Abstract
The function and regulation of lipid metabolic genes are essential for plant male reproduction. However, expression regulation of lipid metabolic genic male sterility (GMS) genes by noncoding RNAs is largely unclear. Here, we systematically predicted the microRNA regulators of 34 maize white brown complex members in ATP-binding cassette transporter G subfamily (WBC/ABCG) genes using transcriptome analysis. Results indicate that the ZmABCG26 transcript was predicted to be targeted by zma-miR164h-5p, and their expression levels were negatively correlated in maize B73 and Oh43 genetic backgrounds based on both transcriptome data and qRT-PCR experiments. CRISPR/Cas9-induced gene mutagenesis was performed on ZmABCG26 and another lipid metabolic gene, ZmFAR1. DNA sequencing, phenotypic, and cytological observations demonstrated that both ZmABCG26 and ZmFAR1 are GMS genes in maize. Notably, ZmABCG26 proteins are localized in the endoplasmic reticulum (ER), chloroplast/plastid, and plasma membrane. Furthermore, ZmFAR1 shows catalytic activities to three CoA substrates in vitro with the activity order of C12:0-CoA > C16:0-CoA > C18:0-CoA, and its four key amino acid sites were critical to its catalytic activities. Lipidomics analysis revealed decreased cutin amounts and increased wax contents in anthers of both zmabcg26 and zmfar1 GMS mutants. A more detailed analysis exhibited differential changes in 54 monomer contents between wild type and mutants, as well as between zmabcg26 and zmfar1. These findings will promote a deeper understanding of miRNA-regulated lipid metabolic genes and the functional diversity of lipid metabolic genes, contributing to lipid biosynthesis in maize anthers. Additionally, cosegregating molecular markers for ZmABCG26 and ZmFAR1 were developed to facilitate the breeding of male sterile lines.
Highlights
Anther and pollen development are critical for male reproduction of flowering plants.Plant microspore mother cells are surrounded by an anther wall consisting of four layers, from outer to inner including epidermis, endothecium, middle layer, and tapetum [1].developing pollen grains are protected from external abiotic stresses and biotic attacks by the anther cuticle that is an extracellular lipidic layer covering anther’s outer surface
The present study focuses on the potential posttranscriptional regulation of ZmABCG26 by zma-miR164h-5p during maize anther development and evaluation of ZmABCG26 and
ZmABCG26 has been predicted as one of the targets of zma-miR164h-5p by bioinformatics analysis based on transcriptomes of maize anthers [42]
Summary
Anther and pollen development are critical for male reproduction of flowering plants. In Arabidopsis, the ATP-binding cassette subfamily G26 (AtABCG26) encoded a key transporter protein for pollen exine formation by transferring lipid precursors and polyketides [18,19]. Fatty acyl reductases (FARs) catalyze the NADPH-dependent reaction for the conversion of fatty acyl-coenzyme A (CoA) or acyl carrier protein (ACP) to a primary fatty alcohol Fatty alcohols and their derivatives are major components of the lipidic anther cuticle and pollen wall [32]. In bread wheat (Triticum aestivum L.), TaTAA1 encoded by Triticum aestivum anther 1 was the first reported alcohol-forming fatty acyl–CoA reductase responsible for male gametophyte development [39]. Even though the major catalytic steps of FARs in lipid biosynthesis have been revealed, our knowledge on the function and regulation of FAR family members involved in plant male reproduction is still limited. These findings will facilitate a greater understanding of the miRNA-regulated lipid metabolic network in maize anthers and may promote the application of lipid metabolic GMS genes in molecular breeding and hybrid seed production in maize
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