Evaluation of altered starch mutants and identification of candidate genes responsible for starch variation in wheat
BMC Plant Biology volume 23, Article number: 377 (2023)
Induction of mutation through chemical mutagenesis is a novel approach for preparing diverse germplasm. Introduction of functional alleles in the starch biosynthetic genes help in the improvement of the quality and yield of cereals.
In the present study, a set of 350 stable mutant lines were used to evaluate dynamic variation of the total starch contents. A megazyme kits were used for measuring the total starch content, resistant starch, amylose, and amylopectin content. Analysis of variance showed significant variation (p < 0.05) in starch content within the population. Furthermore, two high starch mutants (JE0173 and JE0218) and two low starch mutants (JE0089 and JE0418) were selected for studying different traits. A multiple comparison test showed that significant variation in all physiological and morphological traits, with respect to the parent variety (J411) in 2019–2020 and 2020–2021. The quantitative expression of starch metabolic genes revealed that eleven genes of JE0173 and twelve genes of JE0218 had consistent expression in high starch mutant lines. Similarly, in low starch mutant lines, eleven genes of JE0089 and thirteen genes of JE0418 had consistent expression in all stages of seed development. An additional two candidate genes showed over-expression (PHO1, PUL) in the high starch mutant lines, indicating that other starch metabolic genes may also contribute to the starch biosynthesis. The overexpression of SSII, SSIII and SBEI in JE0173 may be due to presence of missense mutations in these genes and SSI also showed overexpression which may be due to 3-primer_UTR variant. These mutations can affect the other starch related genes and help to increase the starch content in this mutant line (JE0173).
This study screened a large scale of mutant population and identified mutants, could provide useful genetic resources for the study of starch biosynthesis and genetic improvement of wheat in the future. Further study will help to understand new genes which are responsible for the fluctuation of total starch.
The quality of wheat flour evaluates based on endosperm starch contents and its composition. The endosperm starch that is approximately 70% dry weight of seed has significant effect on the yield and quality with showing capacity of sink tissues in wheat . The starch granule is developed by composition of amylose (25%) and amylopectin (75%) . By using different breeding approaches or through mutations in genes, we can change the percentage of amylose and amylopectin in resources and improve the quality of wheat. Such as suppression of GBSSI gene to produce partial waxy wheat to create less amylose content which help in production of good quality noodles . By using different approaches such EMS treatment, gene editing and functional genomics tools provide high starch wheat varieties [4, 5]. Similarly, with the increment of amylose content in wheat help to provide additional health benefit such as obesity, cardio-vascular and brain tissues disease .
Glucose-1-phosphate play important role in the amyloplast during starch biosynthesis. The initiation of starch biosynthesis by ADP-glucose pyrophosphorylases (AGPase) with help of other enzymes (SSs, SBEs, GBSS, ISA) in the seed . The formation of amylose initiated by granule bound starch synthase (GBSS) and amylopectin formation done with the coordination of starch synthase, starch branching enzymes and debranching enzymes . Different genes involved in the modulation of amylose and amylopectin ratio during starch biosynthesis by using different breeding and biotechnological approaches in which gene editing, transposon, insertion, and RNAi . There was decline in total starch and amylopectin content due to missense mutation in the TaAGPL1-A, while overexpression of TaGPL1 and TaGPS1 helped to increase total starch and thousand grain weight in wheat . A waxy wheat produced due to knockout of all three genes of GBSSs and all the starch mainly composed of amylopectin in the endosperm of wheat . Through RNAi for SSI in wheat help to increase the concentration amylose content. Similarly, inactivation of SSII also increased the amylose content in wheat  The mutation in TaSSIVb-D had no significant affect at the reserve starch but there was less granules in leaves . Similarly, due to double mutant (agp.L-B1/ssIVb-D), grain starch and amylopectin contents were less than other single mutants in wheat . The formation of starch is a complex process and identified candidate genes with unknown factors help in the formation of reserve starch in the cereal crops .
Phenotypic variation has been produced in cereal crops through chemical agent. One of the most frequently use chemical agent is ethyl methane sulphonate (EMS) in cereal crops . EMS works as an alkylating agent by affecting Guanine (G), due to which mispairing with thiamine (T) instead of cytosine (C) and transition happen from G/C to A/T. EMS mutation is preferable than other biotechnological approaches because it provides multiple alleles of a specific gene within the small population. Many starch biosynthesis genes have been used to produce novel variations through EMS-induced mutagenesis. Through EMS mutation, partial and complete waxy wheat had been produced by targeting the GBSSI gene . Furthermore, mutation in SSIIa, SBEIIa and SBEIIb used to produce high and low starch mutants . To find out new genes/QTL is a challenging task from the natural population . So for this there is need to make near isogenic lines or other functional genomic tools such as genome editing .
