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Heterogeneous drought stress (HED) is prevalent in drought-resistant practices such as deficit irrigation, root zone irrigation and strip intercropping. The mechanisms and improvement of crop drought resistance under HED are not fully understood. This study used double-root grafted Nandou 12 (ND12) soybean seedlings to simulate HED treatment under controlled conditions. Seedlings were transplanted into root cups with waterproof partitions to establish different soil moisture treatments: sufficient irrigation (SI) with 80 % soil moisture on both sides, HED with 80 % on one side and 40 % on the other, and homogeneous drought stress (HOD) with 60 % on both sides. The results indicated that soybean plants treated with HED exhibited healthier growth compared to those treated with HOD. Photosynthesis rate (Pn), stomatal conductance (cond.), transpiration rate (Tr), and relative water content (RWC) decreased by 10.24 %, 43.90 %, 152.66 %, and 8.69 % in HED-treated plants, respectively, but dry biomass increased by 3.38 %. Conversely, HOD conditions led to a significant decline in these parameters. Metabolomic and transcriptomic analyses revealed significant changes in the biosynthesis and signaling pathways of key phytohormones and metabolites, including abscisic acid (ABA), gibberellin (GA), jasmonic acid (JA), isoflavones, starch, and sugars. In HED-treated plants, GmNCED downregulation resulted in 54.22 % less ABA than HOD. GA levels increased under HED with upregulation of GmGA3OX1 and GmGA3OX2. JA content in HED-treated roots was 90.90 % higher than in HOD-treated roots. Isoflavones concentration including genistein (73.01 %), genistin (63.63 %), malonylgenistin (20.58 %), malonylgenistin (65 %), diadzin (38.15 %), and malonyldiadzin (47.61 %) levels, were significantly higher in HED-treated plants. Antioxidant enzyme activities indicated a 20 % increase in peroxidase (POD) activity under HED, while malondialdehyde (MDA) content was 27 % higher in HOD-treated plants, indicating greater oxidative stress. Chlorophyll content remained stable, and starch concentration increased by 33.33 % in HED-treated plants compared to HOD-treated plants. HED enhances phytohormonal responses and metabolic adjustments in soybean plants, boosting photosynthetic efficiency, antioxidant capabilities, growth, and drought resilience. This regulatory mechanism balances growth promotion and drought resistance, highlighting HED potential in improving crop resilience.

The γ-aminobutyric acid (GABA) is a widely distributed neurotransmitter in living organisms, known for its inhibitory role in animals. GABA exerts calming effects on the mind, lowers blood pressure in animals, and enhances stress resistance during the growth and development of plants. Enhancing GABA content in plants has become a focal point of current research. In plants, GABA is synthesized through two metabolic pathways, the GABA shunt and the polyamine degradation pathway, with the GABA shunt being the primary route. Extensive studies have investigated the regulatory mechanisms governing GABA synthesis. At the genetic level, GABA production and degradation can be modulated by gene overexpression, signaling molecule-induced expression, transcription factor regulation, and RNA interference. Additionally, at the level of transporter proteins, increased activity of GABA transporters and proline transporters enhances the transport of glutamate and GABA. The activity of glutamate decarboxylase, a key enzyme in GABA synthesis, along with various external factors, also influences GABA synthesis. This paper summarizes the biological functions, metabolic pathways, and regulatory mechanisms of GABA, providing a theoretical foundation for further research on GABA in plants.

The production of amylose is facilitated by granule-bound starch synthase (GBSS). Despite its importance, the specific protein interactions involving barley grain-bound starch synthase Ia (HvGBSSIa) remain poorly understood. To elucidate this, we engineered a pET-32a-HvGBSSIa prokaryotic expression vector for specific expression in E. coli Rosetta cells. A rabbit anti-HvGBSSIa polyclonal antibody was generated and employed to enrich HvGBSSIa-binding proteins from barley grains through immunoprecipitation. The isolated complexes were then resolved through SDS-PAGE, and the constituent proteins were identified using mass spectrometry coupled with database searches. Our results confirmed the successful preparation of a highly specific polyclonal antibody against HvGBSSI. Furthermore, differential expression of HvGBSSIa was assessed across various barley tissues and developmental stages of the grain, revealing peak expression at 25 days post-flowering. Proteins interacting with HvGBSSIa, including sucrose synthase and starch branching enzyme, were identified through co-immunoprecipitation. This study lays the groundwork for further detailed analyses of the HvGBSSIa protein complex in barley.

