Transcriptional reprogramming of heat-sensitive maize anther development under heat stress

Transcriptional reprogramming of heat-sensitive maize anther development under heat stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcriptional reprogramming of heat-sensitive maize anther development under heat stress Jing Wang, Sen Wang, Long Zhang, Jiwei Yang, Jingjing Li, Putong Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8488369/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Apr, 2026 Read the published version in BMC Plant Biology → Version 1 posted 12 You are reading this latest preprint version Abstract Heat stress during anther development severely impairs pollen fertility and substantially reduces yield. However, how heat stress reshapes transcriptional programs at specific anther developmental stages and which regulatory mechanisms underlie stage-dependent sensitivity remain unclear. Here, we used the heat-sensitive inbred line Zheng641 as a model to characterize fine-scale transcriptional responses to heat stress. Morphological analyses showed that elevated temperatures markedly reduced tassel branch number and pollen viability and delayed tapetal degradation. High-resolution transcriptomic profiling revealed that heat stress mainly activated the pathways related to fatty acid biosynthesis and carbohydrate metabolism during the early stage of pollen development, whereas late-stage heat stress induced amino acid transport and biosynthesis while repressing cell wall modification and protein folding. Phase‐specific transcription factor analysis indicated that bHLHs transcription factor were significantly enriched before the tetrad stage, AP2/ERFs in microspore stage, MYBs in pollen mature stage. Furthermore, we explored Zm00001eb296990 could improve thermotolerance by the phenotypic analysis of transgenic overexpressed lines. Notably, the function-loss of Zm00001eb296990 significantly reduced pollen viability and seed set under heat stress. Together, these findings provide a high-resolution transcriptomic framework for understanding the molecular basis of maize anther sensitivity to heat and identify promising targets for enhancing thermotolerance in maize breeding. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction With the intensification of global warming, extreme high-temperature events are occurring more frequently, leading to significant crop losses worldwide and posing a serious threat to global food security and agricultural sustainability. It has been reported that for every 1 ℃ increase in global average temperature, yields of the four major crops—maize, wheat, rice, and soybeans—decline by 7.4%, 6.0%, 3.2%, and 3.1%, respectively [1-3]. Maize, one of the world’s most important food and feed crops, is particularly vulnerable to heat stress (HS) across multiple developmental stages [4, 5]. During the seedling stage, temperatures exceeding 30 °C disrupt water relations, impair root development, and reduce photosynthetic efficiency [6-8]. In the reproductive phase, temperatures above 35°C shorten the reproductive period, reduce pollen fertility, and inhibit silk elongation, ultimately decreasing kernel numbers and yield [9-11]. During pollination and grain filling, temperatures over 35°C inhibit fertilization, causing a significant restriction of early pollen tube growth [12]. In major maize-growing regions such as China's Huang-Huai-Hai Plain, HS has emerged as a frequent natural disaster affecting maize production safety [13]. Therefore, a mechanistic and developmentally resolved understanding of how maize anthers perceive and integrate heat stress signals is essential for identifying robust targets for thermotolerance breeding. Pollen plays a pivotal role in maize reproduction and productivity by facilitating the formation and delivery of male sperm cells to the female gametophyte. However, pollen viability is severely compromised under elevated temperatures, negatively impacting yield and quality [9, 14, 15]. In flowering plants, normal anther development is essential for pollen viability. Anther development begins with stamen primordia initiation and differentiation of somatic and meiotic cell layers. Pollen mother cells (PMCs) undergo meiosis to form four haploid microspores connected by a thickened exine layer [16, 17]. Key processes such as tapetum formation and apoptosis, meiosis, callose synthesis and degradation, and pollen wall formation are tightly linked to pollen development and are particularly sensitive to environmental stresses, especially heat. In Arabidopsis , early heat-induced lesions in male gametogenesis occur during meiosis, with elevated temperatures increasing crossover frequency and disrupting chromosome segregation and cytokinesis. Heat stress also perturbs cytoskeletal dynamics and spindle orientation in Arabidopsis and tobacco [18-20]. At later stages of anther development, heat reduces starch and soluble sugar contents in anther wall cells and pollen grains, thereby decreasing pollen viability [21]. Elevated temperatures disrupt reactive oxygen species (ROS) homeostasis in the anther sporogenous layer, triggering premature programmed cell death (PCD). For example, rice exposed to heat stress during male meiosis exhibits sharp increases in ROS and superoxide dismutase (SOD) activity [22]. These observations highlight that multiple, temporally distinct cellular processes—spanning meiosis, tapetal PCD, carbohydrate allocation, and mitochondrial energy supply—collectively define the developmental windows most vulnerable to heat stress [23]. At the molecular and metabolic levels, heat stress disrupts pollen development by affecting phytohormone metabolism and signaling, protein folding in the endoplasmic reticulum, ROS homeostasis, and lipid and carbohydrate metabolism [24]. Tapetal cells are the main source of auxin for pollen maturation; heat stress suppresses key auxin biosynthesis genes such as YUCCA2 and YUCCA , disrupting microspore auxin homeostasis and inducing male sterility [25]. Jasmonic acid biosynthesis genes acyl-CoA oxidase ( ACO ) and allene oxide cyclase ( AOCs ) are also induced by heat and contribute to male sterility [26]. Heat stress leads to accumulation of misfolded or unfolded proteins in the cytosol and endoplasmic reticulum, causing pollen abortion and male sterility. For example, Arabidopsis bzip28/bzip60 double mutants, deficient in the unfolded protein response (UPR), exhibit increased heat sensitivity [27]. NADPH oxidases (Rbohs), major ROS producers, mitigate ROS overaccumulation and enhance heat tolerance in rice by increasing starch synthase activity [28]. Lipid and carbohydrate metabolism are vital for male reproduction and are disrupted by heat stress [21, 29]. Mutants of rice OsGPT1 (glucose-6-phosphate/phosphate translocator) and OsHXK5 (hexokinase) fail to accumulate starch granules in pollen grains, resulting in male sterility [30, 31]. Similarly, heat stress in maize impairs sugar-to-starch conversion and reduces starch granule size and number, lowering pollen viability [11]. Disruption of lipid metabolism in tapetum and pollen walls under heat stress also leads to male sterility, as shown by GhACO2 knockout in cotton which reduces fatty acid content and accelerates tapetum degradation [26, 32]. Given that pollen viability is determined by a cascade of developmentally coordinated events and is highly sensitive to temperature fluctuations, understanding how heat stress reshapes transcriptional programs throughout anther development becomes essential. Previous studies have largely centered on transient or acute heat treatments, identifying heat-induced male sterility genes or characterizing isolated developmental stages[33-36]. However, these studies provide limited insight into the continuous, progressive transcriptional adjustments that occur throughout anther development under field-relevant HS conditions. Despite extensive work on HS-induced male sterility, the stage-specific regulatory mechanisms that govern pollen viability under prolonged heat exposure remain insufficiently understood. To address this gap, we used the heat-sensitive inbred line Zheng641 as a representative model to investigate stage-specific transcriptional responses under field-based HS. Using tassel branch number and pollen viability as indicators, we defined HS conditions, followed by transcriptome profiling to elucidate gene regulatory networks underlying maize anther development under heat stress. This study advances our understanding of the molecular mechanisms by which HS affects anther development and provides a foundation for identifying genes and pathways associated with heat tolerance. Materials and Methods Plant materials and anthers collection The maize inbred line Zheng641 was grown with staggered sowing in the experimental fields of the Henan Academy of Agricultural Sciences (Yuanyang, China; 113.792°E, 35.108°N). The first sowing occurred on 10 May 2023, followed by four additional sowings at 10-day intervals, designated as SD1–SD5. For each sowing stage, four rows with 15 plants per row were established with a row spacing of 0.60 m and a plant spacing of 0.25 m. Temperatures are recorded daily by miniature weather stations installed in the fields. Plants with synchronized development from tasseling to pollen shedding were selected daily. spikelets were gently collected and placed into RNase-free, pre-labeled EP tubes. Anthers were rapidly detached from spikelets on ice, and their lengths were measured. Based on length, anthers were grouped into five developmental stages: 1.0 mm, 2.0 mm, 2.5 mm, 3.5 mm, and 4.5 mm. All samples were stored at −80 °C. Each sample consisted of pooled anthers collected from at least three plants. Pollen viability and microscopy Before flowering, pollination bags were placed on uniformly growing tassels. Following flowering, fresh pollen from three plants per sowing stage was collected daily by shaking the tassels. Pollen was stained with I2-KI stain solution (Solarbio, G4801), observed, and imaged using an inverted microscope (Nikon Ti2-U, Japan). Pollen grains staining black were considered viable; unstained grains were considered non-viable. Pollen viability was calculated as the percentage of viable pollen out of the total observed per microscopic field. For morphological analysis, fresh pollen was fixed in electron microscope fixative (Servicebio, G1102) at room temperature for 2 hours and stored at 4 °C. Samples were washed three times with 0.1 M sodium phosphate buffer (15 min each), post-fixed with 1% osmium tetroxide, dehydrated in a graded ethanol series (30–100%), and dried using a critical point dryer (Quorum, K850). Pollen grains were sputter-coated with gold-palladium (IXRF, MSP-2S) and imaged using a scanning electron microscope (SEM; HITACHI SU8010, Tokyo, Japan). cDNA library preparation and illumina sequencing Based on prior field and viability results, the SD2 and SD4 developmental stages were selected for transcriptome sequencing. Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. RNA concentration and purity were assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and integrity was confirmed with an RNA Nano 6000 Assay Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). One microgram of RNA per sample was used for library preparation with the Hieff NGS Ultima Dual-mode mRNA Library Prep Kit (Yeasen Biotechnology, Shanghai, China). Libraries were indexed and sequenced by Biomarker Technologies Co., Ltd. (Beijing, China). Transcriptome sequencing data analysis Raw FASTQ reads were initially processed with in-house Perl scripts to remove adapters, poly-N sequences, and low-quality reads. Clean reads were aligned to the maize reference genome (Zm-B73-REFERENCE-NAM-5.0) using HISAT2 (v2.2.1). Transcript assembly and identification of known and novel transcripts were performed using StringTie with reference annotation-based transcript (RABT) assembly. Gene function was annotated using the following databases: NR (ftp://ftp.ncbi.nih.gov/blast/db/); Pfam (http://pfam.xfam.org/); KOG (http://www.ncbi.nlm.nih.gov/KOG/) COG(http://www.ncbi.nlm.nih.gov/COG/); Swiss-Prot (http://www.uniprot.org/); GO (http://www.geneontology.org/). Gene expression levels were quantified as fragments per kilobase of transcript per million mapped reads (FPKM), using the formula: FPKM = (cDNA Fragments) / ((Mapped Fragments in millions) × (Transcript Length in kb)). Differential expression analysis between groups was performed using DESeq2 (v1.30.1), and genes with a P -value < 0.01 and fold change ≥ 2 were considered differentially expressed. Quantitative real-time PCR (qRT-PCR) Total RNA from anthers at different developmental stages was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Quantitative PCR was conducted using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) in a 20 μL reaction on a CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA). The cycling conditions were: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Relative gene expression was calculated using the 2 −ΔΔCt method. A maize ubiquitin gene ( GRMZM2G102471 ) was used as internal control. Primer sequences used for verification are listed in (Supplementary Table S8). Analysis of the heat stress treatment Phenotype of candidate gene In Arabidopsis , the background material used in this study was the Col-0 ecotype. The overexpression vector was pCMBIA1300-35S-GFP, and the transgenic lines were generated via Agrobacterium-mediated floral dip transformation. After the appearance of the first flowers, plants were subjected to heat treatment in a growth room at 37°C with comparable light intensity conditions for 8 h, while the other control plants were grown at 22°C. After 8 h of heat treatment, plants were transferred to 22°C and continued to grow for 10 d. The siliques furthest along in development were selected for phenotypic analysis and length measurement. The relative shortening of silique rate was calculated as follows: [(silique length without heat stress-silique length with heat stress)/ silique length without heat stress] × 100%. Siliques were further divided into three major categories: fully fertile (Type I, >10 mm in length), partially sterile (Type II, 5-10 mm in length), and completely sterile (Type III, <5 mm in length). In maize, the zmagp2 mutant (accession number Mu178054256) was obtained from the China Mu resource (ChinaMu; http://chinamu.jaas.ac.cn/cindex.html). Homozygous zmagp2 mutants were generated through repeated self-pollination of heterozygous plants and subsequently confirmed by PCR using the primers listed in Table S8. During the peak pollen-shedding stage of B73 and zmagp2 mutant, old anthers were removed from the tassels, which were immediately bagged with pollination bags to prevent foreign pollen contamination. At 18:00 daily, tassels with uniform pollen-shedding were selected, excised, immersed in clean water, and transported to the laboratory. These were transferred separately to a control incubator (25°C, dark 12 h; 28°C, light 3 h) or a heat treatment incubator (25°C, dark 12 h; 38°C, light 3 h) for treatment. Following treatment, pollens were collected: one portion received TTC staining, with viability quantified via microscopic counting; the remaining were pollinated onto original female parents (ears pre-bagged with selfing bags). Pollinated plants were maintained under natural conditions until maturity and harvest. The seed setting rate was calculated as follows: (Actual number of kernels / Theoretical number of kernels) × 100%. Statistical Analysis Statistical analyses were performed using GraphPad and Microsoft Excel. Differences between two groups were assessed using a two-sided Student’s t -test. For comparisons among multiple groups, one-way ANOVA followed by Tukey’s multiple comparison test was used. All data represent means ± standard deviation (SD) from three biological replicates. The effective accumulated temperature (EAT) refers to the total temperature accumulated during the maize growth period, reflecting thermal conditions for development. In this study, a base temperature of 10 °C was used to calculate EAT during anther development. The formula was: EAT = (Daily Maximum Temperature + Daily Minimum Temperature) / 2 − Base Temperature (10 °C). Results Morphological observation of Zheng641 across staggered sowing dates To assess factors influencing seed set in maize, two key traits were evaluated: tassel branch number, a major determinant of pollen production, and pollen viability, defined as the proportion of viable pollen grains relative to the total. The heat-sensitive inbred line Zheng641, characterized by abundant tassel branches and high pollen production, was planted using staggered sowing across five sowing dates (SD1–SD5), beginning on 10 May 2023 with subsequent sowings at 10-day intervals (Fig. 1a). Morphological analyses revealed that SD4 exhibited the most severe reduction in tassel branch number (Fig. 1b), accompanied by a substantial increase in nonviable pollen grains (Fig. 1c). Statistical evaluation further showed that the tassel branching index was lowest in SD4, reaching a value of 14 (Fig. 1d). Pollen viability also differed significantly among sowing dates, with the highest value (81.8%) observed at SD2 and the lowest (38.2%) at SD4 (Fig. 1e). To examine the influence of temperature, climatic conditions during the growth period were monitored, and daily maximum temperature and effective accumulated temperature—an indicator of cumulative thermal input critical for maize development—were calculated from anther initiation to pollen maturation. Average daily maximum temperatures increased progressively from SD1 to SD5, with SD4 exhibiting both the highest maximum temperature and the greatest effective accumulated temperature (Supplementary Fig. S1). These combined thermal conditions account for the pronounced reductions in tassel branching and pollen viability observed in SD4. Collectively, these results indicate that SD4 experienced the most severe heat stress, whereas SD1 and SD2 were least affected. Because SD1 was influenced by notable cooling during anther development, SD2 was selected as the control and SD4 as the heat-stress treatment for subsequent experiments. Anther development of Zheng641 under heat stress To investigate the effects of heat stress on anther development, anthers with lengths of 1.0, 2.0, 2.5, 3.5, and 4.5 mm, as well as mature pollen grains, were collected and examined microscopically. Light microscopy of semi-thin sections showed that 1.0-mm anthers were undergoing initial differentiation of sporogenous cells into distinct somatic layers. Meiotic progression followed: meiocytes reached the pachytene stage in 2.0-mm anthers, and tetrads formed in 2.5-mm anthers. By the 3.5-mm stage, meiosis was complete and tetrads had separated into mononuclear microspores. At the 4.5-mm stage, microspores initiated mitosis and tapetal cells had largely degenerated (Fig. 2a-b). Notably, in SD2, 4.5-mm microspores exhibited typical sickle-shaped morphology accompanied by normal tapetal degradation (Fig. 2a). In contrast, anthers in SD4 showed delayed tapetal degradation, thickened epidermal and endothecial layers, and reduced pollen chamber volume (Fig. 2b). Scanning electron microscopy revealed plump, structurally intact pollen in SD2 (Fig. 2c), whereas pollen in SD4 appeared shriveled with disrupted exine ornamentation (Fig. 2d), consistent with impaired tapetum-mediated nutrient supply under heat stress. Together, these observations demonstrate that heat stress disrupts tapetal degradation and causes abnormal pollen development in Zheng641. Global analysis of transcriptome data To elucidate the molecular mechanisms underlying the heat sensitivity of Zheng641, RNA-seq was performed on anthers from the control (SD2) and heat-stressed (SD4) plants across five developmental stages (1.0–4.5 mm), with three biological replicates per condition, yielding a total of 30 libraries. Sequencing produced high-quality reads (Q30 > 93%) with consistent GC content, ensuring robust downstream analyses. Clean reads were mapped to the maize reference genome (Zm-B73-REFERENCE-NAM-5.0) with alignment efficiencies ranging from 80.01% to 88.21% (Supplementary Table S1). Gene expression distributions showed no systematic bias (Supplementary Fig. S2a), and biological replicates displayed strong correlations (r > 0.97) (Supplementary Fig. S2b), confirming data reliability. Principal component analysis (PCA) revealed clear separation of samples by both developmental stage and treatment, indicating extensive transcriptomic reprogramming under heat stress (Fig. 3a). Using FPKM values and filtering differentially expressed genes (DEGs) by │fold change│ ≥ 2 and FDR < 0.01, we identified stage-specific DEGs between SD2 and SD4: 10,465 (5,750 upregulated, 4,715 downregulated) at 1.0 mm; 2,802 (1,927 upregulated, 875 downregulated) at 2.0 mm; 2,947 (955 upregulated, 1,992 downregulated) at 2.5 mm; 3,118 (1,327 upregulated, 1,791 downregulated) at 3.5 mm; and 5,438 (2,749 upregulated, 2,689 downregulated) at 4.5 mm (Fig. 3b; Fig. S3). Transcript clusters during anther development under heat stress To investigate the transcriptional dynamics of maize anther development under heat stress, fuzzy k-means clustering was applied to 16,674 dynamically expressed genes from Zheng641 anthers under control (SD2) and heat-stress (SD4) conditions. Twelve co-expression clusters were identified, among which four major heat-response patterns—NR, IR, LR, and ER—revealed stage-specific sensitivity of anther development to heat stress, with early and late developmental stages exhibiting the strongest transcriptomic alterations (Fig. 3c-n). Based on their expression responses at different developmental stages, these clusters were categorized into four patterns: a) No Response (NR): Clusters 1 and 2 showed stage-specific expression but no detectable heat-stress effect, both peaking at stage C (Supplementary Table S2). b) Initial Response (IR): Clusters 3, 4, and 5 responded specifically to heat stress during early anther development; Clusters 3 and 4 were upregulated, whereas Cluster 5 was downregulated (Supplementary Table S3). c) Late Response (LR): Clusters 6, 7, and 8 responded during late development; Clusters 6 and 7 were upregulated, while Cluster 8 was downregulated (Supplementary Table S4). d) Entire-stage Response (ER): Clusters 9, 10, 11, and 12 were affected throughout the entire course of anther development (Supplementary Table S5). Gene Ontology (GO) and KEGG pathway enrichment analyses were performed for IR clusters to characterize functional shifts under heat stress (Fig. S4). Cluster 4 was enriched in fatty acid and carbohydrate metabolism, indicating early activation of metabolic compensation pathways, whereas Cluster 7 was enriched for reproductive processes, suggesting heat-induced suppression of early gametogenesis (Fig. S4a-b). KEGG analysis further revealed enrichment of plant hormone signal transduction and starch/sucrose metabolism in Cluster 4, while tryptophan metabolism was enriched in Cluster 7 (Fig. S4c-d). These findings suggest that early pollen development under heat stress involves activation of metabolic and hormone-related pathways alongside suppression of reproductive programs. GO and KEGG