In the present study, a set of 350 stable mutant lines were screened for total starch content. The parent variety of this stable population is J411. Further, two high starch mutant lines and two low starch mutant lines were used to study quantitative gene expression patterns of 19 starch metabolic pathway genes during seed development. The exome sequence had been done to know the allelic variations in these selected lines. Overall workflow is given in the Fig. 1.
Screening of mutants with altered seed starch content
Through starch analysis, it’s indicated that the total starch contents in mutant lines were stable and consistent. Similarly, analysis of variance showed significant variation between the mutant lines. Most of lines showed significant variations (60%) for total starch contents from the parent variety. The lowest value for total starch content was 48.2% and the highest value in the mutant lines was 80.6%, while the value of WT (J411) was 67.1%. Gluten, Protein, and hardness of seed also showed variation within the population. The lowest seed hardness value was 92% and highest was 120% while the value of WT was 96%. The maximum mutant lines whose values were 39% for gluten while the minimum value for protein was 14%. The results of these 350 lines are given in Fig. 2.
Identification of high and low starch lines
Two high starch (JE0173 and JE0218) and two low starch (JE0089 and JE0418) mutant lines had been selected from mutant population. The average low starch was 50% and 52% in two mutant lines JE0089 and JE0418 respectively for both year data while highest starch was 76% and 77% in mutant lines JE0173 and JE0218 respectively, and average value of J411 was 66% in 2020 and 2021. These lines showed significant difference from J411 in two years data. Further, amylose content of JE0089 was significantly lower in both year of data while amylose content of high starch mutant line JE0218 was higher as compared to WT but there was lower amylose content for JE0173 mutant line. Similarly, amylopectin content of all mutant lines showed significant difference from WT. The resistant starch of high starch mutant lines showed significant difference from WT and both lines had higher resistant starch contents than low starch mutant lines. Highest resistant starch was observed in the JE0218 line. There was significant difference in all mutant lines as compared to WT for digestible starch. Protein contents was also higher in the high starch mutant lines. Similarly, gluten was high in high starch lines and less in less starch lines. Clear and significant differences (p < 0.05) were recorded in the case of physiological and biochemical parameters. The results of two years selected lines have been given in the Table 1.
Phenotypic characteristics of mutants
By taking pictures of the WT(J411) and mutant plants of wheat at heading stage, the mature plants were investigated for different biological traits, and the mature grains were photographed (Fig. 3). Grain length and grain width showed in the images of low starch mutants (JE0089 and JE0418) and high starch mutants (JE0173 and JE0218) with comparison of WT (J411). Clear and significant differences (p < 0.05) were recorded in the case of morphological parameters. The results showed that low starch mutants and high starch mutants had significant effects on the four yield-related traits in wheat, namely, TGW, grain width, grain length and grain area in two years data. The grain width of JE0218 was higher than all mutant lines, while the Grain length of JE0173 was higher than all mutants. The TGW was higher of both high starch mutants in which the average TGW of JE0173 was 45.4 g and JE0218 had 46.6 g, while WT had 40.2 g. The gluten content of JE0218 was also higher than all other lines in both year of data (Table 2; Fig. 3).
Impact at morphology of starch granules in selected mutants
TM4000 scanning electron microscopy was used to find out the difference of grain morphology between WT (J411) and high starch mutants (JE0173, JE0218) and low starch mutants (JE0089, JE0418) (Fig. 4). The pure starch was extracted from the seeds by crushing them in the grinder. It was observed that in the low starch mutants (JE0089 and JE0418) that the starch granules were reduced and decreased in numbers with less A-granules and B-granules (Fig. 4). Similarly, there was significant difference in size and number of grains as compared to WT in the high starch mutant lines (JE0173 and JE0218). The number of A- granules and B-granules were higher in the high starch mutant lines.
