【Objective】 The AP2/ERF (APETALA2/ethylene responsive factor) superfamily is a group of transcription factors that play important regulatory roles in plant growth and development, as well as in response to adverse environmental stressors. The AP2/ERF transcription factors are widely present and have many members in plants. Exploring the function of AP2/ERF family gene on grain size provides important genetic resources for regulating grain shape in rice. 【Method】OsDREB1J gene (LOC_Os08g43200) was cloned by homologous recombination, and its basic characteristics, tissue expression characteristics, and the relative expression patterns under plant hormones were analyzed by bioinformatics and qRT-PCR. The transactivation activity and subcellular localization of OsDREB1J were analyzed by yeast heterologous expression, transient expression of rice protoplasts and tobacco. The overexpression and knockout mutant transgenic rice plants of OsDREB1J were obtained by genetic transformation system, and the grain size phenotypes were analyzed by phenotypic analysis technology. 【Result】Subcellular localization analysis showed that OsDREB1J was localized in the nucleus. Bioinformatics showed that the full-length coding sequence of OsDREB1J was 711 bp, encoding 236 amino acids. OsDREB1J protein had no transmembrane structure, and the molecular weight of 27.47 kDa, the theoretical isoelectric point of 5.54, and had a conserved AP2 domain unique to the AP2/ERF family. The cis-acting elements analysis of OsDREB1J promoter showed that the promoter contained cis-acting elements related to hormone response, light and stresses response. The qRT-PCR analysis showed that OsDREB1J was expressed in different tissues of rice with no tissue specificity, and the relative expression level in panicle was the highest. At the same time, OsDREB1J was induced or reduced by different hormone. Transcriptional activation analysis showed that the full-length of OsDREB1J has no transcriptional activity, but the C-terminal fragment was sufficient for the transactivation ability. Phenotypic analysis showed that the grain length, length-width ratio and thousand grain weight of osdreb1j mutant were significantly higher than those of ZH11, OsDREB1J overexpression transgenic rice plants displayed opposite phenotypes, while changing the expression of OsDREB1J did not affect rice grain width. These results show that OsDREB1J may affect grain size by regulating cell length rather than cell proliferation and cell expansion. 【Conclusion】In conclusion, OsDREB1J may be involved in regulating rice grain size through hormone signaling pathway.
【Background】 Cotton is one of the most important crops globally. The application of bioengineering technology has greatly improved the efficiency of molecular breeding. However, current cotton genetic transformation faces challenges such as genotype dependency, lengthy timelines, and limited transformation methods.【Objective】This study aims to establish an efficient Agrobacterium rhizogenes-mediated genetic transformation system for cotton to expand genetic breeding methodologies.【Method】Using the common cotton receptor varieties WC and R18 as primary materials and mRUBY as a reporter gene, the root inducing process mediated by A. rhizogenes was optimized through screening hormone combinations (types and concentrations), analyzing differences in explant types and genotype-specific rooting systems. A stable genetic transformation system was subsequently developed and applied to gene editing.【Result】The addition of naphthaleneacetic acid (NAA) and lovastatin to the root inducing medium (RIM) promoted more efficient root formation compared to NAA alone or combinations of NAA+indole-3-butyric acid (IBA) or NAA+Lovastatin+IBA. The optimal concentrations for inducing hairy roots were both 2 mg·L-1 for NAA and lovastatin. Cotyledons were the most effective explants for root induction: WC cotyledons, cotyledon nodes, and hypocotyls exhibited rooting efficiencies of 398%, 72%, and 39%, respectively. Cotyledons required the shortest induction time (7 d), 3 d shorter than cotyledon nodes and 8 d shorter than hypocotyls. Cotyledons were also the optimal explants for R18, their rooting capacity differed. Genotype comparisons revealed that 20 days post-infection (dpi), the rooting efficiencies per cotyledon were 398% (WC), 116% (R18), 199% (NDM8), 103% (XLZ61), 57% (Gb-1), and 0 (Gb-2). Upland cotton varieties (WC, R18, NDM8, and XLZ61) exhibited rooting efficiencies above 100%, while sea island cotton varieties (Gb-1, Gb-2) were below 100%. Notably, Gb-2 began to root at 35 dpi. Receptor varieties of upland cotton generally showed slightly higher rooting efficiency than production varieties. There was a certain difference between the positive rate of genetic transformation and the rooting rate. The positive rates of NDM8, XLZ61, Gb-1 and Gb-2 at 20 dpi were 59.8%, 16.0%, 38.5% and 0, respectively. Using positive roots as explants, non-embryogenic and embryogenic callus induction yielded transgenic mRUBY-expressing plants, establishing a complete genetic transformation system. The intensity of plant coloration correlated positively with mRUBY expression levels. Additionally, cotton plants with edited GhGI genes were successfully obtained.【Conclusion】The study optimized the A. rhizogenes-mediated root induction process in cotton and established a robust genetic transformation system. This system was successfully applied to gene editing, generating transgenic cotton plants expressing mRUBY and edited GhGI genes.
