Metabolite Profiling and Biological Activity Assessment of Paeonia ostii Anthers and Pollen Using UPLC-QTOF-MS.

Paeonia ostii is an important economic oil and medicinal crop. Its anthers are often used to make tea in China with beneficial effects on human health. However, the metabolite profiles, as well as potential biological activities of P. ostii anthers and the pollen within anthers have not been systematically analyzed, which hinders the improvement of P. ostii utilization. With comprehensive untargeted metabolomic analysis using UPLC-QTOF-MS, we identified a total of 105 metabolites in anthers and pollen, mainly including phenylpropanoids, polyketides, organic acids, benzenoids, lipids, and organic oxygen compounds. Multivariate statistical analysis revealed the metabolite differences between anthers and pollen, with higher carbohydrates and flavonoids content in pollen and higher phenolic content in anthers. Meanwhile, both anthers and pollen extracts exhibited antioxidant activity, antibacterial activity, α-glucosidase and α-amylase inhibitory activity. In general, the anther stage of S4 showed the highest biological activity among all samples. This study illuminated the metabolites and biological activities of anthers and pollen of P. ostii, which supports the further utilization of them.

Mitigation of UV-B Radiation Stress in Tobacco Pollen by Expression of the Tardigrade Damage Suppressor Protein (Dsup).

Pollen, the male gametophyte of seed plants, is extremely sensitive to UV light, which may prevent fertilization. As a result, strategies to improve plant resistance to solar ultraviolet (UV) radiation are required. The tardigrade damage suppressor protein (Dsup) is a putative DNA-binding protein that enables tardigrades to tolerate harsh environmental conditions, including UV radiation, and was therefore considered as a candidate for reducing the effects of UV exposure on pollen. Tobacco pollen was genetically engineered to express Dsup and then exposed to UV-B radiation to determine the effectiveness of the protein in increasing pollen resistance. To establish the preventive role of Dsup against UV-B stress, we carried out extensive investigations into pollen viability, germination rate, pollen tube length, male germ unit position, callose plug development, marker protein content, and antioxidant capacity. The results indicated that UV-B stress has a significant negative impact on both pollen grain and pollen tube growth. However, Dsup expression increased the antioxidant levels and reversed some of the UV-B-induced changes to pollen, restoring the proper distance between the tip and the last callose plug formed, as well as pollen tube length, tubulin, and HSP70 levels. Therefore, the expression of heterologous Dsup in pollen may provide the plant male gametophyte with enhanced responses to UV-B stress and protection against harmful environmental radiation.

OsSRF8 interacts with OsINP1 and OsDAF1 to regulate pollen aperture formation in rice.

In higher plants, mature male gametophytes have distinct apertures. After pollination, pollen grains germinate, and a pollen tube grows from the aperture to deliver sperm cells to the embryo sac, completing fertilization. In rice, the pollen aperture has a single-pore structure with a collar-like annulus and a plug-like operculum. A crucial step in aperture development is the formation of aperture plasma membrane protrusion (APMP) at the distal polar region of the microspore during the late tetrad stage. Previous studies identified OsINP1 and OsDAF1 as essential regulators of APMP and pollen aperture formation in rice, but their precise molecular mechanisms remain unclear. We demonstrate that the Poaceae-specific OsSRF8 gene, encoding a STRUBBELIG-receptor family 8 protein, is essential for pollen aperture formation in Oryza sativa. Mutants lacking functional OsSRF8 exhibit defects in APMP and pollen aperture formation, like loss-of-function OsINP1 mutants. OsSRF8 is specifically expressed during early anther development and initially diffusely distributed in the microsporocytes. At the tetrad stage, OsSRF8 is recruited by OsINP1 to the pre-aperture region through direct protein-protein interaction, promoting APMP formation. The OsSRF8-OsINP1 complex then recruits OsDAF1 to the APMP site to co-regulate annulus formation. Our findings provide insights into the mechanisms controlling pollen aperture formation in cereal species.

Fatty acid de novo biosynthesis in plastids: Key enzymes and their critical roles for male reproduction and other processes in plants.

