Qiqi ZHANG

Crop Research Institute, Anhui Academy of Agricultural Sciences/Anhui Province Key Laboratory of Crop Quality Improvement, Hefei 230031, China

Abstract In this paper, the research progress of wheat grain hardness was reviewed from the aspects of wheat grain hardness formation, determination methods, biochemical genetic basis, puroindoline mutation type and distribution and its effect on wheat processing quality, and the frontier of transgenic research was introduced, in order to provide basic theoretical support for controlling and improving wheat grain hardness.

Key words Wheat, Grain hardness, Puroindoline

1 Introduction

China is the largest country in wheat production and consumption in the world, and occupies a pivotal position in the world grain market. In recent years, with the development of agricultural production and the increase of agricultural products, the commodity rate of wheat is getting higher and higher, and people in urban and rural areas put forward higher and higher requirements for wheat quality. Grain hardness is not only one of the important bases for wheat classification and pricing in wheat markets at home and abroad, but also one of the important breeding target traits. Some high-quality wheat varieties popularized and planted in China have reached the level of first-class bread wheat in protein content and gluten strength. However, because of its insufficient hardness, its processing quality and commercial value have been greatly reduced. Grain hardness is an important target character for wheat quality improvement, which determines wheat milling and other processing qualities by affecting flour yield, flour grain size, water content and the number of damaged starch grains. According to the endosperm, common wheat is divided into hard wheat and soft wheat. Hard wheat flour has large grain size, high damaged starch content and strong water absorption capacity, so it is suitable for making bread, high-quality noodles and other foods. Soft wheat flour has small grain size, low damaged starch content and weak water absorption capacity, so it is suitable for making sweet foods such as biscuits and pastries[1-2].

2 Wheat hardness and its formation

Endosperm accounts for about 80% of wheat grain volume, and its main components are starch granules and storage proteins. Starch granules are synthesized in the amyloplast, and the seed storage protein is precipitated in the encapsulated membrane protein body. During the wheat endosperm cell maturation, the protein bodies fuse to form a continuous protein matrix, while the starch granules can remain intact because they are surrounded by the residual amyloplast membrane. Barlow et al tested the hardness of starch granule and stored protein matrix respectively, and there was no significant difference between soft wheat and hard wheat. When wheat grains are immature, they are all horny. In the process of maturation, due to the loss of water, the binding ability of starch grains to protein matrix in endosperm gradually changes. The combination ability of the two is strong in hard wheat endosperm and can maintain horny endosperm. In soft wheat endosperm, the combination ability of the two is weak and easy to separate. After maturity, farinaceous endosperm is often formed, thus causing the difference in hardness values between hard wheat and soft wheat[3]. Bechteletal. put immature hard wheat at room temperature and low temperature, respectively. After drying, it was found that the seeds placed at room temperature formed hard endosperm, while those placed at low temperature formed soft endosperm. This further verified that the endosperm hardness was formed in the process of grain dehydration and drying. Therefore, after grinding, soft wheat has more free starch grains and fine flour, while hard wheat adheres to the protein matrix because of the strong binding ability between starch grains and protein matrix. More damaged starch granules are formed in the grinding process, and the flour is rougher than soft wheat[4]. According to the study of Stenvertetal., grain hardness is mainly determined by the continuity and structure of protein matrix and the ability of starch granules to be coated. In hard wheat, the protein matrix is continuous and starch granules are trapped in it, so it is difficult to separate the two. In soft wheat, the protein matrix is not continuous, and the starch grains coated with it are easy to leak out from the surrounding gaps, resulting in the weakening of the binding ability of the two. Thus it can be seen that the grain hardness is mainly determined by the adhesion between starch and protein matrix and the continuity of protein matrix coated with starch grains[5].

3 Determination method of wheat hardness

At present, according to the difference of pressure, grinding power consumption, grinding time and absorbance, the hardness is mainly determined by hardness percentage method, pressure method, grinding method and near-infrared method. And grain index method, near-infrared spectroscopy, single-grain characteristics determination method and so on are the most widely used. The system for measuring the characteristics of single grain was established by the Grain Market Research Office of the United States and the Swedish Perten Instrument Company. It crushes the grain by the action of pressure, and then determines the hardness of the sample by the sensing stress of the sensor. The 1 000-grain weight, diameter, hardness index and water content can be measured at the same time. Generally, the wheat with hardness index less than 40 is soft wheat, the wheat with hardness index more than 60 is hard wheat, and the wheat with hardness index of 40-60 is mixed wheat. This method has the advantages of fast detection, simple operation, reliable data and good repeatability. In recent years, it has been widely used in wheat market classification and has become an important instrument for testing hardness.