Sorghum is the main food crop in arid and semi-arid regions of the world, which is of great significance to food security, marginal land use and dietary structure in arid and semi-arid regions. Since the first generation of grain hybrid sorghum was introduced in China in 1958, in order to adapt to mechanized harvesting and reduce labor costs, the plant height of cultivated hybrid sorghum has experienced the change process of high stalk, middle stalk, middle dwarf and dwarf. the green revolution of Chinese grain hybrid sorghum has been completed in the past two decades. This paper summarizes the reasons, history and current situation of grain sorghum dwarfing breeding in China. It shows the trend of decreasing plant height and increasing yield of sorghum varieties in China in the past 60 years. The important germplasms created in the process of green revolution of grain sorghum in China were listed. Through the analysis of genetic relationship, it was found that the dwarf source of restorer line in China came from Chinese local variety Sanchisan, and the dwarf source was traced back to Tx3197B due to the utilization of foreign germplasm Tx3197 A, Tx3197 B. The cloning, variation sites, dwarfing mechanism of sorghum dwarf genes dw1, dw2 and dw3, which play an important role in the green revolution of sorghum, and the contributions of predecessors in exploring new plant height QTLs were reviewed. The dwarfing mechanism of sorghum was different from that of gibberellin regulation system (GA) in rice and wheat. dw1 reduced plant height by regulating the brassinosteroid system (BR) to shorten the length of internodes. dw2 and dw3 encode KIPK protein kinase and auxin efflux transporter (ABCB1), respectively, which regulate the transport of auxin (IAA) to shorten the length of internodes and reduce plant height. The dwarfing genes of dw1, dw2 and dw3 had multiple effects on maturity, spike length, spike grain weight, leaf area while reducing plant height. The distribution and application of dw1, dw2 and dw3 dwarf genes in backbone sterile lines and restorer lines were analyzed by molecular markers and sequencing techniques. It was found that the dwarf genes used more in sorghum restorer lines in China were only dw3, and the combination of dw1dw3 and dw2dw3 formed by dw1, dw2 and dw3 was more widely used in sterile lines. The problems and solutions of sorghum green revolution in China were discussed. It is expected to provide guidance for further improving the process of sorghum green revolution in China and cultivating new germplasm and new varieties with major breakthroughs in yield and stress resistance.
