Preparation of Polyclonal Antibody against ZmBT1 Protein and Its Application in Hormone-Regulated Starch Synthesis

Liu, Lun; Qing, Yun; Shoaib, Noman; Di, Rong; Liu, Hanmei; Li, Yangping; Hu, Yu‐Feng; Huang, Yubi; Yu, Guowu

doi:10.3390/agronomy13071805

Cited by 3 publications

(2 citation statements)

References 51 publications

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“…The protein was extracted from the developing Zea mays seeds according to the protocol described in [ 45 ]. SDS-PAGE was performed using a Vertical Electrophoresis System (Bio-Rad, Shanghai, China).…”

Section: Methodsmentioning

confidence: 99%

Site-Directed Mutations at Phosphorylation Sites in Zea mays PHO1 Reveal Modulation of Enzymatic Activity by Phosphorylation at S566 in the L80 Region

Shoaib,

Mughal,

Liu

et al. 2023

Plants

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Starch phosphorylase (PHO) is a pivotal enzyme within the GT35-glycogen–phosphorylase (GT; glycosyltransferases) superfamily. Despite the ongoing debate surrounding the precise role of PHO1, evidence points to its substantial influence on starch biosynthesis, supported by its gene expression profile and subcellular localization. Key to PHO1 function is the enzymatic regulation via phosphorylation; a myriad of such modification sites has been unveiled in model crops. However, the functional implications of these sites remain to be elucidated. In this study, we utilized site-directed mutagenesis on the phosphorylation sites of Zea mays PHO1, replacing serine residues with alanine, glutamic acid, and aspartic acid, to discern the effects of phosphorylation. Our findings indicate that phosphorylation exerts no impact on the stability or localization of PHO1. Nonetheless, our enzymatic assays unveiled a crucial role for phosphorylation at the S566 residue within the L80 region of the PHO1 structure, suggesting a potential modulation or enhancement of PHO1 activity. These data advance our understanding of starch biosynthesis regulation and present potential targets for crop yield optimization.

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Section: Methodsmentioning

confidence: 99%

Site-Directed Mutations at Phosphorylation Sites in Zea mays PHO1 Reveal Modulation of Enzymatic Activity by Phosphorylation at S566 in the L80 Region

Shoaib,

Mughal,

Liu

et al. 2023

Plants

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“…After one week of acclimation, rabbits were immunized subcutaneously with 500 µg of purified recombinant GST-Sh2 fusion protein emulsified with 500 µL of Freund's complete adjuvant at a ratio of 1:1 (v/v). Two weeks after the first immunization, the rabbits were boosted with five additional subcutaneous injections with 500 µg of the purified protein mixed with 500 µL of Freund's incomplete adjuvant per injection at a ratio of 1:1 every week [43]. Venous blood samples were collected after three injections.…”

Section: Rabbit Breeding Anti-serum Preparation and Antibody Purifica...mentioning

confidence: 99%

Serine 31 Phosphorylation-Driven Regulation of AGPase Activity: Potential Implications for Enhanced Starch Yields in Crops

Yu,

Mou,

Shoaib

et al. 2023

IJMS

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ADP-Glc pyrophosphorylase (AGPase), which catalyzes the transformation of ATP and glucose-1-phosphate (Glc-1-P) into adenosine diphosphate glucose (ADP-Glc), acts as a rate-limiting enzyme in crop starch biosynthesis. Prior research has hinted at the regulation of AGPase by phosphorylation in maize. However, the identification and functional implications of these sites remain to be elucidated. In this study, we identified the phosphorylation site (serine at the 31st position of the linear amino acid sequence) of the AGPase large subunit (Sh2) using iTRAQTM. Subsequently, to ascertain the impact of Sh2 phosphorylation on AGPase, we carried out site-directed mutations creating Sh2-S31A (serine residue replaced with alanine) to mimic dephosphorylation and Sh2-S31D (serine residue replaced with aspartic acid) or Sh2-S31E (serine residue replaced with glutamic acid) to mimic phosphorylation. Preliminary investigations were performed to determine Sh2 subcellular localization, its interaction with Bt2, and the resultant AGPase enzymatic activity. Our findings indicate that phosphorylation exerts no impact on the stability or localization of Sh2. Furthermore, none of these mutations at the S31 site of Sh2 seem to affect its interaction with Bt2 (smaller subunit). Intriguingly, all S31 mutations in Sh2 appear to enhance AGPase activity when co-transfected with Bt2, with Sh2-S31E demonstrating a substantial five-fold increase in AGPase activity compared to Sh2. These novel insights lay a foundational groundwork for targeted improvements in AGPase activity, thus potentially accelerating the production of ADP-Glc (the primary substrate for starch synthesis), promising implications for improved starch biosynthesis, and holding the potential to significantly impact agricultural practices.

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Preparation of Barley AGPS2b Antibody and Its Application in Hormone Regulation Research

Xi,

Zhou,

Guo

et al. 2024

Agriculture

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ADP-glucose pyrophosphorylase (AGPase), which is a key enzyme in the starch biosynthesis pathway, plays a critical role in barley grain development. Despite its importance, the regulatory mechanisms governing AGPase expression, particularly the influence of plant hormones, remain poorly understood in barley. To address this, we identified and characterized the HvAGPS2b gene, which encodes the AGPase small subunit. The full-length HvAGPS2b gene was cloned from the barley database and expressed as a recombinant protein using the pET-30a system. Polyclonal antibodies were prepared against HvAGPS2b to facilitate detailed analysis. Our findings revealed that HvAGPS2b, as a small subunit of the rate-limiting enzyme AGPase, is integral to the later stages of grain development. Furthermore, RT-qPCR and Western blotting analyses showed that the phytohormones ABA, GA, ETH, and BR significantly upregulated the expression of AGPase small subunits. These results underscore the vital role of plant hormones in modulating AGPS2b expression, thereby influencing grain development. This study provides significant insights into the hormonal regulation of starch biosynthesis and establishes a foundation for further investigation into the functional dynamics of AGPase in barley.

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Preparation of Polyclonal Antibody against ZmBT1 Protein and Its Application in Hormone-Regulated Starch Synthesis

Cited by 3 publications

References 51 publications

Site-Directed Mutations at Phosphorylation Sites in Zea mays PHO1 Reveal Modulation of Enzymatic Activity by Phosphorylation at S566 in the L80 Region

Site-Directed Mutations at Phosphorylation Sites in Zea mays PHO1 Reveal Modulation of Enzymatic Activity by Phosphorylation at S566 in the L80 Region

Serine 31 Phosphorylation-Driven Regulation of AGPase Activity: Potential Implications for Enhanced Starch Yields in Crops

Preparation of Barley AGPS2b Antibody and Its Application in Hormone Regulation Research

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