Plants possess intricate defense mechanisms to resist cadmium (Cd) stress, including strategies like metal exclusion, chelation, osmoprotection, and the regulation of photosynthesis, with antioxidants playing a pivotal role. The application of nitrogen (N) and phosphorus (P) fertilizers are reported to bolster these defenses against Cd stress. Several studies investigated the effects of N or P on Cd stress in non-woody plants and crops. However, the relationship between N, P application, and Cd stress resistance in valuable timber trees remains largely unexplored. This study delves into the Cd tolerance mechanisms of Phoebe zhennan, a forest tree species, under various treatments: Cd exposure alone, combined Cd stress with either N or P and Cd stress with both N and P application. Our results revealed that the P application enhanced root biomass and facilitated the translocation of essential nutrients like K, Mn, and Zn. Conversely, N application, especially under Cd stress, significantly inhibited plant growth, with marked reductions in leaf and stem biomass. Additionally, while the application of P resulted in reduced antioxidant enzyme levels, the combined application of N and P markedly amplified the activities of peroxidase by 266.36%, superoxide dismutase by 168.44%, and ascorbate peroxidase by 26.58% under Cd stress. This indicates an amplified capacity of the plant to neutralize reactive oxygen species. The combined treatment also led to effective regulation of nutrient and Cd distribution in roots, shoots, and leaves, illustrating a synergistic effect in mitigating toxic impact of N. The study also highlights a significant alteration in photosynthetic activities under different treatments. The N addition generally reduced chlorophyll content by over 50%, while P and NP treatments enhanced transpiration rates by up to 58.02%. Our findings suggest P and NP fertilization can manage Cd toxicity by facilitating antioxidant production, osmoprotectant, and root development, thus enhancing Cd tolerance processes, and providing novel strategies for managing Cd contamination in the environment.

Sheath blight (ShB) of maize, as a soil-borne disease caused by Rhizoctonia solani AG1-IA, is one of the main obstacles for maintaining the sustainable production of maize. R. solani has a wide host range and low-resistance sources, there is a lack of resistant resources against ShB, and the damage caused by ShB cannot be effectively controlled. To effectively protect crops against ShB, it is necessary to combine information about its pathogenicity and about the disease prevention and control of the pathogenic fungus and to identify areas that require more in-depth research. While progress has been made in the identification of disease-related genes in corn and pathogens, their mechanisms remain unclear. Research related to disease control involves the use of agronomic methods, chemical control, biological control, and genetic improvement. Breeding and identification of high-resistant maize varieties are key and difficult points in the control of maize sheath blight. This article reviews the research progress of the symptoms, the pathogen’s biological characteristics, the infection process, the pathogenic mechanism, and comprehensive control of maize sheath blight disease and provides future research directions of maize sheath blight in China. It aims to provide reliable technical routes and research ideas for future crop-disease-resistance research, especially to sheath blight of maize.

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.

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.

ADP-Glc pyrophosphorylase (AGPase) is a pivotal enzyme catalyzing the conversion of ATP and glucose-1-phosphate (Glc-1-P) to adenosine diphosphate glucose (ADP-Glc), thereby serving as a rate-limiting factor in starch biosynthesis in crops. Although previous investigations have suggested phosphorylation-based regulation of AGPase in maize, the explicit modulation mechanisms have yet to be elucidated. This research evaluated the effect of point mutations at phosphorylation sites (identified using iTRAQTM AB SCIEX, Framingham, MA, USA) on the subcellular localization and activity of the AGPase small subunit Bt2, and its interaction with the large subunit Sh2, in maize. Despite the induction of point mutations, subcellular localization of the Bt2 subunit remained unaltered, primarily within the cytoplasm and nucleus. The interaction between Bt2 and Sh2 subunits continued, mainly in the chloroplast. Notably, an increase in AGPase activity was observed in the case of simulated phosphorylation point mutations, whereas dephosphorylation activity significantly diminished relative to the wild type. These findings demonstrate that point mutations do not affect the subcellular localization of the Bt2 subunit or its interaction with the Sh2 subunit, but substantially modulate AGPase activity. This study provides critical insights into the role of point mutations in enhancing AGPase activity, thus potentially accelerating the production of ADP-Glc, the primary substrate for starch synthesis, promising implications for improved starch biosynthesis in maize.