Fatty acid de novo biosynthesis in plant plastids is initiated from acetyl-CoA and catalyzed by a series of enzymes, which is required for the vegetative growth, reproductive growth, seed development, stress response, chloroplast development and other biological processes. In this review, we systematically summarized the fatty acid de novo biosynthesis-related genes/enzymes and their critical roles in various plant developmental processes. Based on bioinformatic analysis, we identified fatty acid synthase encoding genes and predicted their potential functions in maize growth and development, especially in anther and pollen development. Finally, we highlighted the potential applications of these fatty acid synthases in male-sterility hybrid breeding, seed oil content improvement, herbicide and abiotic stress resistance, which provides new insights into future molecular crop breeding.

Autophagy-mediated degradation of integumentary tapetum is critical for embryo pattern formation.

Autophagy modulates the degradation and recycling of intracellular materials and contributes to male gametophyte development and male fertility in plants. However, whether autophagy participates in seed development remains largely unknown. Here, we demonstrate that autophagy is crucial for timely programmed cell death (PCD) in the integumentary tapetum, the counterpart of anther tapetum, influencing embryo pattern formation and seed viability. Inhibition of autophagy resulted in delayed PCD of the integumentary tapetum and defects in embryo patterning. Cell-type-specific restoration of autophagic activities revealed that the integumentary tapetum plays a non-autonomous role in embryo patterning. Furthermore, high-throughput, comprehensive lipidomic analyzes uncovered an unexpected seed-developmental-stage-dependent role of autophagy in seed lipid metabolism: it contributes to triacylglycerol degradation before fertilization and to triacylglycerol biosynthesis after fertilization. This study highlights the critical role of autophagy in regulating timely integumentary tapetum PCD and reveals its significance in seed lipid metabolism and viability.

Distribution of exchangeable Ca2+ during the process of Larix decidua Mill. pollination and germination.

The involvement of Ca2+ ions in angiosperms sexual processes is well established, while in gymnosperms, such knowledge remains limited and is still a topic of discussion. In this study, we focused on Larix decidua, using Alizarin-red S staining and the pyroantimonate method to examine the tissue and subcellular distribution of free and loosely bound Ca2+ ions at different stages of the male gametophyte's development and its interaction with the ovule. Our findings show that in larch, both the germination of pollen grains and the growth of pollen tubes occur in an environment rich in Ca2+. These ions play a crucial role in the adhesion of the pollen grain to the stigmatic tip and its subsequent movement to the micropylar canal. There is a significant presence of free and loosely bound Ca2+ ions in both the fluid of the micropylar canal and the extracellular matrix of the nucellus. As the pollen tube extends through the nucellus, we observed a notable accumulation of Ca2+ ions just above the entry to the mature archegonium, a region likely crucial for the male gametophyte's directional growth. Meanwhile, the localized presence of free and loosely bound Ca2+ ions within the egg cell cytoplasm may inhibit the pollen tubes growth and rupture, playing an important role in fertilization.

Allergenicity and structural properties of new Cor a 1 isoallergens from hazel identified in different plant tissues.

The hazel allergen Cor a 1 is a PR-10 protein, closely related to the major birch pollen allergen Bet v 1. Hazel allergies are caused by cross-reactive IgE antibodies originally directed against Bet v 1. Despite the importance of PR-10 proteins in allergy development, their function and localization in the plant remain largely elusive. Therefore, the presence of Cor a 1 mRNA and proteins was investigated in different tissues, i.e., the female flower, immature and mature nuts, catkins, and pollen. Four yet unknown Cor a 1 isoallergens, i.e., Cor a 1.0501-1.0801, and one new Cor a 1.03 variant were discovered and characterized. Depending on the isoallergen, the occurrence and level of mRNA expression varied in different tissues, suggesting different functions. Interestingly, Cor a 1.04 previously thought to be only present in nuts, was also detected in catkins and pollen. The corresponding Cor a 1 genes were expressed in Escherichia coli. The purified proteins were analysed by CD and NMR spectroscopy. Immunoblots and ELISAs to determine their allergenic potential showed that the new proteins reacted positively with sera from patients allergic to birch, hazel and elder pollen and were recognized as novel isoallergens/variants by the WHO/IUIS Allergen Nomenclature Sub-Committee.