4 Biochemical characteristics and genetic basis of wheat hardness

4.1 Friabilin and puroindolineA major breakthrough in the study of the biochemical basis of grain hardness is the discovery of friabilin. Greenwelletal.[6]isolated a 15 kD protein from the surface of washed wheat starch and separated it by SDS-PAGE. This protein was rich in soft wheat water-washed starch, relatively less in hard wheat starch, and completely missing in hard wheat. They found that the gene that expressed the protein was on chromosome 5D, and the protein was later named friabilin. There was a corresponding relationship between grain hardness and friabilin content in 53 of 56 materials, and the expression of friabilin in protein band of soft wheat was about 10 times higher than that of hard wheat. After protein separation and identification by electrophoresis and chromatography, it was found that friabilin may be composed of multiple protein components. Morrisetal.[7]found two proteins with a molecular weight difference of only 0.7 kD when conducting SDS-PAGE analysis of friabilin whole protein (no β-mercaptoethanol was added in the extract). With the gradual deepening of the study of friabilin protein, its composition is also known. Blochetetal. isolated a protein soluble in TritonX-114 from wheat flour. After reversed-phase high performance liquid chromatography (RP-HPLC) analysis, six main peaks appeared. Among them, the amino acid group of three main peaks was purine sulfur, and two of the other three peaks "peak5" and "peak7" had high homology, and the content of "peak5" was high. Based on the sequence analysis of "peak5", it was found that there was a region rich in tryptophan, which was named "puroindoline". Marionetal. designed an oligonucleotide based on the puroindoline sequence and used it as a probe to isolate two cDNA clone families. Sequencing results showed that one corresponded to "peak5" and was named puroindoline a (Pina) and the other corresponded to "peak7" and was named puroindoline b (Pinb). The homology of Pina and Pinb protein sequences was about 60%. The study of the structure of puroindoline protein lags behind, and the tertiary structure is not clear at present. After comparing puroindoline with lipid transfer protein (LTP) with known tertiary structure, Marionetal. found that the two sequences had high homology except for the corresponding tryptophan-rich region. Using the tertiary structure of LTP as a template for structural analysis, it can be inferred that the puroindoline protein is likely to contain four helices. Each helix is connected by a loop, and the Trp-rich region is located in the loop between the first helix and the second helix, connected by a disulfide bond.

4.2 Types and distribution of puroindoline mutationsHardness is controlled by a major gene (Ha) and some minor genes. The major genes are dominant and located on the short arm of chromosome 5D, while the minor genes are located on the short arms of chromosome 5A and 5D. Ha encodes a 15kD protein complex Friabilin. The difference in grain hardness of wheat varieties is caused by different variants of Pina and Pinb genes. Non-expression of Pina or mutation of Pinb gene sequence will lead to the hardening of wheat endosperm texture. The difference in grain hardness of wheat varieties is caused by different variation types of Pina and Pinb genes. After studying two hard wheat varieties, Gautieretal. found that there was a mutation in one site of the Pinb gene relative to the wild type sequence, which led to the transformation of Gly at position 46 in the corresponding amino acid sequence into Ser, and this type of mutant gene was named Pinb-D1b. Girouxetal. found the complete deletion type of Pina protein in hard wheat variety Falcon, and named it Pina-D1b, corresponding to the wild type named Pina-D1a. Subsequently, Pinb-D1c, Pinb-D1d, Pinb-D1e, Pinb-D1f and Pinb-D1g were also named. Recently, Morrisetal. found six new Pina mutation types and seven new Pinb mutation types in Aegilops: Pina-D1c, Pina-D1d, Pina-D1e, Pina-D1f, Pina-D1g, Pina-D1h; Pinb-D1h, Pinb-D1i, Pinb-D1j, Pinb-D1k, Pinb-D1m, Pinb-D1n, Pinb-D1o. These were multiple point mutations, showing soft nature[8-10].

