Anther Middle Layer Fatty Acid Synthesis is Essential for Pollen Development and Fertility Restoration at Low Temperature in Arabidopsis

The middle layer of the anther, a narrow and enigmatic cell layer, has received limited research attention, leaving its functional role poorly understood. Through screening fertility restorers of a thermosensitive male-sterile mutant npu-2, we discovered that de novo fatty acid synthesis in the middle layer plays a critical role in pollen development. Compared to the tapetum, the middle layer contains more mature plastids and a more complete set of de novo fatty acid synthesis enzymes, with FatA1 and KAS2 specifically accumulating in this layer during early anther development. Enhancing fatty acid synthesis in this layer restored fertility in several thermosensitive male-sterile mutants, highlighting its critical role in pollen development. Additionally, acyl-CoA supplementation experiments revealed that C18 fatty acids play a critical role in fertility restoration. Furthermore, low temperatures activate fatty acid biosynthesis, while disrupting this pathway prevents fertility recovery under low temperatures, demonstrating that elevated fatty acid levels represent a general mechanism for fertility restoration at low temperature. This study highlights the middle layer's pivotal role in fatty acid synthesis and its regulatory influence on pollen development and temperature-sensitive fertility transitions.