Exo84c-regulated degradation is involved in the normal self-incompatible response in Brassicaceae.

The self-incompatibility system evolves in angiosperms to promote cross-pollination by rejecting self-pollination. Here, we show the involvement of Exo84c in the SI response of both Brassica napus and Arabidopsis. The expression of Exo84c is specifically elevated in stigma during the SI response. Knocking out Exo84c in B. napus and SI Arabidopsis partially breaks down the SI response. The SI response inhibits both the protein secretion in papillae and the recruitment of the exocyst complex to the pollen-pistil contact sites. Interestingly, these processes can be partially restored in exo84c SI Arabidopsis. After incompatible pollination, the turnover of the exocyst-labeled compartment is enhanced in papillae. However, this process is perturbed in exo84c SI Arabidopsis. Taken together, our results suggest that Exo84c regulates the exocyst complex vacuolar degradation during the SI response. This process is likely independent of the known SI pathway in Brassicaceae to secure the SI response.

Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth.

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

Rice transcriptional repressor OsTIE1 controls anther dehiscence and male sterility by regulating JA biosynthesis.

Proper anther dehiscence is essential for successful pollination and reproduction in angiosperms, and jasmonic acid (JA) is crucial for the process. However, the mechanisms underlying the tight regulation of JA biosynthesis during anther development remain largely unknown. Here, we demonstrate that the rice (Oryza sativa L.) ethylene-response factor-associated amphiphilic repression (EAR) motif-containing protein TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) INTERACTOR CONTAINING EAR MOTIF PROTEIN1 (OsTIE1) tightly regulates JA biosynthesis by repressing TCP transcription factor OsTCP1/PCF5 during anther development. The loss of OsTIE1 function in Ostie1 mutants causes male sterility. The Ostie1 mutants display inviable pollen, early stamen filament elongation, and precocious anther dehiscence. In addition, JA biosynthesis is activated earlier and JA abundance is precociously increased in Ostie1 anthers. OsTIE1 is expressed during anther development, and OsTIE1 is localized in nuclei and has transcriptional repression activity. OsTIE1 directly interacts with OsTCP1, and overexpression of OsTCP1 caused early anther dehiscence resembling that of Ostie1. JA biosynthesis genes including rice LIPOXYGENASE are regulated by the OsTIE1-OsTCP1 complex. Our findings reveal that the OsTIE1-OsTCP1 module plays a critical role in anther development by finely tuning JA biosynthesis and provide a foundation for the generation of male sterile plants for hybrid seed production.

Mediator kinase subunit CDK8 phosphorylates transcription factor TCP15 during tomato pollen development.

Pollen development in flowering plants has strong implications for reproductive success. Pollen DNA can be targeted to improve plant traits for yield and stress tolerance. In this study, we demonstrated that the Mediator subunit CYCLIN-DEPENDENT KINASE 8 (CDK8) is a key modulator of pollen development in tomato (Solanum lycopersicum). SlCDK8 knockout led to significant decreases in pollen viability, fruit yield, and fruit seed number. We also found that SlCDK8 directly interacts with transcription factor TEOSINTE BRANCHED1-CYCLOIDEA-PCF15 (SlTCP15) using yeast two-hybrid screens. We subsequently showed that SlCDK8 phosphorylates Ser 187 of SlTCP15 to promote SlTCP15 stability. Phosphorylated TCP15 directly bound to the TGGGCY sequence in the promoters of DYSFUNCTIONAL TAPETUM 1 (SlDYT1) and MYB DOMAIN PROTEIN 103 (SlMYB103), which are responsible for pollen development. Consistently, disruption of SlTCP15 resembled slcdk8 tomato mutants. In sum, our work identified a new substrate of Mediator CDK8 and revealed an important regulatory role of SlCDK8 in pollen development via cooperation with SlTCP15.

Reactive oxygen species are signaling molecules that modulate plant reproduction.