【Objective】 Optimal reduction of nitrogen (N) fertilizer application is a sustainable management strategy in rice production. The effects of lowering N fertilizer input on grain yield and rice quality of early and late-season dual-use rice in South China were investigated, which could provide a theoretical basis for high-quality and high-yield cultivation and nitrogen management of the ‘Simiao Rice’. 【Method】 A two-year in-situ field trial was carried out at the Dafeng Experimental Base of the Guangdong Academy of Agricultural Sciences from 2022 to 2023, two early and late-season dual-use ‘Simiao Rice’ (19Xiang and Nanjingxiangzhan) were used as test cultivars, and a two-factor split-plot experimental design was adopted. The main plots were a 20% reduced N fertilizer application rate treatment (RN) and the conventional N fertilizer application rate treatment (CN). The split plots were rice varieties, to analyze the changing characteristics of early and late-season dual-use rice yield and quality under RN conditions. 【Result】Compared with CN, RN did not change the grain yield in the late season, but significantly decreased the grain yield in the early season by an average of 11.7% in the two years. The decrease in grain yield under RN conditions was related to the decline in total spikelet. In the early season, RN did not affect milled rice rate, chalky grain rate, and chalkiness, but significantly reduced head rice rate by an average of 3.30% in the two years. RN had no effect on the hardness of cooked rice in the early season, but significantly reduced its protein content, stickiness and taste value of cooked rice, with an average reduction of 0.61%, 12.80% and 2.80%, respectively, and significantly increased its amylose content by an average of 1.23%. RN did not influence the milled rice rate, head rice rate, chalky grain rate, chalkiness, amylose and protein content, and the hardness, stickiness, and taste value of cooked rice in the late season. In addition, the relevant analysis showed that the decrease in head rice rate of RN treatment in the early season might be related to the decrease in protein content, while the decrease in stickiness and taste value was related to the increase in amylose content. 【Conclusion】RN decreased the grain yield, milling quality, and eating quality in the early season, while did not alter the appearance quality in the early season, the grain yield, milling quality, appearance quality, and eating quality in the late season. Therefore, in the production of early and late-season dual-use rice, it was necessary to ensure an adequate N supply in the early season to maintain grain yield and rice quality, while reducing N fertilizer by 20% in the late season could still achieve stable and high-quality rice production under current N fertilizer application levels. Keywords:
【Objective】In terms of the issues of yield instability and quality deterioration caused by improper water and fertilizer application, the effects of reduced irrigation combined with organic and inorganic nitrogen fertilization on the yield and quality of silage maize in arid irrigated regions of Northwest China were investigated, so as to identify optimal water and fertilizer management practices for achieving high yield and superior quality in silage maize cultivation in the irrigated areas. 【Method】 From 2021 to 2022, a field experiment based on two-factor split-plot design was carried out at the Oasis Agricultural Experimental Base of Gansu Agricultural University. The main factor was two irrigation levels, respectively, including I1 conventional irrigation reduction 20 % irrigation was 324 mm, and I2 conventional irrigation is 405 mm, and drip irrigation was used. The sub-factor included five different fertilization regimes: F1, 100% chemical nitrogen fertilizer; F2, 75% chemical nitrogen fertilizer+25% organic fertilizer; F3, 50% chemical nitrogen fertilizer+50% organic fertilizer; F4, 25% chemical nitrogen fertilizer+75% organic fertilizer; and F5, 100% organic fertilizer. The effects of different water and fertilizer management practices on the yield, grain quality, and stalk quality of silage maize were analyzed, and the comprehensive evaluation of the yield and quality of silage maize was performed using factor analysis.【Result】Reducing irrigation alone led to a decrease in the yield and quality of silage maize. However, the combined application of organic-inorganic nitrogen fertilizers helped to enhance the potential for simultaneously improving both yield and quality under reduced irrigation conditions. Notably, the combination of reduced 20% irrigation with 75% chemical nitrogen fertilizer+25% organic fertilizer (I1F2) demonstrated significant advantages. The I1F2 treatment significantly increased fresh and hay yields of silage maize, with fresh and dry grass yields improving by 9.9% and 12.7% over conventional irrigation combined with 100% chemical nitrogen fertilization (the control treatment, I2F1), respectively. Meantime, the I1F2 treatment was able to maintain a relatively high grain and stover quality of silage maize. Compared with I2F1, the I1F2 treatment increased protein and fat contents of grain by 17.4% and 20.5%, and increased essential amino acids content too, with phenylalanine, valine, leucine, isoleucine, tryptophan, threonine, lysine, and methionine rose by 17.4%, 13.9%, 19.4%, 17.9%, 23.1%, 30.0%, 44.5%, and 22.0%, respectively. The I1F2 treatment increased crude protein, crude fat, and soluble sugar contents in the stover by 13.9%, 19.1%, and 15.6% over I2F1, respectively, while decreasing neutral detergent fiber content by 13.5%, thereby improving relative feed value by 14.0%. Factor analysis also revealed that the I1F2 treatment had the highest composite applicability index, which was beneficial for increasing both the yield and quality of silage maize.【Conclusion】The combination of 20% reduced irrigation with 75% chemical nitrogen fertilizer+25% organic nitrogen fertilizer was the optimal water and nitrogen management practice for simultaneously enhancing both the yield and quality of silage maize in the Northwest irrigation areas.
