The pollen wall is a specialized extracellular cell wall that protects male gametophytes from various environmental stresses and facilitates pollination. Here, we reported that bHLH010 and bHLH089 together are required for the development of the pollen wall by regulating their specific downstream transcriptional and metabolic networks. Both the exine and intine structures of bhlh010 bhlh089 pollen grains were severely defective. Further untargeted metabolomic and transcriptomic analyses revealed that the accumulation of pollen wall morphogenesis-related metabolites, including polysaccharides, glyceryl derivatives, and flavonols, were significantly changed, and the expression of such metabolic enzyme-encoding genes and transporter-encoding genes related to pollen wall morphogenesis was downregulated in bhlh010 bhlh089 mutants. Among these downstream target genes, CSLB03 is a novel target with no biological function being reported yet. We found that bHLH010 interacted with the two E-box sequences at the promoter of CSLB03 and directly activated the expression of CSLB03. The cslb03 mutant alleles showed bhlh010 bhlh089–like pollen developmental defects, with most of the pollen grains exhibiting defective pollen wall structures.

Faithful pollen wall formation requires tight control of both the amplitude and duration of tapetal transcriptional activity. However, how such dynamic restraint is achieved during anther development remains poorly understood. Here, we identify a phosphorylation-dependent regulatory module in which SBI-PP2A-C1/C5 constrains the functional activity window of the tapetal transcription factor bHLH089 to safeguard male fertility. PP2A-C1 and PP2A-C5 physically associate with bHLH089 and limit its transcriptional output by reducing bHLH089 phosphorylation and nuclear retention, thereby restricting its nuclear accumulation and effective activity. Genetic analyses reveal that precise PP2A-C1/C5 dosage is critical for pollen wall integrity, as both reduced PP2A-C function and PP2A-C1/C5 overexpression disrupt tapetal transcriptional homeostasis and compromise male fertility. Mechanistically, Ser28 and Ser58 function as conserved phosphorylation-dependent determinants of bHLH089 nuclear retention and are required for PP2A-C1/C5-sensitive regulation of bHLH089 activity. Together, these findings identify a phosphatase-mediated timing mechanism that integrates transcriptional feedback with post-translational control to constrain bHLH089-driven transcriptional amplification within a self-limiting tapetal activity window required for tapetal homeostasis, pollen wall formation, and male fertility.

Elevated temperatures, including warm ambient temperature (WAT) and high ambient temperature (HAT), profoundly influence plant growth and development. While WAT promotes thermomorphogenesis and stimulates growth, HAT induces stress responses that can inhibit growth. Using Arabidopsis thaliana , we uncovered both shared and distinct molecular mechanisms underlying plant responses to these temperature conditions. We demonstrated that both WAT and HAT activate genes associated with the DNA damage response (DDR), but they differ in timing and intensity. While WAT encourages progression into the S phase of the cell cycle and enhances cell division, HAT induces G2/M arrest, indicating cell cycle inhibition caused by DNA damage accumulation and impaired DDR proteins. Notably, alternative splicing (AS), particularly intron retention (IR), emerged as a pivotal regulatory layer under heat stress. Prolonged high temperatures uniquely enriched IR events in genes involved in DNA repair and chromatin organization. Integrated analyses of AS and transcriptional changes revealed coordinated regulation, with IR events dominating genes that exhibit simultaneous upregulation. These findings highlight AS as an essential regulatory mechanism in plant responses to heat stress. Conserved responses to elevated temperatures in tobacco and tomato further suggest the presence of shared adaptive pathways across plant species. Our results provide novel insights into plant adaptation to fluctuating temperatures and highlight opportunities for engineering heat-resilient crops to mitigate the impacts of climate change.

Plant basic helix-loop-helix (bHLH) transcription factors constitute one of the largest and most functionally diverse transcription factor families in plants. Through a conserved bHLH domain, they bind E-box cis-elements or dimerize with partner proteins, coupling developmental and metabolic programs to environmental cues. However, a systematic synthesis of their multilayered stress responses remains lacking. Here, we review the structural features, genome-wide distribution, and evolutionary diversification of plant bHLH transcription factors, with emphasis on their regulatory functions under low-temperature, drought, salinity, and iron-deficiency conditions. We further integrate their roles in abscisic acid, jasmonic acid, and light signaling, as well as in anthocyanin and terpenoid biosynthesis, biotic stress, and immune responses. We also discuss emerging evidence linking bHLH factors to reproductive development, particularly anther and pollen formation under heat stress. Mechanistically, we summarize the multilayered regulatory network from four perspectives: transcriptional cascades, post-translational modifications, cooperative regulation by multiple transcription factors, and growth-defense trade-offs. Finally, we highlight current gaps, including limited functional validation in non-model/specialty economic crop species, underexplored combined-stress mechanisms, and lagging breeding translation, and propose future research directions. This review provides an integrated framework for plant stress biology and outlines evidence-based perspectives for future stress-tolerant crop improvement.