Reactive oxygen species (ROS) can serve as signaling molecules that are essential for plant growth and development but abiotic stress can lead to ROS increases to supraoptimal levels resulting in cellular damage. To ensure efficient ROS signaling, cells have machinery to locally synthesize ROS to initiate cellular responses and to scavenge ROS to prevent it from reaching damaging levels. This review summarizes experimental evidence revealing the role of ROS during multiple stages of plant reproduction. Localized ROS synthesis controls the formation of pollen grains, pollen-stigma interactions, pollen tube growth, ovule development, and fertilization. Plants utilize ROS-producing enzymes such as respiratory burst oxidase homologs and organelle metabolic pathways to generate ROS, while the presence of scavenging mechanisms, including synthesis of antioxidant proteins and small molecules, serves to prevent its escalation to harmful levels. In this review, we summarized the function of ROS and its synthesis and scavenging mechanisms in all reproductive stages from gametophyte development until completion of fertilization. Additionally, we further address the impact of elevated temperatures induced ROS on impairing these reproductive processes and of flavonol antioxidants in maintaining ROS homeostasis to minimize temperature stress to combat the impact of global climate change on agriculture.

Antioxidant and anti-Alzheimer's potential of Tetragonisca angustula (Jataí) stingless bee pollen.

Alzheimer's disease (AD) is considered the leading cause of dementia in the elderly worldwide. It results in progressive memory loss and impairment of cognitive and motor skills, leading to a high degree of disability and dependence. The development of AD is associated with the accumulation of senile plaques in the brain, caused by the amyloidogenic pathway of the disease. Several genetic and biochemical events are linked to AD development, with oxidative stress being one of them. Due to the scarcity of drugs aimed at treating AD, antioxidant compounds are increasingly studied as therapeutic targets for the disease. In this study, we investigate the antioxidant and anti-Alzheimer potential of the Tetragonisca angustula (Jataí) pollen extract in a Drosophila melanogaster Alzheimer's model. For this purpose, we utilized a D. melanogaster AD-like model, which expresses genes related to the amyloidogenic pathway of Alzheimer's disease. We explored the floral origin of the collected pollen, conducted phytochemical prospecting, and evaluated its antioxidant capacity in vitro. In vivo experiments involved assessing the survival and climbing ability of the D. melanogaster AD-like model with various concentrations of the pollen extract. Our findings revealed that the pollen extract of Tetragonisca angustula exhibits a significant antioxidant response and high concentrations of important phytochemicals, such as flavonoids and polyphenols. Furthermore, it enhanced the survival rate of D. melanogaster, and across all concentrations tested, it improved the climbing ability of the flies after 15 days of treatment with methanolic pollen extract. Additionally, the pollen extract reduced the neurodegeneration index in histopathological analysis. Thus, our study demonstrates the potential of Tetragonisca angustula pollen as an important subject for further investigation, aiming to isolate molecules that could potentially serve as therapeutic targets for Alzheimer's disease.

IiAGL6 participates in the regulation of stamen development and pollen formation in Isatis indigotica.

The AGL6 (AGMOUSE LIKE 6) gene is a member of the SEP subfamily and functions as an E-class floral homeotic gene in the development of floral organs. In this study, we cloned IiAGL6, the orthologous gene of AGL6 in Isatis indigotica. The constitutive expression of IiAGL6 in Arabidopsis thaliana resulted in a late-flowering phenotype and the development of curly leaves during the vegetative growth period. Abnormal changes in floral organ development were observed during the reproductive stage. In woad plants, suppression of IiAGL6 using TRV-VIGS (tobacco rattle virus-mediated virus-induced gene silencing) decreased the number of stamens and led to the formation of aberrant anthers. Similar changes in stamen development were also observed in miRNA-AGL6 transgenic Arabidopsis plants. Yeast two-hybrid and BiFC tests showed that IiAGL6 can interact with other MADS-box proteins in woad; thus, playing a key role in defining the identities of floral organs, particularly during stamen formation. These findings might provide novel insights and help investigate the biological roles of MADS transcription factors in I. indigotica.

Functional Characterization of CsSWEET5a, a Cucumber Hexose Transporter That Mediates the Hexose Supply for Pollen Development and Rescues Male Fertility in Arabidopsis.

