simeone MC, Giroux MJ, Babb SL, Kidwell KK. In: Crop Science, 2001, 41: 218-228.

Grain hardness ("hard" or "soft" kernel texture) is the single most important trait in determining the utilization and marketing of wheat (Triticum aestivum L.). Puroindoline a and b proteins represent the molecular basis for this trait. This study surveyed the prevalence of puroindoline hardness mutations (alleles) among North American spring and winter wheat varieties with emphasis on those that are historically important. Each variety was assessed for kernel texture using the Single Kernel Characterization System; Hardness alleles were defined by puroindoline gene sequence and the presence or absence of puroindoline a protein on polyacrylamide gels. A total of 90 spring wheats were examined: nine were soft and possessed wild-type ("soft") puroindoline sequences, 10 were mixed hardness, and the remaining 71 were uniformly hard. Of these hard spring wheats, 18 carried the Pina-D1b hardness allele (null for puroindoline a protein), 47 the Pinb-D1b allele (Gly-46–Ser-46), and four the Pinb-D1c allele (Leu-60–Pro-60). Two hard spring wheats possessed a new allele, designated Pinb-D1e, which involves a single nucleotide change in Trp-39 to a "stop" codon. Lastly, among the spring wheats, a new hardness allele was found in the hard component of the variety `Utac' which was mixed. This allele, Pinb-D1f, also involved a single nucleotide change such that Trp-44 became a "stop" codon. A total of 62 winter wheat varieties were examined, of which five were soft and three were of mixed hardness. Of interest, the three mixed hardness wheats were `Turkey', `Kharkof', and `Weston'. The hard component of each carried the Pinb-D1b allele. Of the 54 remaining wheats, all of which were hard, all but two carried this same Pinb-D1b allele. `Chiefkan' winter wheat carried the same Pinb-D1e allele as `Canadian Red' and `Gehun' spring wheats. `Andrews' hard red winter wheat possessed a new allele, designated Pinb-D1g, which was a single nucleotide change in Cys-56 to a "stop" codon. In conclusion, hard grain phenotype results from one of various mutations in either of the puroindoline proteins. To-date, seven hardness alleles have been discovered and characterized in hexapoid wheat. All but one occur in the puroindoline b gene coding sequence and result from single nucleotide changes. These molecular markers are useful in characterizing lineages and analyzing ancestral relationships.

7. Association of puroindoline sequence type and grain hardness in hard red spring wheat Giroux MJ, Talbert L, Habernicht DK, Lanning S, Hemphill A, Martin JM. In: Crop Science, 2000, 40: 370-374.

Wheat (Triticum aestivum L.) endosperm texture is a primary determinant of milling and end-product quality. Friabilin, a marker protein for grain hardness, is composed of two proteins, puroindoline a and b (pinA and pinB, respectively). Hard-textured wheats have variant alleles consisting of a glycine-to-serine change in pinB (pinB-D1b) or the complete absence of pinA (pinA-D1b). Our objectives were to examine the influence of pinA and pinB alterations on grain hardness from populations among elite hard red spring wheat cultivars differing in puroindoline alteration, and to measure associations of grain hardness with kernel weight and grain protein concentration. Fifty F3:6 progenies from three pinA-D1b x pinB-D1b, one pinB-D1b x pinB-D1b, and one pinA-D1b x pinA-D1b type crosses were evaluated with their parents in two field experiments. Lines classified as pinA-D1b were significantly harder than lines classified as pinB-D1b when averaged across the three segregating populations. This difference was significant (P < 0.05) in one of the three populations. Significant genetic variation existed for grain hardness, protein concentration, and kernel weight within puroindoline classes and among lines from crosses not segregating for pinA-D1b vs. pinB-D1b. Significant positive correlations were observed in all five populations for grain hardness and protein concentration (r=0.46-0.79). Our results indicate that most of the genetic variation in grain hardness among the populations studied was due to factors other than pinA and pinB, as the pinA-D1b vs. pinB-D1b difference explained <12% of the variation in grain hardness in these hard wheat populations.

8. Substitutions and deletions of genes related to grain hardness in wheat and their effect on grain texture Tranquilli G, Heaton J, Chicaiza O, Dubcovsky J. In: Crop Science, 2002, 42: 1812-1817.

The Hardness (Ha) locus on chromosome 5D is the main determinant of grain texture in hexaploid wheat (Triticum aestivum L.). Puroindoline (Pina-D1, Pinb-D1) and Grain Softness Protein (Gsp-D1) genes are tightly linked at this locus. Additional copies of the Gsp-1 gene are present on chromosomes 5A and 5B. Mutations in the Pina-D1 and Pinb-D1 genes have been individually associated with grain hardness, but it is not known if mutations at both loci may further increase hardness or if additional copies may reduce it. In addition, there is no clear evidence of the effect of the Gsp-1 genes on grain texture. To answer these questions, we compared the effect of different dosages of puroindoline and Gsp-1 genes on grain texture. Isogenic substitution and deletion lines for homoeologous group 5 in ‘Chinese Spring’ (CS) were evaluated in two replicated field trials with 13 blocks each. Deletions or allelic variants of Gsp-A1 and Gsp-B1 did not produce significant effects on grain texture, suggesting that these genes do not have a critical role in grain hardness. Simultaneous deletions of Pina-D1 and Pinb-D1 in deletion line 5DS-2 and substitution line CS (Red Egyptian 5D) resulted in significantly higher hardness index values than all other lines including CS (Timstein 5D) carrying a single Pina-D1 deletion (P = 0.02). The incorporation of additional copies of Pina-A^m1 and Pinb-A^m1 from T. monococcum L. in recombinant substitution line 5A/5A^m in CS resulted in significantly softer grains than those from the CS control (P < 0.01).

