Extreme temperature conditions seriously impair male reproductive development in plants; however, the molecular mechanisms underlying the response of anthers to extreme temperatures remain poorly described. The transcription factor PHYTOCHROME-INTERACTING FACTOR4 (PIF4) acts as a hub that integrates multiple signaling pathways to regulate thermosensory growth and architectural adaptation in plants. Here, we report that SlPIF4 in tomato (Solanum lycopersicum) plays a pivotal role in regulating cold tolerance in anthers. CRISPR/Cas9-generated SlPIF4 knockout mutants showed enhanced cold tolerance in pollen due to reduced temperature sensitivity of the tapetum, while overexpressing SlPIF4 conferred pollen abortion by delaying tapetal programmed cell death (PCD). SlPIF4 directly interacts with SlDYT1, a direct upstream regulator of SlTDF1, both of which (SlDYT1 and SlTDF1) play important roles in regulating tapetum development and tapetal PCD. Moderately low temperature (MLT) promotes the transcriptional activation of SlTDF1 by the SlPIF4-SlDYT1 complex, resulting in pollen abortion, while knocking out SlPIF4 blocked the MLT-induced activation of SlTDF1. Furthermore, SlPIF4 directly bind to the canonical E-box sequence in the SlDYT1 promoter. Collectively, these findings suggest that SlPIF4 negatively regulates cold tolerance in anthers by directly interacting with the tapetal regulatory module in a temperature-dependent manner. Our results shed light on the molecular mechanisms underlying the adaptation of anthers to low temperatures.

The development of viable pollen determines male fertility, and is crucial for reproduction in flowering plants. Phytochrome interacting factor 3 (PIF3) acts as a central regulator of plant growth and development, but its relationship with pollen development has not been determined.Through genetic, histological and transcriptomic analyses, we identified an essential role for SlPIF3 in regulating tomato (Solanum lycopersicum) pollen development. Knocking out SlPIF3 using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 resulted in pollen mitosis I arrest, and a failure to form viable pollen. We further demonstrated that both glutamate synthase 1 (SlGLT1) and cell wall invertase 9 (SlCWIN9), involved in auxin and sugar homeostasis, respectively, colocalised with SlPIF3 in the anthers and were directly regulated by SlPIF3. Knockout of either SlGLT1 or SlCWIN9 phenocopied the pollen phenotype of SlPIF3 knockout (Slpif3) lines.Slpif3 fertility was partially restored by exogenous auxin indole-3-acetic acid in a dosedependent manner.This study reveals a mechanism by which SlPIF3 regulates pollen development and highlights a new strategy for creating hormone-regulated genic male sterile lines for tomato hybrid seed production.

Plant height is a key agronomic trait regulated by several phytohormones like gibberellins (GAs) and auxin. However, little is known about how cytokinin (CK) participates in this process. Here, we report that SlRR6, a type-A response regulator in CK signaling pathway, positively regulates plant height in tomato. SlRR6 was induced by exogenous kinetin and GA3, but inhibited by indole-3-acetic acid (IAA). Knockout of SlRR6 reduced tomato plant height through shortening internode length, while overexpression of SlRR6 caused higher plant due to increased internode number. Cytological observation of longitudinal stems showed that both knockout and overexpression of SlRR6 generated larger cells, but significantly reduced cell numbers in each internode. Further studies demonstrated that overexpression of SlRR6 enhanced GA accumulation and lowered IAA content, along with expression changes in GA- and IAA-related genes. Exogenous paclobutrazol and IAA treatments restored the increased plant height phenotype in SlRR6-overexpressing lines. Yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays showed that SlRR6 interacts with a small auxin up RNA protein SlSAUR58. Moreover, SlSAUR58-overexpressing plants were dwarf with decreased internode length. Overall, our findings establish SlRR6 as a vital component in the CK signaling, GA, and IAA regulatory network that controls plant height.