At present, a large number of puroindoline allelic variants have been found in common wheat. There are 10 allelic variation types in Pina and 18 allelic variation types in Pinb. There are significant differences in milling and food processing quality among wheat varieties with different puroindoline genotypes. The results showed that Pinb-D1b type wheat was slightly better than wild type and Pina-D1b type wheat in terms of milling and processing quality of steamed bread, noodles and bread. Based on the detection of puroindoline molecular markers, the distribution of different variation types was also studied. Xiaetal. found that Pinb-D1b accounted for 70.2% of hard wheat in the hardness investigation of 251 wheat samples in China, and found that 10 varieties had new mutations, named Pinb-D1p. This also indicated that Pinb-D1b was the most common type of mutation in hard wheat. Generally speaking, there are fewer types of Pina deletions and more types of Pinb mutations, and Pinb-D1b is the most widely distributed in different countries. The puroindoline gene is widely found in most crops ofTriticumaestivumandAveneaeDumort., but lacking in maize and rice. The study of Lillemoetal. showed that the gene existed in Einkorn,Triticumurartu,AegilopstauschiiandAegilopsspeltoidesby reverse PCR, but tetraploid hard wheat with hard endosperm lacked this protein. This was probably due to the deletion of the puroindoline gene in the tetraploid wheat varieties during seed domestication about 9 000 years ago, while the soft hexaploid wheat hybrid betweenT.dicoccumandAegilopstauschiiretained the gene. In addition, puroindoline protein was also found in the washed starch of soft rye, triticale and barley, which was similar to that in wheat.

5 Effect of hardness on wheat processing quality

The study on the molecular basis of grain hardness has important guiding significance for the improvement of wheat quality. The studies of Dubreiletal. showed that the addition of puroindoline protein in wheat flour had a significant effect on its rheological properties and bread processing quality. The flour of two wheat varieties FA and EC was recombined. After 0.1% puroindoline protein was added to the flour, it was found that the dough strength and extensibility of EC and FAEC (FA and EC mixed at a ratio of 1∶1) decreased by 8%, 7.4% and 33.8% and 30%, respectively, while FA increased by 40% and 28.5%, respectively. This was beneficial to improving the bread structure and making the opening size distribution even, but it would cause the bread volume to decrease. Martinetal. used 149 recombinant inbred lines crossed by Pina-D1b and Pinb-D1b as materials to study the effects of different variants of Pina and Pinb genes on milling quality and bread quality traits. They found that the wheat grain of Pinb-D1b group was significantly softer than that of Pina-D1b group, the flour yield, milling rate and milling score were higher, the volume of bread was larger, the ash content of flour was lower, but the score of bread structure was lower. Caneetal. studied wheat varieties in southern Australia and analyzed the effects of different types of hard wheat on quality. The results showed that the PSI value and water absorption of wheat with Pina-D1b genotype were higher than those of wheat with Pinb-D1b genotype, but the flour yield and dough formation time were significantly lower and shorter than those of the latter, respectively. He also believed that it was possible to predict the time of dough formation based on the puroindoline gene in the future, which could be used to improve the quality of wheat[11-14].

6 Transgenic study on hardness of wheat

With the rapid development of biotechnology, some progress has been made in the research on transgenic wheat grain hardness. Girouxetal. transferred wild type Pinb-D1a into hard wheat with Pinb-D1b. The transgenic grains showed soft nature, the content of friabilin in washed starch increased significantly, and the grain hardness and the number of damaged starch grains decreased significantly. Beecheretal. transferred the Pinb-D1a sequence of Chinese wild type into hard wheat and made it expressed in endosperm. The hardness of SKCS was reduced from 70 to 25, which was the same as that of typical soft wheat, and the damaged starch percentage was also reduced from 3.71% to 1.69%. Krishnamurthyetal. transferred wild type Pina and Pinb genes into rice, and the endosperm texture of transgenic rice became soft obviously. Hoggetal. transferred wild type gene of wheat hardness into hard red winter wheat. The analysis of Northern hybridization results showed that the grain hardness became softer with the increase of transcripts, and the range of change was related to the expression sequence of original genes and foreign genes. He also believed that the decrease of wheat grain hardness with the increase of Pinb content was greater than that due to the increase of Pina content. The content of friabilin was related to the expression of wild type Pina and Pinb, but not to the total puroindoline protein. The interaction between Pina and Pinb formed friabilin, and the two determined the value of grain hardness. At present, some experts have cloned Chinese wheat hardness major genes Pina and Pinb, and successfully constructed wheat hardness major gene overexpression vector, RNA interference expression vector, fusion gene expression vector and antisense expression vector, and transferred the overexpression vector into conventional varieties by pollen tube pathway method[15]. The above studies show that it is feasible to improve the tissue structure of grains by regulating the expression of puroindoline in crops. This makes it possible for people to control the grain hardness and make the hardness of wheat and other crops develop towards the predetermined goal.