Expression pattern of starch metabolic genes in high starch mutant lines
The comparatives expression of nineteen genes in high starch mutant lines (JE0173 and JE0218) showed that eleven genes of JE0173 and twelve genes of JE0218 had consistent expression in all stages of seed development (Fig. 5). While eight genes of JE0173 and seven genes of JE0218 expression pattern was inconsistent in four stages of seed development. From these eleven genes of JE0173, eight genes showed overexpression at four seed development stages and three genes (AMY, BMY, SUT) showed reduced expression in all stages while, from 12 genes of JE0218, nine genes showed overexpression and three genes (AMY, ISA3, SUT) had reduced expression (Fig. 5).
Expression pattern of starch metabolic genes in low starch mutant lines
The low starch mutant’s expression analysis had been conducted. The quantitative gene expression analysis showed that from nineteen genes of starch biosynthesis, eleven genes of JE0089 and thirteen genes of JE0418 had consistent expression in four stages of seed development (Fig. 6). Five genes (GBSSI, SSI, SBEI, ISA2, AGPase S) of JE0089 showed reduced expression and six genes (SSIII, SBEII, AMY, ISAI, PHO1, PHO2) had over expression from these eleven genes. Similarly, from the thirteen genes of JE0418, six genes (SSI, SBEI, ISA2, SUT AGPaseS, SSIV) had reduced expression while seven genes (GBSSII, SSIII, SBEII, BMY, ISA1, PHO1, SSII) had over-expression in all seed development stages. The expression pattern of remaining starch synthesis genes in both mutant lines were inconsistent by showing high and reduced expression in different stages of seed development. In the present study, the low expression of TaRSR1 in the low starch mutant lines may be this gene had no effect in starch metabolism. This differential expression analysis in four mutants give the idea that there is involvement of other starch metabolic pathway genes such as phosphorylase, isoamylase etc., in the starch biosynthesis.
Comparison of SNPs and indels of the wheat mutant lines
Exon sequencing had been done for selected high starch mutant lines (JE0173 and JE0218) and low starch mutant lines (JE0089 and JE0418). It was depicted that in the mutant lines, the number of SNPs were more in all four mutant lines than Indels. The number of SNPs in JE0418 was higher than other three mutants. But Indels were almost in equal amount in all mutant lines. The different types of SNPs between the mutants suggested that there were more transitions than transversions in the four mutants (figure S1). There were also fewer synonymous mutations in all mutant lines (figure S1).
From four mutant lines, twenty-two variants were identified in nineteen starch biosynthesis genes. Ten variants were discovered in JE0173 in which four were missense mutation (SSIII, SBEI, GBSSI, SSII) and four were synonymous mutation (ISA1, PHO2, TaRSR1, SBEIII) while remaining two were 3_prime_UTR variant (SSI) and splice region variant (SSII). JE0218 had two variants that consist of one synonymous (AMY) and other was missense (TaRSR1) mutations. Similarly, JE0089 and JE0418 had six and four variants respectively. (Table 3). Three missense (SSI, PHO2, PUL) mutations in JE0089 and two (SBEIII, SSII) in the JE0418 had been observed, while two were synonymous in both lines.
The overexpression of SSII, SSIII and SBEI in JE0173 may be due to presence of missense mutations in these genes and SSI also showed overexpression which may be due to 3-primer_UTR variant. These mutations can affect the other starch related genes and help to increase the starch content in this mutant line (JE0173). Similarly, in JE0218, there was missense mutation in TaRSRI and its showed overexpression as compared to WT (J411). In the low starch mutant line JE0089, SSI and PUL had reduced expression and these genes had missense mutation. PUL also had missense mutation, but the expression of this gene was inconsistent. So, these genes which showed reduced expression and other starch related genes might be the responsible for low starch contents in this mutant line. Similarly, in JE0418, SBEIII had reduced expression due to missense mutation and this mutation might be responsible for low starch content in this mutant line.
Modification of starch composition in wheat help to increase starch content and yield of the crop which ultimately effect on the public health due to good quality of flour. Different chemical mutagenesis have been used in different crop for studying the functional genomic and breeding . Different research has proved that using mutants with altered phenotypes help to know the function of specific gene in an organism. In the present study, 350 stable mutants were used for screening total starch contents and find out low starch mutant lines and high starch mutant lines from this stable mutant population. Different literature proved that EMS treated lines were used to find out mutations for candidate genes in diploid, tetraploid, and hexaploid wheat [17,18,19]. Alteration of starch contents in different cereal crops especially in wheat is getting more attention because starch play important role in the food and non-food applications. By using EMS mutant stable lines which contained multiples variations in gene pool can help us to find out the functional mutations in the candidate genes which can be difficult to find in natural population due to limited variations in natural population . These mutagenesis can directly applied to superior elite cultivars and backcrosses with parent variety help to remove unnecessary mutation by introducing novel allele in to the new line .
