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Acta Agronomica Sinica ›› 2025, Vol. 51 ›› Issue (6): 1514-1525.doi: 10.3724/SP.J.1006.2025.41076

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Functional dissection of sucrose synthase gene TaSUS2 regulating grain starch synthesis and quality in wheat

WU Mei-Juan1,2(), ZHANG Yin-Hui2, LI Yuan-Hao2, LIU Hai-Xia2, HUANG Yi-Lin2, LI Tian2, LIU Hong-Xia2, ZHANG Xue-Yong2, HAO Chen-Yang1,2,*(), GUO Jie1,*(), HOU Jian2,*()   

  1. 1College of Agronomy, Shanxi Agricultural University / Key Laboratory of Sustainable Dryland Agriculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Jinzhong 030801, Shanxi, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / State Key Laboratory of Crop Gene Resources and Breeding / National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing 100081, China
  • Received:2024-11-10 Accepted:2025-03-26 Online:2025-06-12 Published:2025-03-31
  • Contact: *E-mail: haochenyang@caas.cn;E-mail: nxgj1115326@sxau.edu.cn;E-mail: houjian@caas.cn
  • Supported by:
    the National Key Research and Development Program of China(2023YFD1200404)

Abstract:

Wheat is one of the world’s most important cereal crops, and improving yield remains a key goal in wheat breeding. Grain weight is a major determinant of yield, and starch is the primary component of wheat grains. To investigate the function of TaSUS2, a key enzyme gene in the starch synthesis pathway, we amplified its full-length cDNA sequence from the wheat genome and performed gene editing in the cultivar Kenong 199 (KN199). This resulted in the generation of two homozygous diploid mutants (KO-1 and KO-2) and one homozygous triploid mutant (KO-3). Phenotypic analysis of the transgenic lines revealed that TaSUS2 mutant grains exhibited pronounced wrinkling and a significant reduction in grain weight compared to the wild type. Additionally, the total starch content, amylose content, absolute starch content, and the diameter of A-type starch granules in the endosperm were significantly reduced in TaSUS2 mutant grains. These findings confirm that TaSUS2 plays a crucial role in starch synthesis and grain weight determination. Transcriptome analysis indicated that multiple enzyme-encoding genes involved in starch biosynthesis were upregulated in TaSUS2-KO-3 grains at 21 days post-anthesis (DPA). Furthermore, genotyping of a natural population of 145 wheat accessions using the TaSUS2-2A-CAPS marker revealed that TaSUS2 was significantly associated with starch content, wet gluten content, protein content, and sedimentation value. Notably, the TaSUS2-2A-Hap-G haplotype was identified as a favorable allele for these quality traits. Overall, this study provides valuable insights into the biological function of TaSUS2 and offers novel genetic resources for molecular breeding aimed at improving wheat yield and quality.

Key words: wheat, TaSUS2, grain weight, starch content, quality

Table 1

Primers used in this study"

引物用途
Primer use
引物名称
Primer name
引物序列
Primer sequence (5′-3′)
基因扩增 Gene amplification SUS2-2A-F ATGAACAAACGCGGCT
SUS2-2A-R TCATTTGCCCGAGGTC
SUS2-2B-F ATGATCTGCTTCCCAAAGCA
SUS2-2B-R TCATTTGCCCGAGGTC
CAPS标记 CAPS marker SUS2-2A-M1F GCAATAGTTCCGTGCTCCTGTG
SUS2-2A-M1R AGAAATACGCAAGGCAACCAT
SUS2-2A-M2F CACGCCAACCTTTCTTCTTCC
SUS2-2A-M2R TCGCTTAACCGTGTCTCACA

Fig. 1

Gene editing sites of TaSUS2 The editing site of TaSUS2 in Kenong 199. WT (wild-type) shows the targeted region of sgRNA on the third exon of TaSUS2. PAM sequences are highlighted in red. KO-1, KO-2, and KO-3 indicate the genotypes of three mutants, with the following “+” and “-” indicating base insertions or deletions caused by CRISPR/Cas9 editing, and the numbers indicate the number of inserted or deleted bases. The number at the right side of sequences indicates the number of amino acids translated by the gene."

Fig. 2

Phenotypic and quality traits of TaSUS2-KO grains A: grain morphology of WT and TaSUS2-KO. Scale bar: 5 mm. B-F: TGW, GL, GW, GT and YP of WT and TaSUS2-KO. TGW: thousand-grain weigh; GL: grain length; GW: grain width; GT: grain thickness; YP: yield per plant. G: starch granule morphology and starch synthesis of endosperm at 20 DPA in WT and TaSUS2-KO. Scale bar: 20 μm. H: total starch content of grains in WT and TaSUS2-KO. I: amylose content of grains in WT and TaSUS2-KO. J: amylopectin content of grains in WT and TaSUS2-KO. K: absolute starch content of grains in WT and TaSUS2-KO. L: diameter of A-type starch granules in WT and TaSUS2-KO. M: total protein content of grains in WT and TaSUS2-KO. **: P < 0.01; ***: P < 0.001; ns: no significant difference."

Fig. 3

Transcriptome analysis of TaSUS2-KO grains A: comparison of the number of grain transcriptomic differential genes at 7, 14 and 21 DPA in WT and TaSUS2-KO-3. B: GO enrichment of major differential expressed genes at 21 DPA in TaSUS2-KO-3. C: differential expression genes involved in starch synthesis pathway at 21 DPA in TaSUS2-KO-3. DPA: days post-anthesis. SUS: sucrose synthase; FRK: fructokinase; PGI: phosphoglucoisomerase; PGM: phosphoglucomutase; UGPase: UDP-glucose pyrophosphorylase; AGPase: ADP-glucose pyrophosphorylase; BT1: BRITTLE1; GBSSI: granule-bound starch synthase I; SS: starch synthase; SBE: starch branching enzyme; ISA: isoamylase; PUL: pullulanase."