analyses of LR clusters showed that Cluster 6 (446 genes) was enriched for amino acid transmembrane transport and biosynthesis, whereas Cluster 11 (1,925 genes) was enriched for heat response, cell wall modification, unfolded protein response, and protein folding (Fig. S5). KEGG pathways enriched in Cluster 6 included indole alkaloid biosynthesis and galactose metabolism (Fig. S5a-b), while pentose/glucuronate interconversions and protein processing in the endoplasmic reticulum were enriched in Cluster 11 (Fig. S5c-d). Together, these results indicate that heat stress suppresses cell wall remodeling and disrupts protein folding during late pollen development, consistent with the observed impaired tapetum degradation and thickened anther walls under heat stress. Differentially expressed genes under heat stress To identify key heat-responsive genes during anther development, DEGs were analyzed across five developmental stages (1.0 mm-4.5 mm) under control (SD2) and heat stress (SD4). Venn diagram analysis revealed 6,065, 618, 981, 718, and 2,607 unique DEGs at stages 1.0 mm through 4.5 mm, respectively, with 57 DEGs shared across all stages (Fig. 4a, Supplementary Table S6). Gene Ontology (GO) enrichment analysis was performed on these 57 common DEGs, and the results showed that biological processes related to photosynthesis, pectin catabolic process, and cell wall modification were significantly enriched (Fig. 4b). Notably, 11 DEGs were associated with carbohydrate transport/metabolism. Expression analysis of these 11 genes showed that Zm00001eb236200 ( PG-like ), Zm00001eb424030 ( PME1 ), Zm00001eb165010 ( PME43 ), Zm00001eb270510 , and Zm00001eb222250 were downregulated by heat stress, while Zm00001eb018720 , Zm00001eb080840 , Zm00001eb092540 , Zm00001eb164390 , Zm00001eb197410 , and Zm00001eb200910 were upregulated during anther development (Fig. 4c). Since pectin degradation mediated by pectin methylesterases (PMEs) and polygalacturonases (PGs) is essential for programmed tapetum degradation [37], the downregulation of PME1 , PME43 , and PG-like genes suggests attenuated pectin modification, which is essential for controlled tapetum degeneration, thereby explaining the delayed tapetal breakdown observed morphologically under heat stress. To validate RNA-seq results, qRT-PCR was performed on eight carbohydrate transport/metabolism-related DEGs: Zm00001eb398090 , Zm00001eb400780 , Zm00001eb371910 , Zm00001eb283530 , Zm00001eb152540 , Zm00001eb183000 , Zm00001eb378140 , and Zm00001eb304130 . For example, Zm00001eb398090 ( Ms45 ), encoding strictosidine synthase involved in male gametophyte cell wall development [38], was highly expressed and downregulated by heat stress at stage 2.0 mm (Fig. S6a). Zm00001eb400780 , Zm00001eb371910 (encoding xyloglucan endotransglucosylase/hydrolase 12), and Zm00001eb283530 were upregulated at stage 2.5 mm (Fig. S6b). Zm00001eb152540 and Zm00001eb183000 (encoding sucrose transporter 6) were upregulated at stage 3.5 mm (Fig. S6c). Zm00001eb378140 and Zm00001eb304130 were downregulated at stage 4.5 mm (Fig. S6d). Expression trends matched RNA-seq data, confirming data reliability. Identification of differentially expressed transcription factors (TFs) Gene expression patterns are largely regulated by TFs. We identified differentially expressed TFs in maize anthers under heat stress at each developmental stage: 448 TFs (137 up, 311 down) at 1.0 mm stage; 45 (32 up, 13 down) at 2.0 mm stage; 69 (17 up, 52 down) at 2.5 mm stage; 50 (24 up, 26 down) at 3.5 mm stage; and 72 (42 up, 30 down) at 4.5 mm stage, spanning 54, 21, 24, 26, and 27 TF families, respectively (Fig. 5a–e, Supplementary Table S7). TF profiling revealed stage-specific regulatory shifts, where bHLH TFs dominate early responses, AP2/ERFs regulate mid-stage events, and MYBs control late pollen maturation, highlighting a coordinated transcriptional cascade underlying heat-induced development disruption. Specific TFs of notes include: bHLH47 ( Zm00001eb059460 ) and bHLH68 ( Zm00001eb129520 ), downregulated, and bHLH51 ( Zm00001eb208200 ) and bHLH152 ( Zm00001eb244380 ), upregulated at 1.0 mm stage. MYC7 ( Zm00001eb024330 ) and bHLH106 ( Zm00001eb315910 ), upregulated at 2.0 mm stage. bHLH125 ( Zm00001eb375660 ) and bHLH127 ( Zm00001eb228320 ), downregulated at 2.5 mm stage (Fig. 5f). EREB50 ( Zm00001eb119540 ) and EREB156 ( Zm00001eb432090 ), upregulated, and EREB202 ( Zm00001eb099980 ) and EREB52 ( Zm00001eb145420 ), downregulated at 3.5 mm stage (Fig. 5g). MYB128 ( Zm00001eb129490 ), MYB15 ( Zm00001eb148320 ), MYB163 ( Zm00001eb366540 ), and MYB74 ( Zm00001eb369190 ), upregulated at 4.5 mm stage (Fig. 5h). These TFs reflect stage-specific regulatory responses to heat stress and warrant further functional validation. Function verification of candidate genes To verify the biological functions of candidate genes, 9 DEGs (Zm00001eb296990, Zm00001eb270510, Zm00001eb183000, Zm00001eb283530, Zm00001eb421960, Zm00001eb165010, Zm00001eb222250, Zm00001eb236200, Zm00001eb424030) involved in carbohydrate transport and metabolism pathways were selected for functional validation. We constructed corresponding overexpression vectors and performed functional validation in Arabidopsis thaliana . Heat stress treatment significantly affected plant development during the flowering (Fig. S7). Notably, the overexpressing line of Zm00001eb296990 exhibited a greater plant height after heat stress treatment. (Fig. 6a). Silique length, positively correlated with seed number, is a reliable parameter for investigating heat stress effects on plant reproductive development[27]. Accordingly, we used silique length as an indicator to analyze heat stress impact on the reproductive development of different materials. Results showed heat stress significantly reduced silique length in Arabidopsis (Fig. S8a-b). Compared with wild type, the relative shortening rate of silique lengths in lines overexpressing Zm00001eb296990 , exhibited a significantly milder reduction when subjected to heat stress at the reproductive stage (Fig. 6b-c). Furthermore, we classified the siliques into three major categories: fully fertile (Type I), partially sterile (Type II), and completely sterile (Type III). Heat stress significantly increased the percentages of Type II and Type III siliques (Fig. S8c). Notably, compared with the control lines (WT), the Zm00001eb296990-overexpressing line exhibited an increased proportion of Type I siliques, accompanied by a reduced proportion of Type II siliques (Fig. 6d). Zm00001eb296990 encodes ADP-glucose pyrophosphorylase (ZmAGP2) - a key enzyme catalyzing the rate-limiting step in starch biosynthesis. ZmAGP2 overexpression significantly mitigated heat-induced silique abortion, demonstrating a functional role in reproductive heat tolerance, consistent with its position as a starch-biosynthesis regulator. To better characterize the function of ZmAGP2 in maize, the zmagp2 mutant line was identified from the China Mu resource. This mutant carries a Mu insertion (Mu178054256) in the first exon of the ZmAGP2 gene in the B73 genetic background (Fig. S9a). Homozygous mutants harboring this Mu insertion were confirmed by PCR-based genotyping (Fig. S9b). Subsequently, self-pollination was used to obtain homozygous wild-type B73 and homozygous zmagp2 mutant lines. TTC staining and pollen viability assays were performed on maize pollen subjected to heat stress or non-stress conditions. The results showed that heat stress led to a reduction in pollen viability in both B73 and zmagp2 mutant . Notably, compared with B73 exhibiting a 51% decrease in pollen viability, the mutant displayed a more substantial reduction of 68% under heat stress treatment (Fig. 6e, 6g). Consistent with the above results, further analysis of the seed setting rate showed that B73 exhibited a seed setting rate of 72% under normal conditions, which declined to 41% after heat stress. In stark contrast, the zmagp2 mutant had an initial seed setting rate of 59% under non-heat stress conditions, and this value plummeted by 86% to a mere 8% when subjected to heat stress (Fig. 6f, 6h). Collectively, the stronger reduction in pollen viability and seed-setting rate in the zmagp2 mutant indicates that ZmAGP2 is required for maintaining carbohydrate flux under heat stress, and its loss leads to severe reproductive energy deficiency, exacerbating heat sensitivity. Discussion In this study, the heat-sensitive line Zheng641 provided a useful biological model to dissect how elevated temperature disrupts male fertility in maize. Unlike many previous reports that focused solely on extreme heat treatments under controlled conditions[39–41], our field-based staggered-sowing experiments allowed us to capture natural temperature fluctuations and identify a progressive decline in tassel branching and pollen viability (Fig. 1 ). This highlights that even moderately elevated temperatures can compromise pollen productivity before visible heat injury occurs. Although Zheng641 is a heat-sensitive background, similar phenotypic patterns have been documented in a range of tropical, temperate, and subtropical maize lines, suggesting that the developmental processes affected here are broadly conserved across genetic backgrounds. Our results therefore provide a generalizable framework for understanding how gradual seasonal warming impairs male reproductive development and ultimately reduces yield potential. Heat stress impairs maize pollen development by disrupting tapetum degeneration and reducing viability High temperature is known to impair tapetal programmed cell death (PCD)[37, 42, 43], but our stage-resolved analysis further clarifies when and how this disruption occurs. Previous studies have generated RNA sequencing libraries covering 10 key stages of maize anther development, classifying them into four major phases: cell division and expansion, meiosis, pollen maturation, and mature pollen, and identified stage-specific key genes in the inbred line Chang7-2[44].Here, we revealed that heat stress caused abnormal tapetum development, with tight adhesion to the inner epidermis and failure to undergo normal degradation, thickening of epidermis and endodermis layers, and a markedly reduced pollen chamber size (Fig. 2 ). Heat stress caused tight adhesion of the tapetum to the inner epidermis and delayed degradation specifically during meiosis and early pollen maturation, stages in which lipidic precursors and carbohydrate reserves normally accumulate rapidly[45, 46]. Because tapetum-derived metabolites fuel pollen wall biosynthesis and supply energy for microspore expansion, the concurrent suppression of lipid and sugar metabolic pathways provides a mechanistic link between transcriptional reprogramming and cellular abnormalities[30, 47, 48]. We therefore propose that heat stress initiates a cascade in which early mis-regulation of metabolic pathways compromises tapetal function, leading to insufficient nutrient flux to developing microspores and ultimately pollen collapse. This stage-specific vulnerability also explains why minor elevation in temperature disproportionately affects fertility compared with vegetative growth. Heat stress alters lipid and carbohydrate metabolism during anther development The substantial enrichment of DEGs involved in fatty acid biosynthesis and carbohydrate metabolism during early anther development suggests that these pathways constitute a primary metabolic checkpoint for heat tolerance. Previous studies in rice (TDR/UDT1) and Arabidopsis (AMS/DYT1) indicate that tapetal TFs form hierarchical regulatory modules controlling lipidic precursor synthesis and transport[49–51]. Our data reveals that