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Mitochondrial calcium uniporter (MCU) is a conserved calcium ion (Ca2+) transporter in the mitochondrial inner membrane of eukaryotic cells. How MCU proteins regulate Ca2+flow and modulate plant cell development remain largely unclear. Here, we identified the gene GhMCU4encoding a MCU protein that negatively regulates plant development and fiber elongation in cotton (Gossypium hirsutum). GhMCU4expressed constitutively in various tissues with the higher transcripts in elongating fiber cells. Knockdown of GhMCU4in cotton significantly elevated the plant height and root length. The calcium signaling pathway was significantly activated and calcium sensor genes, including Ca2+dependent modulator of interactor of constitutively active ROP (GhCMI1),calmodulin like protein (GhCML46), calcium-dependent protein kinases (GhCPKs), calcineurin B-like protein (GhCBLs), and CBL-interacting protein kinases (GhCIPKs), were dramatically upregulated in GhMCU4-silenced plants. Metabolic processes were preferentially enriched, and genes related to regulation of transcription were upregulated in GhMCU4-silenced plants. The contents of Ca2+and H2O2were significantly increased in roots and leaves of GhMCU4-silenced plants. Fiber length and Ca2+and H2O2contents in fibers were significantly increased in GhMCU4-silenced plants. This study indicated that GhMCU4plays a negative role in regulating cell elongation in cotton, thus expanding understanding in the role of MCU proteins in plant growth and development.

Pentatricopeptide repeat (PPR) proteins encoded by nuclear genomes can bind to organellar RNA and are involved in the regulation of RNA metabolism. However, the functions of many PPR proteins remain unknown in plants, especially in polyploidy crops. Here, through a map-based cloning strategy and Clustered regularly interspaced short palindromic repeats/cas9 (CRISPR/cas9) gene editing technology, we cloned and verified an allotetraploid cotton immature fiber(im) mutant gene (GhImA) encoding a PPR protein in chromosome A03, that is associated with the non-fluffy fiber phenotype. GhImA protein targeted mitochondrion and could bind to mitochondrial nad7mRNA, which encodes the NAD7 subunit of Complex I. GhImA and its homolog GhImD had the same function and were dosage-dependent. GhImAin the immutant was a null allele with a 22 bp deletion in the coding region. Null GhImAresulted in the insufficient GhIm dosage, affected mitochondrial nad7pre-mRNA splicing, produced less mature nad7transcripts, and eventually reduced Complex I activities, up-regulated alternative oxidase metabolism, caused reactive oxygen species (ROS) burst and activation of stress or hormone response processes. This study indicates that the GhIm protein participates in mitochondrial nad7splicing, affects respiratory metabolism, and further regulates cotton fiber development via ATP supply and ROS balance.