Table 2

Differential expression analysis of auxin signaling pathway genes in TaSUS2-KO-3 at 21 days post-anthesis"

生长素信号通路基因ID
Auxin signalling pathway gene ID
生长素信号通路基因注释
Gene annotation of auxin signaling pathway
差异倍数
Difference multiple
TraesCS2D02G322600 ABC transporter B family member 19 0.39
TraesCS2A02G344300 ABC transporter B family member 19 0.32
TraesCS2B02G341800 ABC transporter B family member 19 0.24
TraesCS2D02G548900 Auxin response factor 12 0.37
TraesCS2A02G547800 Auxin response factor 12 0.46
TraesCS2B02G578500 Auxin response factor 12 0.34
TraesCS7A02G252000 Auxin response factor 21 0.45
TraesCS7B02G138900 Auxin response factor 21 0.43
TraesCS7D02G250100 Auxin response factor 21 0.46
TraesCS5D02G045700 Auxin response factor 25 0.36
TraesCS5B02G039800 Auxin response factor 25 0.37
TraesCS5A02G038300 Auxin response factor 25 0.42
TraesCS6A02G138600 Auxin response factor 6 0.38
TraesCS6D02G127600 Auxin response factor 6 0.35
TraesCS6B02G167100 Auxin response factor 6 0.24
TraesCS1A02G077800 Auxin transporter-like protein 3 0.28
TraesCS1D02G079900 Auxin transporter-like protein 3 0.33
TraesCS1B02G095900 Auxin transporter-like protein 3 0.31
TraesCS5B02G104100 Auxin-binding protein 1 0.39
TraesCS5A02G098900 Auxin-binding protein 1 0.41
TraesCS3B02G181500 Auxin-responsive protein IAA17 0.44
TraesCS7A02G331100 Auxin-responsive protein IAA21 0.47
TraesCS3A02G155200 Auxin-responsive protein IAA3 0.48
TraesCS5D02G069300 Auxin-responsive protein IAA30 0.35
TraesCS5B02G058500 Auxin-responsive protein IAA30 0.31
TraesCS5A02G058600 Auxin-responsive protein IAA30 0.40
TraesCS7A02G384600 Auxin-responsive protein SAUR32 0.47
TraesCS1A02G178700 Auxin-responsive protein SAUR32 0.43
TraesCS6A02G141200 Auxin-responsive protein SAUR71 0.15
TraesCS6B02G169500 Auxin-responsive protein SAUR71 0.43
TraesCS4B02G016500 Auxin-responsive protein SAUR71 0.31
TraesCS7D02G191600 Probable auxin efflux carrier component lc 0.33
TraesCS7A02G190600 Probable auxin efflux carrier component lc 0.37

Table 3

Differential expression of starch synthesis-related genes (SSRG) in TaSUS2-KO-3 at 21 days post-anthesis"

淀粉合成相关酶基因
SSRG
差异倍数
Difference multiple
淀粉合成相关酶基因
SSRG
差异倍数
Difference multiple
淀粉合成相关酶基因
SSRG
差异倍数
Difference multiple
SUS2-2A 0.09 AGPL-1B 1.95 SSIIb-6D 0.90
SUS2-2B 0.14 AGPL-1D 2.10 SSIII-1A 1.89
SUS2-2D 0.17 AGPS1-7A 1.51 SSIII-1B 2.22*
SUS1-7A 1.47 AGPS1-7B 2.98* SSIII-1D 1.76
SUS1-7B 1.25 AGPS1-7D 2.50* SBEI-7A 0.79
SUS1-7D 1.33 BT1-6A 2.35* SBEI-7B 1.06
UGP-5A 1.25 BT1-6B 2.03* SBEI-7D 0.92
UGP-5B 1.31 BT1-6D 2.73* SBEIIa-2A 2.11*
UGP-5D 1.19 GBSSI-4A 1.52 SBEIIa-2B 1.90
FRK-7A 1.55 GBSSI-7A 1.98 SBEIIa-2D 1.72
FRK-7B 1.68 GBSSI-7D 1.65 SBEIIb-2A 2.13*
FRK-7D 1.37 SSI-7A 1.60 SBEIIb-2B 2.18*
PGI-1A 0.92 SSI-7B 1.48 SBEIIb-2D 2.57*
PGI-1B 1.31 SSI-7D 1.79 ISA-7A 1.58
PGI-1D 1.28 SSIIa-7A 2.73* ISA-7B 1.66
PGM-4A 1.22 SSIIa-7B 2.02* ISA-7D 1.39
PGM-4B 1.33 SSIIa-7D 2.37* PUL-7A 1.01
PGM-4D 1.28 SSIIb-6A 0.75 PUL-7B 1.64
AGPL-1A 1.47 SSIIb-6B 0.62 PUL-7D 1.96

Fig. 4

TaSUS2-2A-CAPS marker development and significance of difference analysis between haplotypes and quality traits in a 145 natural population A: the TaSUS2-2A-CAPS marker development. The PCR product of TaSUS2-2A-Hap-A amplified by TaSUS2-2A-MF/R was 341 bp after enzyme digestion of Asc I, while the product of TaSUS2-2A-Hap-G was 175 plus 166 bp after enzyme digestion. B-G: significance of difference analysis of different haplotypes of TaSUS2-2A and quality traits in a 145 natural population. B: total starch content (SC); C: amylose content (AC); D: testweight; E: protein content; F: wet gluten content; G: sedimentation value (Sv). ns: no significant difference."

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