heat stress perturbs similar gene networks in maize, implying partial conservation of the metabolic-transcriptional circuitry across species (Fig. 3 – 4 ). The observed mis-regulation of key genes such as Ms45 further supports the notion that heat disrupts the coordination between metabolic flux and tapetal maturation[38]. Integrating these findings, we propose that maize anthers rely on a TF-centered regulatory hub that synchronizes lipid and carbohydrate metabolism with developmental timing, and that heat stress destabilizes this hub, leading to metabolic insufficiency and pollen abortion. This framework provides testable hypotheses for future genetic and biochemical validation. Stage-specific transcriptional regulation of anther development under heat stress The dynamic expression patterns of bHLH, MYB, and AP2/ERF transcription factors highlight that each developmental stage recruits distinct regulatory modules to cope with heat stress (Fig. 5 ). Several of these TFs correspond to orthologs of well-characterized regulators in Arabidopsis and rice[48, 52–54], suggesting that conserved reproductive stress-response circuits operate in maize. The strong induction of AP2/ERF factors at late stages further indicates a shift from developmental regulation toward stress mitigation, potentially coordinating ROS scavenging and cell wall remodeling[43, 55]. Based on these observations, we propose a working model in which heat stress disrupts the early bHLH-MYB regulatory cascade controlling tapetal development, while later activation of AP2/ERF TFs reflects compensatory mechanisms that ultimately prove insufficient to rescue pollen viability. Importantly, the TFs identified here—including bHLH47/68/51/125, MYB15/74/128/163, and EREB50/52/156/202—represent promising targets for breeding heat-resilient varieties through marker-assisted selection or CRISPR-based genome editing. Future studies integrating proteomics, chromatin accessibility profiling, and transgenic validation will be essential to define the upstream signals and downstream effectors of these TFs, and to establish a comprehensive genetic network underlying thermotolerance in maize anthers. Declarations Author Contributions: H.L. and L.W. conceived and designed research. S.W., J.W., P.W. and H.W. conducted experiments. Y.D. and Z.Z. contributed new reagents or analytical tools. J.W., J.Y. and J.L. analyzed data. J.W. writing—original draft preparation. L.Z., H.L. writing—review and editing. All authors read and approved the manuscript. Funding: This study was supported by National Key R&D Program of China (2023YFD1200505), Central Government-Guided Local Science and Technology Development Fund Project of Henan Province (2025ZYYD06), National Key R&D Program of China (2021YFD1200703), Technology Innovation Team Project (2025TD19), Applied Science and Technology Research Projects (2025YG01) and Independent Innovative (2024ZC028) Projects of Henan Academy of Agricultural Science. Data Availability Statemen: Data supporting the findings of this work are available within the paper and its Supplementary Information files. The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request, Original RNA-seq data for this article was deposited in Science Data Bank (https://doi.org/10.57760/sciencedb.28740). Conflicts of Interest: The authors declare no conflict of interest. Consent for publication: Not applicable. 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Supplementary Files SupplementaryMaterials.zip Cite Share Download PDF Status: Published Journal Publication published 21 Apr, 2026 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 09 Mar, 2026 Reviews received at journal 21 Feb, 2026 Reviews received at journal 15 Feb, 2026 Reviewers agreed at journal 08 Feb, 2026 Reviewers agreed at journal 08 Feb, 2026 Reviewers agreed at journal 19 Jan, 2026 Reviewers agreed at journal 16 Jan, 2026 Reviewers invited by journal 13 Jan, 2026 Editor assigned by journal 13 Jan, 2026 Editor invited by journal 13 Jan, 2026 Submission checks completed at journal 12 Jan, 2026 First submitted to journal 12 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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12:46:38","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120651,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8488369/v1/5a45faa3bd3417dacd3b97cf.html"},{"id":100589710,"identity":"68b73191-de2c-45c1-9330-9a6939d5082b","added_by":"auto","created_at":"2026-01-19 12:44:30","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":549594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic analysis of Zheng641 under different sowing dates.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Daily maximum and average temperatures during the growth period of Zheng641. SD1-SD5 indicate sequential delayed sowing dates at 10-day intervals. \u003cstrong\u003eb\u003c/strong\u003e, Tassel morphology of Zheng641 at SD1-SD5. Scale bar = 5 cm. \u003cstrong\u003ec\u003c/strong\u003e, Iodine-potassium iodide (I₂-KI) staining of pollen grains at SD1–SD5. \u003cstrong\u003ed\u003c/strong\u003e, Quantification of tassel branch number across SD1-SD5; at least five plants with uniform growth and comparable inflorescence structures were examined for each sowing date. \u003cstrong\u003ee\u003c/strong\u003e, Statistics of pollen viability at SD1-SD5; 3-5 microscopic fields were analyzed per stage, with \u0026gt;200 pollen grains counted in total. Statistical significance was assessed using Student’s \u003cem\u003et\u003c/em\u003e-test, and significant differences are indicated by asterisks (**\u003cem\u003eP\u003c/em\u003e ≤ 0.01).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8488369/v1/e0c16bf767e4e3553de9a1ff.jpeg"},{"id":100589492,"identity":"8bfe4817-fb27-4780-9703-1eb66f85ef29","added_by":"auto","created_at":"2026-01-19 12:42:52","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":319375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamic microscopic structure of Zheng641 during pollen development.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Transverse sections of anthers from 1.0 mm to 4.5 mm at SD2. \u003cstrong\u003eb\u003c/strong\u003e, Transverse sections of anthers from 1.0 mm to 4.5 mm at SD4. \u003cstrong\u003ec\u003c/strong\u003e, Scanning electron microscopy of pollen grains at SD2. \u003cstrong\u003ed\u003c/strong\u003e, Scanning electron microscopy of pollen grains at SD4. Cell layers are color-coded: PPC, primary peripheral cells (blue); T, tapetum (green); ML, middle layer (purple); EN, endothecium (gray); E, epidermis (orange); AR–Msp, germinal cells from archesporial cells to microspores (pink).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8488369/v1/7574dca39665f1eef8c1ca5e.jpeg"},{"id":100590146,"identity":"7cce6c1d-b79b-4a0c-a334-fc1ddbcb96ac","added_by":"auto","created_at":"2026-01-19 12:47:28","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":551358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic profiling of Zheng641 during anther development in SD2 and SD4.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Principal component analysis (PCA) illustrating relationships among samples. \u003cstrong\u003eb\u003c/strong\u003e, Numbers of up- and downregulated differentially expressed genes (DEGs) identified at each anther developmental stage (FDR \u0026lt; 0.01, |log₂FC| ≥ 2). \u003cstrong\u003ec-n\u003c/strong\u003e, Clustering of dynamically expressed genes during anther development under control (SD2) and heat stress (SD4) conditions. Clusters 1–2 represent NR (\u003cstrong\u003ec-d\u003c/strong\u003e); Clusters 3–5 represent IR (\u003cstrong\u003ee-g\u003c/strong\u003e); Clusters 6–8 represent LR (\u003cstrong\u003eh-j\u003c/strong\u003e); and Clusters 9–12 represent ER patterns (\u003cstrong\u003ek-n\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8488369/v1/00faf49751ceff0dee4b2695.jpeg"},{"id":100596158,"identity":"0073d094-d28b-463c-8664-d839af4a308c","added_by":"auto","created_at":"2026-01-19 13:54:01","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":373901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially expressed genes (DEGs) during anther development in Zheng641.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Venn diagram showing the overlap of DEGs across developmental stages. \u003cstrong\u003eb\u003c/strong\u003e, Gene Ontology (GO) enrichment analysis of 57 common DEGs shared across all five stages. \u003cstrong\u003ec\u003c/strong\u003e, Hierarchical clustering heatmap of 11 DEGs involved in carbohydrate transport and metabolism.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8488369/v1/57c9506a51d84cada115570a.jpeg"},{"id":100589719,"identity":"901e9f05-aec1-4acc-9256-2b46f8094170","added_by":"auto","created_at":"2026-01-19 12:44:33","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":546279,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential expression of transcription factors (TFs) during anther development in Zheng641.\u003c/strong\u003e \u003cstrong\u003ea-e\u003c/strong\u003e, Stage-specific expression profiles of differentially expressed TFs at 1.0 mm (\u003cstrong\u003ea\u003c/strong\u003e), 2.0 mm (\u003cstrong\u003eb\u003c/strong\u003e), 2.5 mm (\u003cstrong\u003ec\u003c/strong\u003e), 3.5 mm (\u003cstrong\u003ed\u003c/strong\u003e), and 4.5 mm (\u003cstrong\u003ee\u003c/strong\u003e). \u003cstrong\u003ef\u003c/strong\u003e, Heatmap of differentially expressed bHLH TFs. \u003cstrong\u003eg\u003c/strong\u003e, Heatmap of differentially expressed AP2/ERF-ERF-7 TFs. \u003cstrong\u003eh\u003c/strong\u003e, Heatmap of differentially expressed MYB TFs.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8488369/v1/b9e7264eea5ea5cd57cdf800.jpeg"},{"id":100590202,"identity":"54adfdd0-2f11-4396-8458-520ac63740a1","added_by":"auto","created_at":"2026-01-19 12:48:12","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":516558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eZmAGP2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eis a key gene regulating heat tolerance in maize.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Whole-plant phenotypes of WT and ZmAGP2-OE lines under control (CK) and heat stress (HS) conditions. Scale bar = 2 cm. \u003cstrong\u003eb\u003c/strong\u003e, Silique phenotypes of WT and ZmAGP2-OE under CK and HS conditions. Scale bar = 1 cm. \u003cstrong\u003ec\u003c/strong\u003e, Statistical analysis of silique length corresponding to panel (b). \u003cstrong\u003ed\u003c/strong\u003e, Statistical analysis of silique fertility, categorized as fully fertile (Type I), partially sterile (Type II), or completely sterile (Type III). \u003cstrong\u003ee\u003c/strong\u003e, TTC staining of pollen from B73 and the \u003cem\u003ezmagp2\u003c/em\u003e mutant. Scale bar = 200 μm. \u003cstrong\u003ef\u003c/strong\u003e, Ear phenotypes of B73 and the \u003cem\u003ezmagp2\u003c/em\u003e mutant. Scale bar = 2 cm. \u003cstrong\u003eg\u003c/strong\u003e, Statistical analysis of pollen viability. \u003cstrong\u003eh\u003c/strong\u003e, Statistical analysis of seed-setting rate. Statistical significance was assessed using Student’s \u003cem\u003et\u003c/em\u003e-test, and significant differences are indicated by asterisks (**\u003cem\u003eP\u003c/em\u003e ≤ 0.01).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8488369/v1/4c262e9cf67ef0fb3c20bddf.jpeg"},{"id":107928307,"identity":"e824c41e-faf0-465f-b210-44193d8e8cf3","added_by":"auto","created_at":"2026-04-27 16:09:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3297145,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8488369/v1/94da8f12-2d0c-4173-8026-1e61f63f6f94.pdf"},{"id":100590115,"identity":"80fd94c3-d794-45cf-b054-6d4aff6c68bd","added_by":"auto","created_at":"2026-01-19 12:47:08","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6763427,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.zip","url":"https://assets-eu.researchsquare.com/files/rs-8488369/v1/22eb61d5f944919b19d2b360.