Pollen cells require large amounts of sugars from the anther to support their development, which is critical for plant sexual reproduction and crop yield. Sugars Will Eventually be Exported Transporters (SWEETs) have been shown to play an important role in the apoplasmic unloading of sugars from anther tissues into symplasmically isolated developing pollen cells and thereby affect the sugar supply for pollen development. However, among the 17 CsSWEET genes identified in the cucumber (Cucumis sativus L.) genome, the CsSWEET gene involved in this process has not been identified. Here, a member of the SWEET gene family, CsSWEET5a, was identified and characterized. The quantitative real-time PCR and ß-glucuronidase expression analysis revealed that CsSWEET5a is highly expressed in the anthers and pollen cells of male cucumber flowers from the microsporocyte stage (stage 9) to the mature pollen stage (stage 12). Its subcellular localization indicated that the CsSWEET5a protein is localized to the plasma membrane. The heterologous expression assays in yeast demonstrated that CsSWEET5a encodes a hexose transporter that can complement both glucose and fructose transport deficiencies. CsSWEET5a can significantly rescue the pollen viability and fertility of atsweet8 mutant Arabidopsis plants. The possible role of CsSWEET5a in supplying hexose to developing pollen cells via the apoplast is also discussed.

Evolution of the thermostability of actin-depolymerizing factors enhances the adaptation of pollen germination to high temperature.

Double fertilization in many flowering plants (angiosperms) often occurs during the hot summer season, but the mechanisms that enable angiosperms to adapt specifically to high temperatures are largely unknown. The actin cytoskeleton is essential for pollen germination and the polarized growth of pollen tubes, yet how this process responds to high temperatures remains unclear. Here, we reveal that the high thermal stability of 11 Arabidopsis (Arabidopsis thaliana) actin-depolymerizing factors (ADFs) is significantly different: ADFs that specifically accumulate in tip-growing cells (pollen and root hairs) exhibit high thermal stability. Through ancestral protein reconstruction, we found that subclass II ADFs (expressed specifically in pollen) have undergone a dynamic wave-like evolution of the retention, loss, and regeneration of thermostable sites. Additionally, the sites of AtADF7 with high thermal stability are conserved in ADFs specific to angiosperm pollen. Moreover, the high thermal stability of ADFs is required to regulate actin dynamics and turnover at high temperatures to promote pollen germination. Collectively, these findings suggest strategies for the adaptation of sexual reproduction to high temperature in angiosperms at the cell biology level.

CRISPR/Cas9-based genome editing of 14 lipid metabolic genes reveals a sporopollenin metabolon ZmPKSB-ZmTKPR1-1/-2 required for pollen exine formation in maize.

Lipid biosynthesis and transport are essential for plant male reproduction. Compared with Arabidopsis and rice, relatively fewer maize lipid metabolic genic male-sterility (GMS) genes have been identified, and the sporopollenin metabolon in maize anther remains unknown. Here, we identified two maize GMS genes, ZmTKPR1-1 and ZmTKPR1-2, by CRISPR/Cas9 mutagenesis of 14 lipid metabolic genes with anther stage-specific expression patterns. Among them, tkpr1-1/-2 double mutants displayed complete male sterility with delayed tapetum degradation and abortive pollen. ZmTKPR1-1 and ZmTKPR1-2 encode tetraketide α-pyrone reductases and have catalytic activities in reducing tetraketide α-pyrone produced by ZmPKSB (polyketide synthase B). Several conserved catalytic sites (S128/130, Y164/166 and K168/170 in ZmTKPR1-1/-2) are essential for their enzymatic activities. Both ZmTKPR1-1 and ZmTKPR1-2 are directly activated by ZmMYB84, and their encoded proteins are localized in both the endoplasmic reticulum and nuclei. Based on protein structure prediction, molecular docking, site-directed mutagenesis and biochemical assays, the sporopollenin biosynthetic metabolon ZmPKSB-ZmTKPR1-1/-2 was identified to control pollen exine formation in maize anther. Although ZmTKPR1-1/-2 and ZmPKSB formed a protein complex, their mutants showed different, even opposite, defective phenotypes of anther cuticle and pollen exine. Our findings discover new maize GMS genes that can contribute to male-sterility line-assisted maize breeding and also provide new insights into the metabolon-regulated sporopollenin biosynthesis in maize anther.

Unlocking a 'lock-key' mechanism governing pollen-pistil interactions.