The deposition of protein increased or decreased due to mutation and there will be significant reduction of amino acids in seed tissues exposed to EMS chemical . The deposition of protein in grain filling stage of wheat is earlier than starch deposition in grain filling stage which normally start after 12 days of anthesis, . The deposition of protein in cereal crops normally initiated 8 to 10 days after anthesis and reaches its peak position in 20 days while deposition of starch start at the same time but its complete deposition need at least 45 days . Hence it is depicted that starch and protein deposition in the cereal is an independent event which controlled by sink and source limited process respectively. Recent studies have proved that not only plant hormone transduction but also starch and sucrose metabolism play significant role in the grain size and grain weight during grain development in the cereal . In the present study, the grain size and 1000 grain weight of high starch lines were higher than the WT. Normally, the total starch contents and TGW normally increase in initial grain filling stage but totally ceased at the end of grain filling stage. Similarly in the present study the correlation between total starch and TGW and between total starch and resistant starch showed similar results as reported in the previous studies .
The key genes of starch biosynthesis such as AGP-S1, SS1, SSIIa, SSIIIa, SBEIIa, SBEIIb, and GBSSIa expression level play significant role in starch biosynthesis and accumulation. Changing of expression pattern of these main genes may have positive affect on the grain weight of the wheat during grain filling stages [25, 26]. In the present result, high starch mutants have higher expression of the main genes (GBSSII, SBEII, AGPaseSS and SSs) as compared to low starch mutants during grain filling stages may lead to accumulation of high starch . Amylose content increase due to overexpression of GBSSI. Similarly, due to silent and null mutants produce waxy and partial waxy wheat with low amount of total starch and lacking amylose contents [28, 29]. With the inhibition of ISA help to reduction in the amylopectin contents in wheat , while silencing of SBEII also increased amylose content with reduced total starch contents . Therefore, these results support that the overexpression of main starch genes help to increase total starch contents in high starch mutant lines.
Amylases (AMY and BMY) with phosphorylases (Pho1 and Pho2) play significant role in starch metabolism along with hydrolysis and degradation . These are the main genes which play important role in the starch structure and their grain morphology. Alteration of starch structure was observed in rice and potato by silencing of these genes . Similarly, overexpression of AMY and BMY fluctuate the starch structure and baking quality in wheat . A negative transcription factor (RSR1) had been observed during co-expression in the rice which negatively regulate the some starch biosynthesis genes and modulate the starch metabolism . Therefore, it can say that the accumulation of amylopectin in low starch mutant may be the overexpression of key genes of starch biosynthesis. The differential expression of starch main genes in low starch mutants and high starch mutants give the idea that the involvement of other starch metabolic genes such as phosphorylase during synthesis of starch.
Material and method
In 2019–2020, a stable mutant population that consist of 350 lines, were grown in the field with three replications at the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing. This mutant population was developed by Institute of Crop Sciences, Chinese Academy of agricultural Sciences by using the ethyl methane sulphonate chemical (EMS) and other mutagens to the J411 variety. J411 is the hexaploidy modern cultivar in China. After selecting the lines based on different traits, stable lines were developed. After maturing, seed samples from each mutant lines were collected and crushed these seeds to make the flour for studying the total starch contents and other physiological traits (table S1). Furthermore, selected lines were again sown in the field in 2020–2021 and collect the seed samples to study the morphological and physiological traits.
Determination of total starch content
For measuring the total starch content of 350 samples, Megazyme kit that was based on AOAC Method 996.11, AACC Method 76-13.01 was used. We followed the instruction mentioned in the kit for measuring the total starch content .
Resistant and digestible starch
A commercial Megazyme kit (K-RSTAR 05/19, Megazyme Int., Wicklow, Ireland) was used for measuring the resistant starch and digestible starch from the flour of the wheat. Instruction was followed according to the kit guidance .
The amylose/amylopectin megazyme kit (K-AMYL 06/18, Megazyme Int., Wicklow, Ireland) was used for measuring the amylose content of wheat flour . The protocol was followed according to the instruction of the manufacturer kit. Amylose and amylopectin were measured in percentage.