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptional reprogramming of heat-sensitive maize anther development under heat stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWith the intensification of global warming, extreme high-temperature events are occurring more frequently, leading to significant crop losses worldwide and posing a serious threat to global food security and agricultural sustainability. It has been reported that for every 1\u0026nbsp;℃\u0026nbsp;increase in global average temperature, yields of the four major crops\u0026mdash;maize, wheat, rice, and soybeans\u0026mdash;decline by 7.4%, 6.0%, 3.2%, and 3.1%, respectively [1-3]. Maize, one of the world\u0026rsquo;s most important food and feed crops, is particularly vulnerable to heat stress (HS) across multiple developmental stages\u0026nbsp;[4, 5]. During the seedling stage, temperatures exceeding 30 \u0026deg;C disrupt water relations, impair root development, and reduce photosynthetic efficiency\u0026nbsp;[6-8]. In the reproductive phase, temperatures above 35\u0026deg;C shorten the reproductive period, reduce pollen fertility, and inhibit silk elongation, ultimately decreasing kernel numbers and yield\u0026nbsp;[9-11]. During pollination and grain filling, temperatures over 35\u0026deg;C inhibit fertilization, causing a significant restriction of early pollen tube growth\u0026nbsp;[12]. In major maize-growing regions such as China\u0026apos;s Huang-Huai-Hai Plain, HS has emerged as a frequent natural disaster affecting maize production safety\u003csup\u003e\u0026nbsp;\u003c/sup\u003e[13]. Therefore, a mechanistic and developmentally resolved understanding of how maize anthers perceive and integrate heat stress signals is essential for identifying robust targets for thermotolerance breeding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePollen plays a pivotal role in maize reproduction and productivity by facilitating the formation and delivery of male sperm cells to the female gametophyte. However, pollen viability is severely compromised under elevated temperatures, negatively impacting yield and quality [9, 14, 15]. In flowering plants, normal anther development is essential for pollen viability. Anther development begins with stamen primordia initiation and differentiation of somatic and meiotic cell layers. Pollen mother cells (PMCs) undergo meiosis to form four haploid microspores connected by a thickened exine layer\u0026nbsp;[16, 17]. Key processes such as tapetum formation and apoptosis, meiosis, callose synthesis and degradation, and pollen wall formation are tightly linked to pollen development and are particularly sensitive to environmental stresses, especially heat. In \u003cem\u003eArabidopsis\u003c/em\u003e, early heat-induced lesions in male gametogenesis occur during meiosis, with elevated temperatures increasing crossover frequency and disrupting chromosome segregation and cytokinesis. Heat stress also perturbs cytoskeletal dynamics and spindle orientation in \u003cem\u003eArabidopsis\u003c/em\u003e and tobacco\u0026nbsp;[18-20].\u0026nbsp;At later stages of anther development, heat reduces starch and soluble sugar contents in anther wall cells and pollen grains, thereby decreasing pollen viability\u0026nbsp;[21].\u0026nbsp;Elevated temperatures disrupt reactive oxygen species (ROS) homeostasis in the anther sporogenous layer, triggering premature programmed cell death (PCD). For example, rice exposed to heat stress during male meiosis exhibits sharp increases in ROS and superoxide dismutase (SOD) activity\u0026nbsp;[22].\u0026nbsp;These observations highlight that multiple, temporally distinct cellular processes\u0026mdash;spanning meiosis, tapetal PCD, carbohydrate allocation, and mitochondrial energy supply\u0026mdash;collectively define the developmental windows most vulnerable to heat stress\u0026nbsp;[23].\u003c/p\u003e\n\u003cp\u003eAt the molecular and metabolic levels, heat stress disrupts pollen development by affecting phytohormone metabolism and signaling, protein folding in the endoplasmic reticulum, ROS homeostasis, and lipid and carbohydrate metabolism\u0026nbsp;[24]. Tapetal cells are the main source of auxin for pollen maturation; heat stress suppresses key auxin biosynthesis genes such as \u003cem\u003eYUCCA2\u003c/em\u003e and \u003cem\u003eYUCCA\u003c/em\u003e, disrupting microspore auxin homeostasis and inducing male sterility [25]. Jasmonic acid biosynthesis genes acyl-CoA oxidase (\u003cem\u003eACO\u003c/em\u003e) and allene oxide cyclase (\u003cem\u003eAOCs\u003c/em\u003e) are also induced by heat and contribute to male sterility [26]. Heat stress leads to accumulation of misfolded or unfolded proteins in the cytosol and endoplasmic reticulum, causing pollen abortion and male sterility. For example, \u003cem\u003eArabidopsis\u003c/em\u003e \u003cem\u003ebzip28/bzip60\u003c/em\u003e double mutants, deficient in the unfolded protein response (UPR), exhibit increased heat sensitivity [27]. NADPH oxidases (Rbohs), major ROS producers, mitigate ROS overaccumulation and enhance heat tolerance in rice by increasing starch synthase activity [28]. Lipid and carbohydrate metabolism are vital for male reproduction and are disrupted by heat stress [21, 29]. Mutants of rice \u003cem\u003eOsGPT1\u003c/em\u003e (glucose-6-phosphate/phosphate translocator) and \u003cem\u003eOsHXK5\u003c/em\u003e (hexokinase) fail to accumulate starch granules in pollen grains, resulting in male sterility [30, 31]. Similarly, heat stress in maize impairs sugar-to-starch conversion and reduces starch granule size and number, lowering pollen viability [11]. Disruption of lipid metabolism in tapetum and pollen walls under heat stress also leads to male sterility, as shown by \u003cem\u003eGhACO2\u003c/em\u003e knockout in cotton which reduces fatty acid content and accelerates tapetum degradation [26, 32].\u003c/p\u003e\n\u003cp\u003eGiven that pollen viability is determined by a cascade of developmentally coordinated events and is highly sensitive to temperature fluctuations, understanding how heat stress reshapes transcriptional programs throughout anther development becomes essential. Previous studies have largely centered on transient or acute heat treatments, identifying heat-induced male sterility genes or characterizing isolated developmental stages[33-36]. However, these studies provide limited insight into the continuous, progressive transcriptional adjustments that occur throughout anther development under field-relevant HS conditions. Despite extensive work on HS-induced male sterility, the stage-specific regulatory mechanisms that govern pollen viability under prolonged heat exposure remain insufficiently understood. To address this gap, we used the heat-sensitive inbred line Zheng641 as a representative model to investigate stage-specific transcriptional responses under field-based HS. Using tassel branch number and pollen viability as indicators, we defined HS conditions, followed by transcriptome profiling to elucidate gene regulatory networks underlying maize anther development under heat stress. This study advances our understanding of the molecular mechanisms by which HS affects anther development and provides a foundation for identifying genes and pathways associated with heat tolerance.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlant materials and anthers collection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe maize inbred line Zheng641 was grown with staggered sowing in the experimental fields of the Henan Academy of Agricultural Sciences (Yuanyang, China; 113.792\u0026deg;E, 35.108\u0026deg;N). The first sowing occurred on 10 May 2023, followed by four additional sowings at 10-day intervals, designated as SD1\u0026ndash;SD5. For each sowing stage, four rows with 15 plants per row were established with a row spacing of 0.60 m and a plant spacing of 0.25 m. Temperatures are recorded daily by miniature weather stations installed in the fields.\u003c/p\u003e\n\u003cp\u003ePlants with synchronized development from tasseling to pollen shedding were selected daily. spikelets were gently collected and placed into RNase-free, pre-labeled EP tubes. Anthers were rapidly detached from spikelets on ice, and their lengths were measured. Based on length, anthers were grouped into five developmental stages: 1.0 mm, 2.0 mm, 2.5 mm, 3.5 mm, and 4.5 mm. All samples were stored at \u0026minus;80 \u0026deg;C. Each sample consisted of pooled anthers collected from at least three plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePollen viability and microscopy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore flowering, pollination bags were placed on uniformly growing tassels. Following flowering, fresh pollen from three plants per sowing stage was collected daily by shaking the tassels. Pollen was stained with I2-KI stain solution (Solarbio, G4801), observed, and imaged using an inverted microscope (Nikon Ti2-U, Japan). Pollen grains staining black were considered viable; unstained grains were considered non-viable. Pollen viability was calculated as the percentage of viable pollen out of the total observed per microscopic field.\u003c/p\u003e\n\u003cp\u003eFor morphological analysis, fresh pollen was fixed in electron microscope fixative (Servicebio, G1102) at room temperature for 2 hours and stored at 4 \u0026deg;C. Samples were washed three times with 0.1 M sodium phosphate buffer (15 min each), post-fixed with 1% osmium tetroxide, dehydrated in a graded ethanol series (30\u0026ndash;100%), and dried using a critical point dryer (Quorum, K850). Pollen grains were sputter-coated with gold-palladium (IXRF, MSP-2S) and imaged using a scanning electron microscope (SEM; HITACHI SU8010, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ecDNA library preparation and illumina sequencing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on prior field and viability results, the SD2 and SD4 developmental stages were selected for transcriptome sequencing. Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) following the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and integrity was confirmed with an RNA Nano 6000 Assay Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). One microgram of RNA per sample was used for library preparation with the Hieff NGS Ultima Dual-mode mRNA Library Prep Kit (Yeasen Biotechnology, Shanghai, China). Libraries were indexed and sequenced by Biomarker Technologies Co., Ltd. (Beijing, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTranscriptome sequencing data analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw FASTQ reads were initially processed with in-house Perl scripts to remove adapters, poly-N sequences, and low-quality reads. Clean reads were aligned to the maize reference genome (Zm-B73-REFERENCE-NAM-5.0) using HISAT2 (v2.2.1). Transcript assembly and identification of known and novel transcripts were performed using StringTie with reference annotation-based transcript (RABT) assembly. Gene function was annotated using the following databases: NR (ftp://ftp.ncbi.nih.gov/blast/db/); Pfam (http://pfam.xfam.org/); KOG (http://www.ncbi.nlm.nih.gov/KOG/) COG(http://www.ncbi.nlm.nih.gov/COG/); Swiss-Prot (http://www.uniprot.org/); GO (http://www.geneontology.org/). Gene expression levels were quantified as fragments per kilobase of transcript per million mapped reads (FPKM), using the formula: FPKM = (cDNA Fragments) / ((Mapped Fragments in millions) \u0026times; (Transcript Length in kb)). Differential expression analysis between groups was performed using DESeq2 (v1.30.1), and genes with a \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.01 and fold change \u0026ge; 2 were considered differentially expressed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantitative real-time PCR (qRT-PCR)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA from anthers at different developmental stages was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Quantitative PCR was conducted using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) in a 20 \u0026mu;L reaction on a CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA). The cycling conditions were: 95 \u0026deg;C for 3 min, followed by 40 cycles of 95 \u0026deg;C for 10 s and 60 \u0026deg;C for 30 s. Relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. A maize ubiquitin gene (\u003cem\u003eGRMZM2G102471\u003c/em\u003e) was used as internal control. Primer sequences used for verification are listed in (Supplementary Table S8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnalysis of the heat stress treatment Phenotype of\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003e\u003cem\u003ecandidate gene\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eArabidopsis\u003c/em\u003e, the background material used in this study was the \u003cem\u003eCol-0\u003c/em\u003e ecotype. The overexpression vector was pCMBIA1300-35S-GFP, and the transgenic lines were generated via Agrobacterium-mediated floral dip transformation. After the appearance of the first flowers, plants were subjected to heat treatment in a growth room at 37\u0026deg;C with comparable light intensity conditions for 8 h, while the other control plants were grown at 22\u0026deg;C. After 8 h of heat treatment, plants were transferred to 22\u0026deg;C and continued to grow for 10 d. The siliques furthest along in development were selected for phenotypic analysis and length measurement. The relative shortening of silique rate was calculated as follows: [(silique length without heat stress-silique length with heat stress)/ silique length without heat stress] \u0026times; 100%. Siliques were further divided into three major categories: fully fertile (Type I, \u0026gt;10 mm in length), partially sterile (Type II, 5-10 mm in length), and completely sterile (Type III, \u0026lt;5 mm in length).\u003c/p\u003e\n\u003cp\u003eIn maize, the \u003cem\u003ezmagp2\u003c/em\u003e mutant (accession number Mu178054256) was obtained from the China Mu resource (ChinaMu; http://chinamu.jaas.ac.cn/cindex.html). Homozygous \u003cem\u003ezmagp2\u003c/em\u003e mutants were generated through repeated self-pollination of heterozygous plants and subsequently confirmed by PCR using the primers listed in Table S8. During the peak pollen-shedding stage of B73 and \u003cem\u003ezmagp2\u003c/em\u003e mutant, old anthers were removed from the tassels, which were immediately bagged with pollination bags to prevent foreign pollen contamination. At 18:00 daily, tassels with uniform pollen-shedding were selected, excised, immersed in clean water, and transported to the laboratory. These were transferred separately to a control incubator (25\u0026deg;C, dark 12 h; 28\u0026deg;C, light 3 h) or a heat treatment incubator (25\u0026deg;C, dark 12 h; 38\u0026deg;C, light 3 h) for treatment. Following treatment, pollens were collected: one portion received TTC staining, with viability quantified via microscopic counting; the remaining were pollinated onto original female parents (ears pre-bagged with selfing bags). Pollinated plants were maintained under natural conditions until maturity and harvest. The seed setting rate was calculated as follows: (Actual number of kernels / Theoretical number of kernels) \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad and Microsoft Excel. Differences between two groups were assessed using a two-sided Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. For comparisons among multiple groups, one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test was used. All data represent means \u0026plusmn; standard deviation (SD) from three biological replicates.\u003c/p\u003e\n\u003cp\u003eThe effective accumulated temperature (EAT) refers to the total temperature accumulated during the maize growth period, reflecting thermal conditions for development. In this study, a base temperature of 10 \u0026deg;C was used to calculate EAT during anther development. The formula was: EAT = (Daily Maximum Temperature + Daily Minimum Temperature) / 2 \u0026minus; Base Temperature (10 \u0026deg;C).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMorphological observation of Zheng641 across staggered sowing dates\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess factors influencing seed set in maize, two key traits were evaluated: tassel branch number, a major determinant of pollen production, and pollen viability, defined as the proportion of viable pollen grains relative to the total. The heat-sensitive inbred line Zheng641, characterized by abundant tassel branches and high pollen production, was planted using staggered sowing across five sowing dates (SD1\u0026ndash;SD5), beginning on 10 May 2023 with subsequent sowings at 10-day intervals (Fig. 1a). Morphological analyses revealed that SD4 exhibited the most severe reduction in tassel branch number (Fig. 1b), accompanied by a substantial increase in nonviable pollen grains (Fig. 1c). Statistical evaluation further showed that the tassel branching index was lowest in SD4, reaching a value of 14 (Fig. 1d). Pollen viability also differed significantly among sowing dates, with the highest value (81.8%) observed at SD2 and the lowest (38.2%) at SD4 (Fig. 1e).\u003c/p\u003e\n\u003cp\u003eTo examine the influence of temperature, climatic conditions during the growth period were monitored, and daily maximum temperature and effective accumulated temperature\u0026mdash;an indicator of cumulative thermal input critical for maize development\u0026mdash;were calculated from anther initiation to pollen maturation. Average daily maximum temperatures increased progressively from SD1 to SD5, with SD4 exhibiting both the highest maximum temperature and the greatest effective accumulated temperature (Supplementary Fig. S1). These combined thermal conditions account for the pronounced reductions in tassel branching and pollen viability observed in SD4. Collectively, these results indicate that SD4 experienced the most severe heat stress, whereas SD1 and SD2 were least affected. Because SD1 was influenced by notable cooling during anther development, SD2 was selected as the control and SD4 as the heat-stress treatment for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnther development of Zheng641 under heat stress\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of heat stress on anther development, anthers with lengths of 1.0, 2.0, 2.5, 3.5, and 4.5 mm, as well as mature pollen grains, were collected and examined microscopically. Light microscopy of semi-thin sections showed that 1.0-mm anthers were undergoing initial differentiation of sporogenous cells into distinct somatic layers. Meiotic progression followed: meiocytes reached the pachytene stage in 2.0-mm anthers, and tetrads formed in 2.5-mm anthers. By the 3.5-mm stage, meiosis was complete and tetrads had separated into mononuclear microspores. At the 4.5-mm stage, microspores initiated mitosis and tapetal cells had largely degenerated (Fig. 2a-b). Notably, in SD2, 4.5-mm microspores exhibited typical sickle-shaped morphology accompanied by normal tapetal degradation\u0026nbsp;(Fig. 2a). In contrast, anthers in SD4 showed delayed tapetal degradation, thickened epidermal and endothecial layers, and reduced pollen chamber volume (Fig. 2b). Scanning electron microscopy revealed plump, structurally intact pollen in SD2 (Fig. 2c), whereas pollen in SD4 appeared shriveled with disrupted exine ornamentation\u0026nbsp;(Fig. 2d), consistent with impaired tapetum-mediated nutrient supply under heat stress. Together, these observations demonstrate that heat stress disrupts tapetal degradation and causes abnormal pollen development in Zheng641.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGlobal analysis of transcriptome data\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular mechanisms underlying the heat sensitivity of Zheng641, RNA-seq was performed on anthers from the control (SD2) and heat-stressed (SD4) plants across five developmental stages (1.0\u0026ndash;4.5 mm), with three biological replicates per condition, yielding a total of 30 libraries. Sequencing produced high-quality reads (Q30 \u0026gt; 93%) with consistent GC content, ensuring robust downstream analyses. Clean reads were mapped to the maize reference genome (Zm-B73-REFERENCE-NAM-5.0) with alignment efficiencies ranging from 80.01% to 88.21% (Supplementary Table S1). Gene expression distributions showed no systematic bias (Supplementary Fig. S2a), and biological replicates displayed strong correlations (r \u0026gt; 0.97) (Supplementary Fig. S2b), confirming data reliability. Principal component analysis (PCA) revealed clear separation of samples by both developmental stage and treatment, indicating extensive transcriptomic reprogramming under heat stress (Fig. 3a). Using FPKM values and filtering differentially expressed genes (DEGs) by\u0026nbsp;│fold change│\u0026nbsp;\u0026ge;\u0026nbsp;2 and FDR \u0026lt; 0.01, we identified stage-specific DEGs between SD2 and SD4: 10,465 (5,750 upregulated, 4,715 downregulated) at 1.0 mm; 2,802 (1,927 upregulated, 875 downregulated) at 2.0 mm; 2,947 (955 upregulated, 1,992 downregulated) at 2.5 mm; 3,118 (1,327 upregulated, 1,791 downregulated) at 3.5 mm; and 5,438 (2,749 upregulated, 2,689 downregulated) at 4.5 mm (Fig. 3b; Fig. S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTranscript clusters during anther development under heat stress\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the transcriptional dynamics of maize anther development under heat stress, fuzzy k-means clustering was applied to 16,674 dynamically expressed genes from Zheng641 anthers under control (SD2) and heat-stress (SD4) conditions. Twelve co-expression clusters were identified, among which four major heat-response patterns\u0026mdash;NR, IR, LR, and ER\u0026mdash;revealed stage-specific sensitivity of anther development to heat stress, with early and late developmental stages exhibiting the strongest transcriptomic alterations (Fig. 3c-n). Based on their expression responses at different developmental stages, these clusters were categorized into four patterns: a) No Response (NR): Clusters 1 and 2 showed stage-specific expression but no detectable heat-stress effect, both peaking at stage C (Supplementary Table S2). b) Initial Response (IR): Clusters 3, 4, and 5 responded specifically to heat stress during early anther development; Clusters 3 and 4 were upregulated, whereas Cluster 5 was downregulated (Supplementary Table S3). c) Late Response (LR): Clusters 6, 7, and 8 responded during late development; Clusters 6 and 7 were upregulated, while Cluster 8 was downregulated (Supplementary Table S4). d) Entire-stage Response (ER): Clusters 9, 10, 11, and 12 were affected throughout the entire course of anther development (Supplementary Table S5).