Pollen-pistil interactions ensure genetic diversity and shape the reproductive success of plants. Lan et al. recently revealed that the interaction among various receptor-like kinases, cell-wall proteins, and stigmatic RALF peptides (sRALFs) or pollen RALF peptides (pRALFs) on the stigma surface govern the penetration of pollen tubes in members of the Brassicaceae.

Heterologous Expression of Two Brassica campestris CCCH Zinc-Finger Proteins in Arabidopsis Induces Cytoplasmic Foci and Causes Pollen Abortion.

The membrane-less organelles in cytoplasm that are presented as cytoplasmic foci were successively identified. Although multiple CCCH zinc-finger proteins have been found to be localized in cytoplasmic foci, the relationship between their specific localization and functions still needs further clarification. Here, we report that the heterologous expression of two Brassica campestris CCCH zinc-finger protein genes (BcMF30a and BcMF30c) in Arabidopsis thaliana can affect microgametogenesis by involving the formation of cytoplasmic foci. By monitoring the distribution of proteins and observing pollen phenotypes, we found that, when these two proteins were moderately expressed in pollen, they were mainly dispersed in the cytoplasm, and the pollen developed normally. However, high expression induced the assembly of cytoplasmic foci, leading to pollen abortion. These findings suggested that the continuous formation of BcMF30a/BcMF30c-associated cytoplasmic foci due to high expression was the inducement of male sterility. A co-localization analysis further showed that these two proteins can be recruited into two well-studied cytoplasmic foci, processing bodies (PBs), and stress granules (SGs), which were confirmed to function in mRNA metabolism. Together, our data suggested that BcMF30a and BcMF30c play component roles in the assembly of pollen cytoplasmic foci. Combined with our previous study on the homologous gene of BcMF30a/c in Arabidopsis, we concluded that the function of these homologous genes is conserved and that cytoplasmic foci containing BcMF30a/c may participate in the regulation of gene expression in pollen by regulating mRNA metabolism.

Heat-responsive microRNAs participate in regulating the pollen fertility stability of CMS-D2 restorer line under high-temperature stress.

Anther development and pollen fertility of cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) restorer lines are susceptible to continuous high-temperature (HT) stress in summer, which seriously hinders the large-scale application of "three-line" hybrids in production. Here, integrated small RNA, transcriptome, degradome, and hormone profiling was performed to explore the roles of microRNAs (miRNAs) in regulating fertility stability in mature pollens of isonuclear alloplasmic near-isogenic restorer lines NH and SH under HT stress at two environments. A total of 211 known and 248 novel miRNAs were identified, of which 159 were differentially expressed miRNAs (DEMs). Additionally, 45 DEMs in 39 miRNA clusters (PmCs) were also identified, and most highly expressed miRNAs were significantly induced in SH under extreme HT, especially four MIR482 and six MIR6300 family miRNAs. PmC28 was located in the fine-mapped interval of the Rf1 gene and contained two DEMs, gra-miR482_L-2R + 2 and gma-miR2118a-3p_R + 1_1ss18TG. Transcriptome sequencing identified 6281 differentially expressed genes, of which heat shock protein (HSP)-related genes, such as HSP70, HSP22, HSP18.5-C, HSP18.2 and HSP17.3-B, presented significantly reduced expression levels in SH under HT stress. Through integrating multi-omics data, we constructed a comprehensive molecular network of miRNA-mRNA-gene-KEGG containing 35 pairs of miRNA/target genes involved in regulating the pollen development in response to HT, among which the mtr-miR167a_R + 1, tcc-miR167c and ghr-miR390a, tcc-miR396c_L-1 and ghr-MIR169b-p3_1ss6AG regulated the pollen fertility by influencing ARF8 responsible for the auxin signal transduction, ascorbate and aldarate metabolism, and the sugar and lipid metabolism and transport pathways, respectively. Further combination with hormone analysis revealed that HT-induced jasmonic acid signaling could activate the expression of downstream auxin synthesis-related genes and cause excessive auxin accumulation, followed by a cascade of auxin signal transduction, ultimately resulting in pollen abortion. The results provide a new understanding of how heat-responsive miRNAs regulate the stability of fertility restoration for CMS-D2 cotton under heat stress.