In order to investigate the crucial role of ZmBT1 in starch accumulation during maize grain development and analyze the expression and distribution of ZmBT1 in various maize tissues, we prepared a polyclonal antibody. Specifically, we successfully expressed the recombinant plasmid pGEX-6p-ZmBT1-C (382-437aa) and purified Gst-ZmBT1-C as the antigen for antibody preparation. Our results confirmed that the ZmBT1 protein in maize tissues can be specifically recognized by the ZmBT1 antibody. Through Western blotting, we observed that the expression protein of ZmBT1 varied by tissues, with the highest content in the grain and endosperm. Furthermore, we employed a combination of Western blotting and quantitative real-time PCR to show that the expression level of ZmBT1 can be influenced by plant hormones. This finding suggests that ZmBT1 plays a critical role in the accumulation of starch and opens up new avenues for functional studies of this protein.

Starch biosynthesis is a complex and highly controlled process that requires coordinated activities among multiple enzymes. Starch synthase III (SSIII) is the largest protein in the starch synthase complex and its function is to lengthen long-chain amylopectin in starch synthesis. It potentially affects the activity of other key enzymes in starch synthesis through protein–protein interactions; therefore, its function and regulation play a predominant role in starch synthesis. In this review, we summarized the main research of SSIII including its biochemical characteristics, structural features, expression atlas, and regulation means. Structural features and expressional analysis indicated that SSIIIa is the main functional protein in maize endosperm rather than SSIIIb-a and SSIIIb-b, even though they are similar in the tertiary structures. The regulation investigation of SSIIIa showed that there are 13 transcription factors that control the transcription of SSIIIa. Interaction network analysis showed that SSIIIa could be involved with ten other key enzymes in starch synthesis. In conclusion, this review considerably extends our understanding of SSIII and provides the theoretical basis for improving starch synthesis by SSIII in maize.

Starch phosphorylase (PHO) is a multimeric enzyme with two distinct isoforms: plastidial starch phosphorylase (PHO1) and cytosolic starch phosphorylase (PHO2). PHO1 specifically resides in the plastid, while PHO2 is found in the cytosol. Both play a critical role in the synthesis and degradation of starch. This study aimed to report the detailed structure, function, and evolution of genes encoding PHO1 and PHO2 and their protein ligand-binding sites in eight monocots and four dicots. “True” orthologs of PHO1 and PHO2 of Oryza sativa were identified, and the structure of the enzyme at the protein level was studied. The genes controlling PHO2 were found to be more conserved than those controlling PHO1; the variations were mainly due to the variable sequence and length of introns. Cis-regulatory elements in the promoter region of both genes were identified, and the expression pattern was analyzed. The real-time quantitative polymerase chain reaction indicated that PHO2 was expressed in all tissues with a uniform pattern of transcripts, and the expression pattern of PHO1 indicates that it probably contributes to the starch biosynthesis during seed development in Zea mays. Under abscisic acid (ABA) treatment, PHO1 was found to be downregulated in Arabidopsis and Hordeum vulgare. However, we found that ABA could up-regulate the expression of both PHO1 and PHO2 within 12 h in Zea mays. In all monocots and dicots, the 3D structures were highly similar, and the ligand-binding sites were common yet fluctuating in the position of aa residues.

Starch phosphorylase is a member of the GT35-glycogen-phosphorylase superfamily. Glycogen phosphorylases have been researched in animals thoroughly when compared to plants. Genetic evidence signifies the integral role of plastidial starch phosphorylase (PHO1) in starch biosynthesis in model plants. The counterpart of PHO1 is PHO2, which specifically resides in cytosol and is reported to lack L80 peptide in the middle region of proteins as seen in animal and maltodextrin forms of phosphorylases. The function of this extra peptide varies among species and ranges from the substrate of proteasomes to modulate the degradation of PHO1 in Solanum tuberosum to a non-significant effect on biochemical activity in Oryza sativa and Hordeum vulgare. Various regulatory functions, e.g., phosphorylation, protein–protein interactions, and redox modulation, have been reported to affect the starch phosphorylase functions in higher plants. This review outlines the current findings on the regulation of starch phosphorylase genes and proteins with their possible role in the starch biosynthesis pathway. We highlight the gaps in present studies and elaborate on the molecular mechanisms of phosphorylase in starch metabolism. Moreover, we explore the possible role of PHO1 in crop improvement.