【Objective】 The effects of different nitrogen forms on filling characteristics, grain quality and yield of summer maize were studied, so as to provide the scientific basis for selecting suitable nitrogen fertilizer types and improving the yield and grain quality of summer maize. 【Method】 The experiment was conducted in Taian, Shandong Province from 2022 to 2023. Denghai 605 (DH605) was selected as the experimental material, with a nitrogen application rate of 210 kg N·hm-2. The experiment included five treatments: amide nitrogen (Urea, UREA), nitrate nitrogen (Calcium nitrate, NN), ammonium nitrogen (Ammonium chloride, AN), co-application of nitrate and ammonium nitrogen (1:1, HH), and urea ammonium nitrate solution with a blend of amide nitrogen, nitrate nitrogen, and ammonium nitrogen (2:1:1, UAN). The effects of different nitrogen forms on the yield and quality of summer maize were investigated by determining the grain filling characteristics, grain quality characteristics and grain capacity of summer maize. 【Result】Compared with the conventional application of amide nitrogen in UREA, both the maize yield and grain quality under NN decreased. The maize yield under AN increased, but the grain quality decreased. HH significantly increased maize yield without affecting grain quality. UAN significantly increased maize yield and improved grain quality. Over the two years, the highest maize yield achieved with the co-application of the three nitrogen forms, significantly increasing by 13.7% to 16.3% compared with UREA. The Next the highest maize yield were from AN and HH, which significantly increased maize yield by 5.2% to 6.8% and 7.3% to 10.6%, respectively, compared with UREA. The maize yield under NN decreased by 5.4% to 5.8% compared with UREA. Compared with UREA, the growth amount at the maximum filling rate (Wmax) under UAN was enhanced by 6.3% to 9.7%, and the active filling period (D) was extended by 7.7% to 10.9%. Both AN and HH increased Wmax and prolonged D, thereby promoting the accumulation of grain weight and increasing yield. The Wmax, D, grain filling rate, and dehydration rate of NN were significantly lower than those in the other treatments. The crude protein content was lower with NN and AN, decreasing by 20.6% to 22.0% and 15.2% to 17.4% than that under UREA, respectively. The rude fat content with NN was significantly higher than that of other treatments, increasing by 23.6% to 30.9% than that under UREA. Compared with UREA, UAN improved grain quality, with total starch and amylopectin content increasing by 4.9% to 5.2% and 11.7% to 14.4%, respectively, compared with UREA, and the ratio of amylopectin to amylose increased by 31.0% to 39.1%. The amylose content decreased by 14.1% to 16.8%. The crude protein content of UAN increased by 11.7% to 24.1%. The grain bulk weight under UAN was significantly higher than that under other treatments. 【Conclusion】Compared with the conventional application of amide nitrogen, the treatment with nitrate nitrogen inhibited grain filling, reduced grain weight, and decreased yield. In contrast, ammonium nitrogen or the co-application of multiple nitrogen forms enhanced the grain filling process, increased grain weight, and thereby improved yield. Furthermore, compared with the application of a single nitrogen form, the co-application of three nitrogen forms could achieve a synergistic improvement in both yield and grain quality.
