A Maize Male Gametophyte-Specific Gene Encodes ZmLARP6c1, a Potential RNA-Binding Protein Required for Competitive Pollen Tube Growth

Abstract

Members of the La-related protein family (LARPs) contain a conserved La module, which has been associated with RNA-binding activity. Expression of the maize gene GRMZM2G323499/Zm00001d018613, a member of the LARP family, is highly specific to pollen, based on both transcriptomic and proteomic assays. This suggests a pollen-specific RNA regulatory function for the protein, designated ZmLARP6c1 based on sequence similarity to the LARP6 subfamily in Arabidopsis. To test this hypothesis, a Ds-GFP transposable element insertion in the ZmLarp6c1 gene (tdsgR82C05) was obtained from the Dooner/Du mutant collection. Sequencing confirmed that the Ds-GFP insertion is in an exon, and thus likely interferes with ZmLARP6c1 function. Tracking inheritance of the insertion via its endosperm-expressed GFP indicated that the mutation was associated with reduced transmission from a heterozygous plant when crossed as a male (ranging from 0.5 to 26.5% transmission), but not as a female. Furthermore, this transmission defect was significantly alleviated when less pollen was applied to the silk, reducing competition between mutant and wild-type pollen. Pollen grain diameter measurements and nuclei counts showed no significant differences between wild-type and mutant pollen. However, in vitro, mutant pollen tubes were significantly shorter than those from sibling wild-type plants, and also displayed altered germination dynamics. These results are consistent with the idea that ZmLARP6c1 provides an important regulatory function during the highly competitive progamic phase of male gametophyte development following arrival of the pollen grain on the silk. The conditional, competitive nature of the Zmlarp6c1::Ds male sterility phenotype (i.e., reduced ability to produce progeny seed) points toward new possibilities for genetic control of parentage in crop production.

Genome-wide investigation of the LARP gene family: focus on functional identification and transcriptome profiling of ZmLARP6c1 in maize pollen

“…All primers used in this study are listed in Supplementary Table S 1 . The Zmlarp6c1::Ds allele used in this study was obtained from the Maize Genetics Cooperation Stock Center as previously described [ 36 ].…”

“…Fresh mature pollen was collected between 11:00 am and 12:00 pm. Pollen was germinated on PGM (0.0005% H 3 BO 3 , 10 mM CaCl 2 , 0.05 mM KH 2 PO 4 , 6% PEG 4000, and 10% sucrose), and observed at 15 or 30 min after plating on the medium as described previously [ 36 ]. Germinated, non-germinated, and ruptured pollen grains were counted at each time point.…”

“…For measurement of the pollen grain diameter, fresh pollen was collected from ZmLARP6c1- OE and WT plants, immediately fixed in ethanol:acetic (3:1) acid solution, and rehydrated for measurement of the pollen grain diameter as described previously [ 36 ]. Four replicates were measured, with at least 150 pollen grains measured per replicate.…”

“…In Arabidopsis , AtLARP6c regulates mRNA post-transcription during guidance of the pollen tube to the embryo sac in the ovule [ 35 ]. In maize, ZmLARP6c1 has an important male-specific function in the haploid gametophyte during the highly competitive phase of pollen tube germination and growth following pollination [ 36 ]. These two studies suggest that LARP6c has conserved molecular and cellular roles in maize and Arabidopsis .…”

“…One of these genes, ZmLARP6c1 , which was specifically and highly expressed in pollen. The Ds transposable element insertional mutant of ZmLARP6c1 was associated with reduced transmission when crossed as a male in our previous studies [ 36 ]. We analyzed the subcellular localization of ZmLARP6c1, generated transgenic lines overexpressing this protein, and determined pollen germination in vitro.…”