\u003c/p\u003e\n\u003cp\u003eGene Ontology (GO) and KEGG pathway enrichment analyses were performed for IR clusters to characterize functional shifts under heat stress (Fig. S4). Cluster 4 was enriched in fatty acid and carbohydrate metabolism, indicating early activation of metabolic compensation pathways, whereas Cluster 7 was enriched for reproductive processes, suggesting heat-induced suppression of early gametogenesis (Fig. S4a-b). KEGG analysis further revealed enrichment of plant hormone signal transduction and starch/sucrose metabolism in Cluster 4, while tryptophan metabolism was enriched in Cluster 7 (Fig. S4c-d). These findings suggest that early pollen development under heat stress involves activation of metabolic and hormone-related pathways alongside suppression of reproductive programs.\u003c/p\u003e\n\u003cp\u003eGO and KEGG analyses of LR clusters showed that Cluster 6 (446 genes) was enriched for amino acid transmembrane transport and biosynthesis, whereas Cluster 11 (1,925 genes) was enriched for heat response, cell wall modification, unfolded protein response, and protein folding (Fig. S5). KEGG pathways enriched in Cluster 6 included indole alkaloid biosynthesis and galactose metabolism (Fig. S5a-b), while pentose/glucuronate interconversions and protein processing in the endoplasmic reticulum were enriched in Cluster 11 (Fig. S5c-d). Together, these results indicate that heat stress suppresses cell wall remodeling and disrupts protein folding during late pollen development, consistent with the observed impaired tapetum degradation and thickened anther walls under heat stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDifferentially expressed genes under heat stress\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify key heat-responsive genes during anther development, DEGs were analyzed across five developmental stages (1.0 mm-4.5 mm) under control (SD2) and heat stress (SD4). Venn diagram analysis revealed 6,065, 618, 981, 718, and 2,607 unique DEGs at stages 1.0 mm through 4.5 mm, respectively, with 57 DEGs shared across all stages (Fig. 4a, Supplementary Table S6). Gene Ontology (GO) enrichment analysis was performed on these 57 common DEGs, and the results showed that biological processes related to photosynthesis, pectin catabolic process, and cell wall modification were significantly enriched (Fig. 4b). Notably, 11 DEGs were associated with carbohydrate transport/metabolism. Expression analysis of these 11 genes showed that \u003cem\u003eZm00001eb236200\u003c/em\u003e (\u003cem\u003ePG-like\u003c/em\u003e), \u003cem\u003eZm00001eb424030\u003c/em\u003e (\u003cem\u003ePME1\u003c/em\u003e), \u003cem\u003eZm00001eb165010\u003c/em\u003e (\u003cem\u003ePME43\u003c/em\u003e), \u003cem\u003eZm00001eb270510\u003c/em\u003e, and \u003cem\u003eZm00001eb222250\u003c/em\u003e were downregulated by heat stress, while \u003cem\u003eZm00001eb018720\u003c/em\u003e, \u003cem\u003eZm00001eb080840\u003c/em\u003e, \u003cem\u003eZm00001eb092540\u003c/em\u003e, \u003cem\u003eZm00001eb164390\u003c/em\u003e, \u003cem\u003eZm00001eb197410\u003c/em\u003e, and \u003cem\u003eZm00001eb200910\u003c/em\u003e were upregulated during anther development (Fig. 4c). Since pectin degradation mediated by pectin methylesterases (PMEs) and polygalacturonases (PGs) is essential for programmed tapetum degradation [37], the downregulation of \u003cem\u003ePME1\u003c/em\u003e, \u003cem\u003ePME43\u003c/em\u003e, and \u003cem\u003ePG-like\u003c/em\u003e genes suggests attenuated pectin modification, which is essential for controlled tapetum degeneration, thereby explaining the delayed tapetal breakdown observed morphologically under heat stress.\u003c/p\u003e\n\u003cp\u003eTo validate RNA-seq results, qRT-PCR was performed on eight carbohydrate transport/metabolism-related DEGs: \u003cem\u003eZm00001eb398090\u003c/em\u003e, \u003cem\u003eZm00001eb400780\u003c/em\u003e, \u003cem\u003eZm00001eb371910\u003c/em\u003e, \u003cem\u003eZm00001eb283530\u003c/em\u003e, \u003cem\u003eZm00001eb152540\u003c/em\u003e, \u003cem\u003eZm00001eb183000\u003c/em\u003e, \u003cem\u003eZm00001eb378140\u003c/em\u003e, and \u003cem\u003eZm00001eb304130\u003c/em\u003e. For example, \u003cem\u003eZm00001eb398090\u003c/em\u003e (\u003cem\u003eMs45\u003c/em\u003e), encoding strictosidine synthase involved in male gametophyte cell wall development [38], was highly expressed and downregulated by heat stress at stage 2.0 mm (Fig. S6a). \u003cem\u003eZm00001eb400780\u003c/em\u003e, \u003cem\u003eZm00001eb371910\u003c/em\u003e (encoding xyloglucan endotransglucosylase/hydrolase 12), and \u003cem\u003eZm00001eb283530\u003c/em\u003e were upregulated at stage 2.5 mm (Fig. S6b). \u003cem\u003eZm00001eb152540\u003c/em\u003e and \u003cem\u003eZm00001eb183000\u003c/em\u003e (encoding sucrose transporter 6) were upregulated at stage 3.5 mm (Fig. S6c). \u003cem\u003eZm00001eb378140\u0026nbsp;\u003c/em\u003eand \u003cem\u003eZm00001eb304130\u003c/em\u003e were downregulated at stage 4.5 mm (Fig. S6d). Expression trends matched RNA-seq data, confirming data reliability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIdentification of differentially expressed transcription factors (TFs)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression patterns are largely regulated by TFs. We identified differentially expressed TFs in maize anthers under heat stress at each developmental stage: 448 TFs (137 up, 311 down) at 1.0 mm stage; 45 (32 up, 13 down) at 2.0 mm stage; 69 (17 up, 52 down) at 2.5 mm stage; 50 (24 up, 26 down) at \u0026nbsp;3.5 mm stage; and 72 (42 up, 30 down) at 4.5 mm stage, spanning 54, 21, 24, 26, and 27 TF families, respectively (Fig. 5a\u0026ndash;e, Supplementary Table S7). TF profiling revealed stage-specific regulatory shifts, where bHLH TFs dominate early responses, AP2/ERFs regulate mid-stage events, and MYBs control late pollen maturation, highlighting a coordinated transcriptional cascade underlying heat-induced development disruption. Specific TFs of notes include: \u003cem\u003ebHLH47\u003c/em\u003e (\u003cem\u003eZm00001eb059460\u003c/em\u003e) and \u003cem\u003ebHLH68\u003c/em\u003e (\u003cem\u003eZm00001eb129520\u003c/em\u003e), downregulated, and \u003cem\u003ebHLH51\u003c/em\u003e (\u003cem\u003eZm00001eb208200\u003c/em\u003e) and \u003cem\u003ebHLH152\u003c/em\u003e (\u003cem\u003eZm00001eb244380\u003c/em\u003e), upregulated at 1.0 mm stage. \u003cem\u003eMYC7\u003c/em\u003e (\u003cem\u003eZm00001eb024330\u003c/em\u003e) and \u003cem\u003ebHLH106\u003c/em\u003e (\u003cem\u003eZm00001eb315910\u003c/em\u003e), upregulated at 2.0 mm stage. \u003cem\u003ebHLH125\u003c/em\u003e (\u003cem\u003eZm00001eb375660\u003c/em\u003e) and \u003cem\u003ebHLH127\u003c/em\u003e (\u003cem\u003eZm00001eb228320\u003c/em\u003e), downregulated at 2.5 mm stage (Fig. 5f). \u003cem\u003eEREB50\u003c/em\u003e (\u003cem\u003eZm00001eb119540\u003c/em\u003e) and \u003cem\u003eEREB156\u003c/em\u003e (\u003cem\u003eZm00001eb432090\u003c/em\u003e), upregulated, and \u003cem\u003eEREB202\u003c/em\u003e (\u003cem\u003eZm00001eb099980\u003c/em\u003e) and \u003cem\u003eEREB52\u003c/em\u003e (\u003cem\u003eZm00001eb145420\u003c/em\u003e), downregulated at 3.5 mm stage (Fig. 5g). \u003cem\u003eMYB128\u003c/em\u003e (\u003cem\u003eZm00001eb129490\u003c/em\u003e), \u003cem\u003eMYB15\u003c/em\u003e (\u003cem\u003eZm00001eb148320\u003c/em\u003e), \u003cem\u003eMYB163\u003c/em\u003e (\u003cem\u003eZm00001eb366540\u003c/em\u003e), and \u003cem\u003eMYB74\u003c/em\u003e (\u003cem\u003eZm00001eb369190\u003c/em\u003e), upregulated at 4.5 mm stage (Fig. 5h). These TFs reflect stage-specific regulatory responses to heat stress and warrant further functional validation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunction verification of candidate genes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify the biological functions of candidate genes, 9 DEGs (Zm00001eb296990, Zm00001eb270510, Zm00001eb183000, Zm00001eb283530, Zm00001eb421960, Zm00001eb165010, Zm00001eb222250, Zm00001eb236200, Zm00001eb424030) involved in carbohydrate transport and metabolism pathways were selected for functional validation. We constructed corresponding overexpression vectors and performed functional validation in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Heat stress treatment significantly affected plant development during the flowering (Fig. S7). Notably, the overexpressing line of Zm00001eb296990\u0026nbsp;exhibited a greater plant height after heat stress treatment. (Fig. 6a). Silique length, positively correlated with seed number, is a reliable parameter for investigating heat stress effects on plant reproductive development[27]. Accordingly, we used silique length as an indicator to analyze heat stress impact on the reproductive development of different materials. Results showed heat stress significantly reduced silique length in Arabidopsis (Fig. S8a-b). Compared with wild type, the relative shortening rate of silique lengths in lines overexpressing \u003cem\u003eZm00001eb296990\u003c/em\u003e, exhibited a significantly milder reduction when subjected to heat stress at the reproductive stage (Fig. 6b-c).\u0026nbsp;Furthermore, we classified the siliques into three major categories: fully fertile (Type I), partially sterile (Type II), and completely sterile (Type III). Heat stress significantly increased the percentages of Type II and Type III siliques (Fig. S8c). Notably, compared with the control lines (WT), the Zm00001eb296990-overexpressing line exhibited an increased proportion of Type I siliques, accompanied by a reduced proportion of Type II siliques (Fig. 6d). Zm00001eb296990 encodes ADP-glucose pyrophosphorylase (ZmAGP2) - a key enzyme catalyzing the rate-limiting step in starch biosynthesis. ZmAGP2 overexpression significantly mitigated heat-induced silique abortion, demonstrating a functional role in reproductive heat tolerance, consistent with its position as a starch-biosynthesis regulator.\u003c/p\u003e\n\u003cp\u003eTo better characterize the function of \u003cem\u003eZmAGP2\u003c/em\u003e in maize, the \u003cem\u003ezmagp2\u003c/em\u003e mutant line was identified from the China Mu resource. This mutant carries a Mu insertion (Mu178054256) in the first exon of the \u003cem\u003eZmAGP2\u003c/em\u003e gene in the B73 genetic background (Fig. S9a). Homozygous mutants harboring this Mu insertion were confirmed by PCR-based genotyping (Fig. S9b). Subsequently, self-pollination was used to obtain homozygous wild-type B73 and homozygous \u003cem\u003ezmagp2\u003c/em\u003e mutant lines. TTC staining and pollen viability assays were performed on maize pollen subjected to heat stress or non-stress conditions. The results showed that heat stress led to a reduction in pollen viability in both B73 and \u003cem\u003ezmagp2 mutant\u003c/em\u003e. Notably, compared with B73 exhibiting a 51% decrease in pollen viability, the mutant displayed a more substantial reduction of 68% under heat stress treatment (Fig. 6e, 6g). Consistent with the above results, further analysis of the seed setting rate showed that B73 exhibited a seed setting rate of 72% under normal conditions, which declined to 41% after heat stress. In stark contrast, the \u003cem\u003ezmagp2\u003c/em\u003e mutant had an initial seed setting rate of 59% under non-heat stress conditions, and this value plummeted by 86% to a mere 8% when subjected to heat stress (Fig. 6f, 6h). Collectively, the stronger reduction in pollen viability and seed-setting rate in the zmagp2 mutant indicates that ZmAGP2 is required for maintaining carbohydrate flux under heat stress, and its loss leads to severe reproductive energy deficiency, exacerbating heat sensitivity.