【Background】 The phenoloxidase (PO) activation system is essential for insect innate immunity, particularly in pathogen defense, with prophenoloxidase-activating protease (PAP) being a key component that directly activates prophenoloxidase (proPO). However, research on these components in the insect PO activation system remains limited.【Objective】 The objectives of this study are to explore the regulatory role of microRNA (miRNA) in the phenoloxidase activation system of Plutella xylostella infected by Metarhizium anisopliae, and to provide new targets and approaches for pest control.【Method】 Bioinformatics was used to identify miRNAs targeting specific mRNAs. Real-time quantitative PCR (qRT-PCR) was employed to assess the transcription levels of miRNAs, PAP2, and PAP3 of P. xylostella at different time points post-infection with M. anisopliae (1×106 CFU/mL). The regulatory effects of miRNAs on PAP2 and PAP3 were evaluated using a dual-luciferase system. Moths were injected with miRNA mimics or inhibitors and infected with M. anisopliae 12 h later. The expression levels of PAP2 and PAP3 were measured by qRT-PCR, while mortality and PO activity were also assessed.【Result】 miR-6497-x, miR-8545-x, novel-m0313-3p, and novel-m0592-5p target PAP3, while novel-m0042-5p, pxy-miR-2756-3p, and miR-9215-x target PAP2. A negative regulatory relationship between miRNAs and their target genes was observed at 24 and 48 h post-infection with M. anisopliae. In vitro experiments confirmed that miR-6497-x, novel-m0313-3p, and novel-m0592-5p negatively regulated PAP3, while miR-9215-x significantly downregulated PAP2. In vivo injection of miR-6497-x mimic led to decreased PAP3 expression, increased larval mortality, and reduced PO activity within 12 to 48 h post-infection. Conversely, injection of miR-6497-x inhibitor resulted in upregulated PAP3 expression, decreased larval mortality, and increased PO activity. However, overexpression or inhibition of miR-9215-x did not significantly affect PAP2 expression, larval mortality, or PO activity compared to the control group during the same period post-infection.【Conclusion】 The miR-6497-x targeting PAP3 was screened and identified. Both in vitro and in vivo experiments confirm that miR-6497-x negatively regulates PAP3, thereby affecting the PO cascade. miR-6497-x plays a crucial role in modulating the immune defense of P. xylostella against M. anisopliae infection, which will provide a theoretical basis for biological control strategies targeting pest immune systems.
【Objective】 This study explored the applicability of the RothC model for simulating soil organic carbon (SOC) dynamics in dryland and paddy fields in Northeast China and evaluated the impact of various calibration methods on simulation performance.【Method】 This study selected one typical dryland and one typical paddy field as long-term experimental sites. The dryland experiment was conducted at the Heilongjiang Agricultural Ecology Experimental Station of the Chinese Academy of Sciences (2004-2015), and the paddy field experiment utilized data from the 850 Farm (2010-2017). At each experimental site, two treatments were selected for model simulation validation and performance evaluation: one with fertilization only, without straw return (NPK), and the other with both fertilization and straw returning (NPKS). For the paddy field soil, in addition to the RothC model, two modified versions, including RothC_p and RothC_0.6, were also selected for suitability evaluation. Three different model calibration methods were employed: the equilibrium method, parameter optimization method, and transfer function method, to analyze the impact of these calibration methods on model simulation performance. Normalized root mean square error (nRMSE), mean difference (MD), and the index of agreement (d) were selected as model evaluation metrics. 【Result】At the Heilongjiang station, organic carbon input exhibited a significant fluctuating trend, with the average annual carbon input under NPK and NPKS treatments being 1.71 and 3.52 t·hm-⊃2;, respectively. In contrast, organic carbon input at the 850 Farm was relatively stable, with the average annual carbon input for NPK and NPKS treatments being 1.89 and 5.90 t·hm-⊃2;, respectively. The simulation validation results from the Heilongjiang station showed that, under different model calibration methods, the nRMSE was consistently below 5%, and the index of agreement (d) ranged from 0.60 to 0.74. This indicated that the model performance was excellent across all calibration methods, and RothC was able to accurately simulate the