Starch and prolamin composition and content are important indexes for determining the processing and nutritional quality of wheat (Triticum aestivum L.) grains. Several transcription factors (TFs) regulate gene expression during starch and protein biosynthesis in wheat. Storage protein activator (TaSPA), a member of the basic leucine zipper (bZIP) family, has been reported to activate glutenin genes and is correlated to starch synthesis related genes. In this study, we generated TaSPA-B overexpressing (OE) transgenic wheat lines. Compared with wild-type (WT) plants, the starch content was slightly reduced and starch granules exhibited a more polarized distribution in the TaSPA-B OE lines. Moreover, glutenin and ω- gliadin contents were significantly reduced, with lower expression levels of related genes (e.g., By15, Dx2, and ω-1,2 gliadin gene). RNA-seq analysis identified 2023 differentially expressed genes (DEGs). The low expression of some DEGs (e.g., SUSase, ADPase, Pho1, Waxy, SBE, SSI, and SS II a) might explain the reduction of starch contents. Some TFs involved in glutenin and starch synthesis might be regulated by TaSPA-B, for example, TaPBF was reduced in TaSPA-B OE-3 lines. In addition, dual-luciferase reporter assay indicated that both TaSPA-B and TaPBF could transactivate the promoter of ω-1,2 gliadin gene. These results suggest that TaSPA-B regulates a complex gene network and plays an important role in starch and protein biosynthesis in wheat.

Wheat aphids are major pests during the seed filling stage of wheat. Plant lectins are toxic to sap-sucking pests such as wheat aphids. In this study, Pinellia pedatisecta agglutinin (ppa), a gene encoding mannose binding lectin, was cloned, and it shared 92.69% nucleotide similarity and 94% amino acid similarity with Pinellia ternata agglutinin (pta). The ppa gene, driven by the constitutive and phloem-specific ribulose bisphosphate carboxylase small subunit gene (rbcs) promoter in pBAC-rbcs-ppa expression vector, was transferred into the wheat cultivar Baofeng104 (BF104) by particle bombardment transformation. Fifty-four T₀ transgenic plants were generated. The inheritance and expression of the ppa gene were confirmed by PCR and RT-PCR analysis respectively, and seven homozygous transgenic lines were obtained. An aphid bioassay on detached leaf segments revealed that seven ppa transgenic wheat lines had lower aphid growth rates and higher inhibition rates than BF104. Furthermore, two-year aphid bioassays in isolated fields showed that aphid numbers per tiller of transgenic lines were significantly decreased, compared with wild type BF104. Therefore, ppa could be a strong biotechnological candidate to produce aphid-resistant wheat.

As one of the microelements, nitrogen play essential roles in cereal production. Although the use of chemical fertilizers has significantly improved the yield of wheat, it has also caused increasingly adverse environmental pollution. Revealing the molecular mechanism manipulating wheat nitrogen use efficiency (NUE), and cultivating wheat germplasms with high nitrogen use efficiency has become important goals for wheat researchers. In this study, we investigated the physiological and transcriptional differences of three wheat cultivars with different NUE under low nitrogen stress. The results showed that, under low nitrogen conditions, the activities of nitrogen metabolism-related enzymes (GS, NR, GDH), antioxidant enzymes (SOD, POD, CAT) and soluble protein contents of ZM366 (high NUE cultivar) were higher than those of JD8 (low NUE cultivar). The hybrid cultivar of ZM366 and JD8 showed mid-parent or over-parent heterosis. Transcriptome analysis revealed that ‘alanine, aspartate and glutamate metabolism’, ‘terpenoid backbone biosynthesis’ and ‘vitamin B6 metabolism’ pathways play key roles in nitrogen use efficiency in wheat. The significant enhancement of the ‘Calvin cycle’ and ‘photorespiration’ in ZM366 contributed to its higher level of carbon metabolism under low nitrogen stress, which is an important attribute differs from the other two varieties. In addition, the activation of ABA signal transduction and biosynthesis pathways also helps to maintain NUE under low- nitrogen conditions. Moreover, bHLH transcription factors were also found to play a positive role in wheat NUE. In conclusion, these results enriched our knowledge of the mechanism of wheat NUE, and provided a theoretical basis for improving wheat NUE and breeding new cultivars.

Sucrose transporters (SUTs) play vital roles in phloem sucrose unloading and transportation in wheat grains. However, the genomic information regarding the SUT gene family and their expression patterns in response to heat stress in grains of male-sterile wheat (Triticum aestivum L.) lines has not been systematically studied. In this study, a thorough examination of the wheat SUT gene family was conducted, focusing on their expression patterns in male-sterile lines under heat stress conditions in grain tissues. A total of 19 SUT genes were identified, with phylogenetic analysis indicating their classification into five distinct groups. Polyploidization was identified as a substantial factor in the expansion of SUT genes, with segmental duplication being the predominant mechanism driving the evolutionary expansion of the SUT gene family in wheat. Transcriptome data indicate that the expression levels of TaSUT1 and TaSUT2 were higher than other SUT genes in grains of male-sterile lines. The TaSUT1 expression showed a gradual decreasing trend, while TaSUT2 showed a reverse trend with the process of grain filling. After heat stress, the TaSUT1 expression in grains of male-sterile lines was first significantly increased and then significantly decreased with the filling stage extension, aligning with the observed trend of sucrose levels, indicating that heat stress may decrease the grain weight by reducing sucrose unloading and transportation process in grains. These results provide a systematic analysis of the SUT gene family and lay a theoretical foundation for us to study the grain filling of male-sterile lines in response to abiotic stress.

High-temperature (HT) stress frequently affects the early and middle stages of grain filling in hybrid seed production regions. Photo-thermo-sensitive male-sterile (PTMS) wheat lines, which play a critical role as female parents in hybrid seed production, face challenges under HT conditions. However, the mechanisms governing grain filling in PTMS lines under HT stress remain poorly understood. This study used the BS253 line to investigate the effects of HT on grain filling, primarily focusing on the transition from sucrose unloading to starch synthesis. The findings indicated that HT significantly reduced the grain starch content and weight by 7.65% and 36.35% at maturity, respectively. Further analysis revealed that the expression levels of TaSUT1 and TaSWEETs in grains initially increased after HT stress, paralleling the rise in sucrose content during the same period. The activities of ADP-glucose pyrophosphorylase, UDP-glucose pyrophosphorylase, granule-bound starch synthase, and soluble starch synthase were markedly decreased, indicating that impaired starch synthesis was a key factor limiting grain filling immediately after HT exposure. A total of 41 key regulatory genes involved in sucrose-to-starch metabolism were identified, with HT significantly reducing the expression of genes associated with pathways from sucrose unloading to starch synthesis during the middle and late stages post-HT. Based on the observed ultrastructural changes in the abdominal phloem and sucrose transporter expression levels under HT, we concluded that limited sucrose supply, degradation, and inhibition of starch synthesis collectively constrained grain filling during these stages. Additionally, 11 heat shock proteins and two catalase genes were identified and significantly upregulated during the initial phase post-HT, suggesting their potential role in enhancing sucrose supply at this critical time. More importantly, seven key genes involved in the sucrose-to-starch pathway were identified by weighted gene co-expression network analysis (WGCNA), which provides target genes for their functional research for starch synthase. These findings provide a comprehensive understanding of how HT limits grain filling, identify several genes involved in the sucrose-to-starch pathway, and offer a novel perspective for future research on HT-restricted grain filling across the entire process from sucrose unloading to starch synthesis in developing grains.