A Comparative miRNAome Analysis Reveals Seven Fiber Initiation-Related and 36 Novel miRNAs in Developing Cotton Ovules

An increasing number of microRNAs （miRNAs） have been shown to play crucial regulatory roles in the process of plant development. Here, we used high-throughput sequencing combined with computational analysis to characterize miRNAomes from the ovules of wild-type upland cotton and a fiberless mutant during fiber initiation. Comparative miR- NAome analysis combined with northern blotting and RACE-PCR revealed seven fiber initiation-related miRNAs expressed in cotton ovules and experimentally validated targets of these miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin and gibberellin signal transduction, actin bundles, and lignin biosynthesis. This paper describes a complex regulatory network consisting of these miRNAs expressed in cotton ovules to coordinate fiber initiation responses. In addition, 36 novel miRNAs and two conserved miRNAs were newly iden- tified, nearly doubling the number of known cotton miRNA families to a total of 78. Furthermore, a chromatin remodeling complex subunit and a pre-mRNA splicing factor are shown for the first time to be miRNA targets. To our knowledge, this study is the first systematic investigation of fiber initiation-related miRNAs and their targets in the developing cotton ovule, deepening our understanding of the important regulatory functions of miRNAs in cotton fiber initiation.

A Malvaceae-specific miRNA targeting the newly duplicated GaZIP1L to regulate Zn^(2+)ion transporter capacity in cotton ovules

MicroRNAs(miRNAs)play critical roles in regulating gene expression in plants,yet their functions underlying cultivated diploid Gossypium arboreum cotton ovule development are largely unknown.Here,we acquired small RNA profiles from G.arboreum ovules and fibers collected at different growth stages,and identified 46 novel miRNAs that accounted for 23.7%of all miRNAs in G.arboreum reported in the latest plant sRNA database.Through analysis of 84(including 38 conserved)differentially expressed G.arboreum miRNAs,we detected 215 putative protein-coding genes in 26 biological processes as their potential targets.A Malvaceae-specific novel miRNA named gar-miRN44 was found to likely regulate cotton ovule growth by targeting to a newly duplicated Zn^(2+)ion transporter gene GaZIP1L.During cotton ovule development,gar-miRN44 transcript level decreased sharply after 10 to 15 days post-anthesis(DPA),while that of the GaZIP1L increased significantly,with a concomitant increase of Zn^(2+)ion concentration in late ovule developmental stages.Molecular dynamics simulation and ion absorption analysis showed that GaZIP1L has stronger Zn2+ion binding ability than the original GaZIP1,indicating that the newly evolved GaZIP1L may be more suitable for maintaining high Zn2+ion transport capacity that is likely required for cotton ovule growth via enhanced cellulose synthase activities.Our systematic miRNA profiling in G.arboreum and characterization of gar-miRN44 not only contribute to the understanding of miRNA function in cotton,but also provide potential targets for plant breeding.