SOC stock changes for both NPK and NPKS treatments in the dryland. When using the M2 method, the nRMSE for NPK and NPKS was the smallest, at 3.46% and 3.09%, respectively. The simulation validation results for the 850 Farm showed that the MD for RothC and RothC_p ranged from -1.47 to -13.41, with nRMSE values between 2.90% and 26.48% and d-values all below 0.1. This indicated that both models significantly overestimated the increase in SOC stocks and were unable to accurately simulate the changes in SOC stocks in the paddy field. For the RothC_0.6 model under the NPK treatment, the MD ranged from -0.08 to 0.44, with nRMSE values between 0.24% and 0.85% and d-values ranging from 0.31 to 0.76. Under the NPKS treatment, the MD ranged from -5.71 to -6.22, with nRMSE values between 11.21% and 12.12% and d-values between 0.12 and 0.13. These results indicated that RothC_0.6 could accurately simulate the dynamic changes in SOC stocks under the NPK treatment but significantly overestimate the changes in SOC stocks under the NPKS treatment.【Conclusion】RothC and RothC_0.6 were suitable for studying the dynamic changes in SOC stocks under dryland and paddy field conditions without straw returning in the Northeast region, respectively, and could accurately simulate the trends in SOC stocks. The impact of different model calibration methods on simulation performance was not significant. However, the transfer function method was simpler to compute, saved model running time, and provided better simulation performance. Therefore, this study recommended prioritizing the use of the transfer function method for model calibration.
【Objective】 This study aimed to elucidate the relationships among bacterial population structure, key species, carbon metabolic functions, and variations in soil physicochemical and biological properties resulting from long-term different fertilization treatments in agricultural soils. 【Method】 The amplicon sequencing technology based on the molecular marker of bacterial 16S rRNA were employed to analyze soil bacterial community structure, ecological networks, potential carbon metabolic functions, and their correlations with soil physicochemical and biological properties after 29 years of continuous application of various fertilization treatments (no fertilization as control, CK; chemical fertilizers, N4; and reduced application of chemical fertilizer combined with organic manure, M2N2) at the Long-term Positioning Experimental Station at Shenyang Agricultural University. 【Result】Different fertilization treatments significantly altered soil physicochemical and biological properties, and bacterial populations, diversity, and abundance of potential carbon metabolic genes. Compared with CK treatment, N4 treatment significantly decreased soil pH value, bacterial abundance and community diversity, which indicated that M2N2 treatment demonstrated a beneficial maintenance effect. Although long-term fertilization practices (both N4 and M2N2) significantly increased soil respiration rates, they also markedly reduced net nitrogen (N) mineralization rates at 0-20 cm soil layer. Furthermore, compared with N4 treatment, M2N2 treatment significantly enhanced soil net N mineralization rates. Soil ammonium N content, net N mineralization rate and pH value were the critical environmental factors influencing soil bacterial populations. Network co-occurrence analysis revealed that Bradyrhizobium elkanii and beta proteobacterium WWH154 were the key bacterial species that maintained the stability of bacterial ecological networks, and about 100 dominant bacterial species co-occurred fully with beta proteobacterium WWH154 and 54% of the species co-occurred with Bradyrhizobium elkanii. Long-term fertilization (N4 and M2N2) increased the relative abundance of two key species by 61.9%-169.4%, especially the M2N2 treatment. The function prediction of carbon metabolic genes showed that N4 treatment reduced the abundance of various carbon metabolism-related genes, such as carbon fixation pathways in prokaryotes, Aminoacyl tRNA biosynthesis and Amino acid related enzymes in soil bacteria, and M2N2 treatment significantly stabilized the carbon metabolic pathways. 【Conclusion】In summary, long-term fertilization altered the physicochemical and biological properties of agricultural soil. Reduced application of chemical fertilizer combined with organic manure enriched key bacterial species and increased the complexity of microbial networks, which would be beneficial to coping with environmental changes, thus maintaining soil ecological functions and increasing crop yield.