9. Expression of wild-type pinB sequence in transgenic wheat complements a hard phenotype. Beecher B, Bettge A, Smidansky E, Giroux, MJ. In: Theoretical and Applied Genetics, 2002, 105: 870-877.

Wheat grain hardness is a major factor in the wheat end-product quality. Grain hardness in wheat affects such parameters as milling yield, starch damage and baking properties. A single locus determines whether wheat is hard or soft textured. This locus, termed Hardness (Ha), resides on the short arm of chromosome 5D. Sequence alterations in the tryptophan-rich proteins puroindoline a and b (PINA and PINB) are inseparably linked to hard textured grain, but their role in endosperm texture has been controversial. Here, we show that the pinB-D1b alteration, common in hard textured wheats, can be complemented by the expression of wild-type pinB-D1a in transformed plants. Transgenic wheat seeds expressing wild-type pinB were soft in phenotype, having greatly increased friabilin levels, and greatly decreased kernel hardness and damaged starch. These results indicate that the pinB-D1b alteration is most likely the causative Ha mutation in the majority of hard wheats.

10. Quantitative Trait Loci Associated with Kernel Traits in a Soft x Hard Wheat Cross. Campbell KG, Bergman CJ, Gualberto DG, Anderson JA, Giroux MJ, Hareland G, Fulcher RG, Sorrells ME, Finney PL.In: Crop Science, 1999, 39:1184-1195.

11. Triticum aestivum puroindolines, two cystine-rich seed proteins: cDNA sequence analysis and developmental gene expression Gautier MF, Aleman ME, Guirao A, Marion D, Joudrier P. In: Plant Molecular Biology, 1994, 25: 43-57.

From a mid-maturation seed cDNA library we have isolated cDNA clones encoding two Triticum aestivum puroindolines. Puroindoline-a and puroindoline-b, which are 55% similar, are basic, cystine-rich and tryptophan-rich proteins. Puroindolines are synthesized as preproproteins which include N- and C-terminal propeptides which could be involved in their vacuolar localization. The mature proteins have a molecular mass of 13 kDa and a calculated isoelectric point greater than 10. A notable feature of the primary structure of puroindolines is the presence of a tryptophan-rich domain which also contains basic residues. A similar tryptophan-rich domain was found within an oat seed protein and a mammalian antimicrobial peptide. The ten cysteine residues of puroindolines are organized in a cysteine skeleton which shows similarity to the cysteine skeleton of other wheat seed cystine-rich proteins. Northern blot analysis showed that puroindoline genes are specifically expressed in T. aestivum developing seeds. No puroindoline transcripts as well as no related genes were detected in Triticum durum. The identity of puroindolines to wheat starch-granule associated proteins is discussed as well as the potential role of puroindolines in the plant defence mechanist

12. Genetic and physical characterization of grain texture-related loci in diploid wheat. Tranquilli G, Lijavetzky D, Muzzi G, Dubcovsky J. In: Molecular and General Genetics, 1999, 262: 846 - 850.

Endosperm texture, i.e. the hardness or softness of the grain, is an important quality criterion in cereals because it determines many grain end-use properties. Grain softness is the dominant trait and is mainly controlled by the Ha locus on the short arm of chromosome 5D in hexaploid bread wheat. Genes for puroindoline a (Pina-D1), puroindoline b (Pinb-D1), and grain softness related protein (Gsp-D1) have been shown to be linked to the Ha locus in different mapping populations and have been associated with the expression of grain softness. The study of the linkage relationships among these genes has been limited by the low level of polymorphism in the D genome of hexaploid Triticum aestivum. In the present study, a highly polymorphic Triticum monococcum mapping population was used to analyze linkage relationships among these three genes. Gsp-A^m1 and Pina-A^m1 were found to be completely linked and lie 0.14 cM distal to Pinb-A^m1 in the distal region of the short arm of chromosome 5A^m. The tight genetic linkage among these three genes was paralleled by their physical proximity within a single 105-kb clone isolated from a T. monococcum bacterial artificial chromosome (BAC) library. A restriction map of this BAC clone showed that Pina-A^m1 is located between Pinb-A^m1 and Gsp-A^m1. Partial sequences of the T. monococcum genes showed a high degree of similarity with their T. aestivum counterparts (> or =94%). Marker-assisted selection strategies based on the tight linkage among Ha-related genes are discussed.