Protein, gluten content and seed hardness
Grain analyzer had been used to measure the protein, gluten, and seed hardness. FOSS InfratecTM 1241 Grain Analyzer (FOSS Analytical AB, Sweden) was used for scanning NIR spectra. WinISI II v1.50 (InfraSoft International LLC, 2000) software was used to find out the final reading . All these traits were measured in the percentage.
Investigation of agronomic traits
Samples of seed from each mutant lines and WT were collected with three replications for measuring the grain width, grain length, grain area and thousand grain weight (TGW). SmartGrain was used for evaluating these traits by following methodology as described in previous studies [38, 39]. TGW was measured in gram, while width and length were measured in millimeters.
Purification of starch for studying grain morphology
Seeds of mutant samples were crushed for extracting pure starch. The methodology was followed for starch purification as describe in somewhere else [11, 40]. Granule morphology was observed using a Nova NanoSEM 450 (FEI) scanning electron 133 microscope (SEM).
Mutant lines exon sequencing
Selected mutants were used to capture the genetic diversity. The 4 mutant pools were subjected to exon capture and sequencing to compare the polymorphisms between different pools. The process of exon sequencing was followed as describe in Irshad et al.,.
Expression analysis of mutant lines
Nineteen genes had been selected for quantitative expression analysis during different grain filling stages. These genes consist of starch synthase, starch branching enzymes, debranching enzymes, amylase, phosphorylase, and transcription factors. These genes play important role in amylose and amylopectin biosynthesis as well as total starch biosynthesis. Thirteen was main starch biosynthesis genes in which one was small subunit of ADP-glucose pyrophosphorylases (AGPaseS), while four starch synthase isoforms (SSI, II, III, IV), three were starch branching isoforms (SBEI, II, III) and remaining consisted of granule bound starch synthase (GBSSI) with three starch debranching isoforms and pullunase (ISA1, ISA2, ISA3 and PUL). Further we selected four starch degrading genes (Pho1, Pho2, BMY and AMY). Two transcription factors were also added for expression analysis that play role in starch biosynthesis (SPA and TaRSR1).
Expression analysis had been done in two low starch mutant lines (JE0089 and JE0418), two high starch mutant lines (JE0173 and JE0218), and the parent wheat variety ‘J411’. Samples of these mutants and parent variety were collected after 6, 12, 18, and 24 days anthesis (DAA). These samples were quickly put in the liquid nitrogen after taking from spike and then shifted to the − 80c refrigerator for RNA extraction. The detailed protocol of RNA extraction, cDNA formation and qRT-PCR followed by as described by Zhang et al., (2019). In addition, Actin (GenBank accession no: AAW78915), a housekeeping gene, was used as an internal control. Similarly, genes and their primer information was obtained from Singh et al., . Three biologicals with two technical replications had been used for quantitative expression of these nineteen genes by using 7500 Fast Real-Time PCR System (Applied Biosystems, Forster City, CA, USA). The ∆∆CT method was used to analyze the relative expression level.
Microsoft excel formulas had been used for calculating the mean and standard deviation. One-way analysis of variance (ANOVA) was used to study variation in total starch, in the replications for the mutant lines. To test the significant difference between mutants and parent variety for the above traits, Dunnett’s test had been performed. For the purpose of these analyses, three biological replications were used for studying all of these traits.
In this study, 350 mutant lines were used and mutants with starch content variations were identified, this useful germplasm can be used for a genome-wide study and help to improve the starch-based nutritional quality in wheat. The gene expression of nineteen starch metabolic genes in two high starch mutants and two low starch mutants indicate that with starch main genes, there may be other genes (phosphorylases, pullulanases and isoamylases) responsible in starch biosynthesis. Further exon sequencing of these diverse mutant lines gives the information for further studies. There is a need to find out about starch metabolic genes through the backcrossing of these mutant lines and a developed NILs population for mapping.
All data generated or analysed during this study are included in this published article [and its supplementary information files].
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All authors acknowledged Dr. Shoaib Ur Rehman, Mian Nawaz Sharif University, Multan, Pakistan for guiding during research work.
The work is supported by the Crop Varietal Improvement and Insect Pests Control by Nuclear Radiation, China Agriculture Research System of MOF and MARA (CARS-03), and the Agricultural Science and Technology Innovation Program (CAAS-ZDRW202002) of China.
The authors declare no competing interests.
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Irshad, A., Guo, H., Xiong, H. et al. Evaluation of altered starch mutants and identification of candidate genes responsible for starch variation in wheat. BMC Plant Biol 23, 377 (2023). https://doi.org/10.1186/s12870-023-04389-3