【Objective】 This study aimed to explore the effects of straw and green manure mulching on soil fertility and crop yield on the dryland in southwest China, so as to provide the theoretical basis and practical guidance for exploring reasonable, efficient and ecologically healthy conservation tillage measures in southwest China. 【Method】 The sweet potato field in the "broad bean/maize/sweet potato" dry three-crop intercropping mode in southwest China was selected as the research object, and four treatments were set up: no mulching (CK), straw mulching (S), straw and milk vetch mulching (S+M), and milk vetch mulching (M). The effects of different treatments on soil characteristics and soil fertility, sweet potato dry matter content in the sweet potato field were studied. 【Result】(1) Compared with no-mulching treatment, straw and milk vetch mulching could improve the physical and chemical properties and biological characteristics of soil in sweet potato field. Among them, straw and milk vetch mulching had the best effect. (2) The comprehensive evaluation of soil fertility based on principal component analysis showed that soil fertility under straw and milk vetch mulching treatment was higher than that under no mulching treatment, and the comprehensive scores of straw and milk vetch mulching treatment were the highest in both rhizosphere and non-rhizosphere soil. (3) S+M treatment significantly improved the dry matter quality of various organs of sweet potato and sweet potato yield, the yield of sweet potato under S+M, S and M treatment was 34.53%, 14.60% and 11.55% higher than that under CK treatment, respectively.【Conclusion】Straw and milk vetch mulching in the dryland, triple cropping systems of southwest China, could effectively improve the physical and chemical properties and biological characteristics of soil, enhance soil fertility, and improve dry matter quality and yield.
【Objective】 MADS-box transcription factors are one of the largest transcription factor families in plants, and play important roles in plant growth, development, and stress responses. Previously, based on transcriptome data of pepper, a heat-responsive gene, Agamous-like MADS-box protein61 (AGL61), was identified as a MADS-box transcription factor. However, the function of CaAGL61 in pepper heat stress remains unknown. In this study, we explored the molecular function of CaAGL61 in heat tolerance of pepper plants, providing the regulatory insights of CaAGL61 as a potential locus for genetic improvement. 【Method】 The SMART online tool was used to predict the conserved domain of CaAGL61, and a phylogenetic tree of AGL61 in pepper and other plant species was constructed using MEGA7. The expression pattern of CaAGL61 was analyzed by quantitative real-time PCR. The subcellular localization of CaAGL61 was examined in tobacco, and its transcriptional regulatory role was investigated using a yeast two-hybrid system (Y2H). The effects of CaAGL61 on heat tolerance of pepper were accessed using transgenic plants generated by virus-induced gene silencing (VIGS) and transient overexpression techniques.【Result】CaAGL61 encodes a protein of 179 amino acids, containing a MADS domain and exhibiting high evolutionary conservation. CaAGL61’s expressional level was relatively high in the flower of pepper, followed by the stem and fruit, and lowest in the root. Further analysis reveals that the expression of CaAGL61 increased along with flower maturation process, peaking in the anther during pollination and fruit setting. High temperature treatment at 45 ℃ significantly upregulates the expression of CaAGL61. Subcellular localization showed that CaAGL61 is located in the nucleus, and yeast transcriptional activation assays indicates that CaAGL61 has transcriptional activation activity. Silencing of CaAGL61 significantly enhances heat tolerance of pepper. Comparing with control, CaAGL61-silenced plants exhibit reduced wilting in plant growth points, lower relative electrolyte leakage, reduced content of malondialdehyde, and accumulation of dead cells and reactive oxygen species (ROS), and increased content of chlorophyll under heat stress. However, transient overexpression of CaAGL61 reduces the heat tolerance of pepper, indicated by more severe heat-stress damage, higher relative electrolyte leakage, increased content of malondialdehyde, and accumulation of dead cells and ROS, and decreased content of chlorophyll compared to control. 【Conclusion】A heat-responsive MADS-box transcription factor gene, CaAGL61, was identified in pepper. This gene negatively regulates pepper heat tolerance by exacerbating oxidative stress.