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, the heat-sensitive line Zheng641 provided a useful biological model to dissect how elevated temperature disrupts male fertility in maize. Unlike many previous reports that focused solely on extreme heat treatments under controlled conditions[39\u0026ndash;41], our field-based staggered-sowing experiments allowed us to capture natural temperature fluctuations and identify a progressive decline in tassel branching and pollen viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This highlights that even moderately elevated temperatures can compromise pollen productivity before visible heat injury occurs. Although Zheng641 is a heat-sensitive background, similar phenotypic patterns have been documented in a range of tropical, temperate, and subtropical maize lines, suggesting that the developmental processes affected here are broadly conserved across genetic backgrounds. Our results therefore provide a generalizable framework for understanding how gradual seasonal warming impairs male reproductive development and ultimately reduces yield potential.\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHeat stress impairs maize pollen development by disrupting tapetum degeneration and reducing viability\u003c/h2\u003e \u003cp\u003eHigh temperature is known to impair tapetal programmed cell death (PCD)[37, 42, 43], but our stage-resolved analysis further clarifies when and how this disruption occurs. Previous studies have generated RNA sequencing libraries covering 10 key stages of maize anther development, classifying them into four major phases: cell division and expansion, meiosis, pollen maturation, and mature pollen, and identified stage-specific key genes in the inbred line Chang7-2[44].Here, we revealed that heat stress caused abnormal tapetum development, with tight adhesion to the inner epidermis and failure to undergo normal degradation, thickening of epidermis and endodermis layers, and a markedly reduced pollen chamber size (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Heat stress caused tight adhesion of the tapetum to the inner epidermis and delayed degradation specifically during meiosis and early pollen maturation, stages in which lipidic precursors and carbohydrate reserves normally accumulate rapidly[45, 46]. Because tapetum-derived metabolites fuel pollen wall biosynthesis and supply energy for microspore expansion, the concurrent suppression of lipid and sugar metabolic pathways provides a mechanistic link between transcriptional reprogramming and cellular abnormalities[30, 47, 48]. We therefore propose that heat stress initiates a cascade in which early mis-regulation of metabolic pathways compromises tapetal function, leading to insufficient nutrient flux to developing microspores and ultimately pollen collapse. This stage-specific vulnerability also explains why minor elevation in temperature disproportionately affects fertility compared with vegetative growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHeat stress alters lipid and carbohydrate metabolism during anther development\u003c/h2\u003e \u003cp\u003eThe substantial enrichment of DEGs involved in fatty acid biosynthesis and carbohydrate metabolism during early anther development suggests that these pathways constitute a primary metabolic checkpoint for heat tolerance. Previous studies in rice (TDR/UDT1) and Arabidopsis (AMS/DYT1) indicate that tapetal TFs form hierarchical regulatory modules controlling lipidic precursor synthesis and transport[49\u0026ndash;51]. Our data reveals that heat stress perturbs similar gene networks in maize, implying partial conservation of the metabolic-transcriptional circuitry across species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The observed mis-regulation of key genes such as \u003cem\u003eMs45\u003c/em\u003e further supports the notion that heat disrupts the coordination between metabolic flux and tapetal maturation[38]. Integrating these findings, we propose that maize anthers rely on a TF-centered regulatory hub that synchronizes lipid and carbohydrate metabolism with developmental timing, and that heat stress destabilizes this hub, leading to metabolic insufficiency and pollen abortion. This framework provides testable hypotheses for future genetic and biochemical validation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStage-specific transcriptional regulation of anther development under heat stress\u003c/h2\u003e \u003cp\u003eThe dynamic expression patterns of bHLH, MYB, and AP2/ERF transcription factors highlight that each developmental stage recruits distinct regulatory modules to cope with heat stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Several of these TFs correspond to orthologs of well-characterized regulators in \u003cem\u003eArabidopsis\u003c/em\u003e and rice[48, 52\u0026ndash;54], suggesting that conserved reproductive stress-response circuits operate in maize. The strong induction of AP2/ERF factors at late stages further indicates a shift from developmental regulation toward stress mitigation, potentially coordinating ROS scavenging and cell wall remodeling[43, 55]. Based on these observations, we propose a working model in which heat stress disrupts the early bHLH-MYB regulatory cascade controlling tapetal development, while later activation of AP2/ERF TFs reflects compensatory mechanisms that ultimately prove insufficient to rescue pollen viability.\u003c/p\u003e \u003cp\u003eImportantly, the TFs identified here\u0026mdash;including bHLH47/68/51/125, MYB15/74/128/163, and EREB50/52/156/202\u0026mdash;represent promising targets for breeding heat-resilient varieties through marker-assisted selection or CRISPR-based genome editing. Future studies integrating proteomics, chromatin accessibility profiling, and transgenic validation will be essential to define the upstream signals and downstream effectors of these TFs, and to establish a comprehensive genetic network underlying thermotolerance in maize anthers.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eH.L. and L.W. conceived and designed research.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eS.W., J.W., P.W.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand H.W. conducted experiments.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eY.D. and Z.Z. contributed new reagents or analytical tools.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eJ.W., J.Y. and J.L.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eanalyzed data.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eJ.W. writing\u0026mdash;original draft preparation. L.Z., H.L. writing\u0026mdash;review and editing.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was supported by National Key R\u0026amp;D Program of China (2023YFD1200505), Central Government-Guided Local Science and Technology Development Fund Project of Henan Province (2025ZYYD06), National Key R\u0026amp;D Program of China (2021YFD1200703), Technology Innovation Team Project (2025TD19), Applied Science and Technology Research Projects (2025YG01) and Independent Innovative (2024ZC028) Projects of Henan Academy of Agricultural Science.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statemen:\u0026nbsp;\u003c/strong\u003eData supporting the findings of this work are available within the paper and its Supplementary Information files. The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request, Original RNA-seq data for this article was deposited in Science Data Bank (https://doi.org/10.57760/sciencedb.28740).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declarations:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhao C, Liu B, Piao S, Wang X, Lobell DB, Huang Y, Huang M, Yao Y, Bassu S, Ciais P\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eTemperature increase reduces global yields of major crops in four independent estimates\u003c/strong\u003e. \u003cem\u003eProceedings of 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\u003cstrong\u003e15\u003c/strong\u003e:10857.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8488369/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8488369/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeat stress during anther development severely impairs pollen fertility and substantially reduces yield. However, how heat stress reshapes transcriptional programs at specific anther developmental stages and which regulatory mechanisms underlie stage-dependent sensitivity remain unclear. Here, we used the heat-sensitive inbred line Zheng641 as a model to characterize fine-scale transcriptional responses to heat stress. Morphological analyses showed that elevated temperatures markedly reduced tassel branch number and pollen viability and delayed tapetal degradation. High-resolution transcriptomic profiling revealed that heat stress mainly activated the pathways related to fatty acid biosynthesis and carbohydrate metabolism during the early stage of pollen development, whereas late-stage heat stress induced amino acid transport and biosynthesis while repressing cell wall modification and protein folding. Phase‐specific transcription factor analysis indicated that \u003cem\u003ebHLHs\u003c/em\u003e transcription factor were significantly enriched before the tetrad stage, \u003cem\u003eAP2/ERFs\u003c/em\u003e in microspore stage, \u003cem\u003eMYBs\u003c/em\u003e in pollen mature stage. Furthermore, we explored \u003cem\u003eZm00001eb296990\u003c/em\u003e could improve thermotolerance by the phenotypic analysis of transgenic overexpressed lines. Notably, the function-loss of \u003cem\u003eZm00001eb296990\u003c/em\u003e significantly reduced pollen viability and seed set under heat stress. Together, these findings provide a high-resolution transcriptomic framework for understanding the molecular basis of maize anther sensitivity to heat and identify promising targets for enhancing thermotolerance in maize breeding.\u003c/p\u003e","manuscriptTitle":"Transcriptional reprogramming of heat-sensitive maize anther development under heat stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 12:33:54","doi":"10.21203/rs.3.rs-8488369/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-10T03:43:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-21T23:59:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-15T09:00:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214839767712049436923340972550726917868","date":"2026-02-09T02:42:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25295561117620946545178534743389347114","date":"2026-02-08T08:57:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93862360053477629727851285066920102521","date":"2026-01-19T05:42:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153430347304679931000888544721208418955","date":"2026-01-16T17:38:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-14T02:25:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-14T02:16:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-13T11:25:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-13T02:25:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-01-13T02:19:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad3a9b1d-7a75-4813-8ee0-ac1ad4558086","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:07:33+00:00","versionOfRecord":{"articleIdentity":"rs-8488369","link":"https://doi.org/10.1186/s12870-026-08732-2","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2026-04-21 15:59:04","publishedOnDateReadable":"April 21st, 2026"},"versionCreatedAt":"2026-01-19 12:33:54","video":"","vorDoi":"10.1186/s12870-026-08732-2","vorDoiUrl":"https://doi.org/10.1186/s12870-026-08732-2","workflowStages":[]},"version":"v1","identity":"rs-8488369","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8488369","identity":"rs-8488369","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}