Decreased Spikelets 4 Encoding a Novel Tetratricopeptide Repeat Domain-Containing Protein Is Involved in DNA Repair and Spikelet Number Determination in Rice

Abstract

Spikelet number per panicle is a determinative factor of rice yield. DNA repair epigenetically alters the DNA accessibility, which can eventually regulate the transcription of the target genes. However, what and how DNA repair genes are related to rice spikelet development remains unknown. Here, we report the map-based cloning of a novel spikelet number gene DES4 encoding a tetratricopeptide domain-containing protein. DES4 is a close ortholog of Arabidopsis BRU1, which is functionally related to axillary meristem development. A single base pair deletion in the last exon of DES4 caused a premature stop of the resulting protein. The des4 mutant exhibited dwarf, reduced tiller, and spikelet numbers phenotypes, as well as hypersensitivity to genotoxic stresses, suggesting its essential role in DNA repair. DES4 is predominantly expressed in young panicles and axillary meristems, and DES4 protein is localized in nucleus. A set of DNA repair genes such as cyclins, KUs (KD subunits) and recombinases were differentially regulated in des4. Meanwhile, rice spikelet number genes LAX1, LAX2, and MOC1 were significantly down-regulated in des4. In morphology, des4 showed more severe reduction of spikelet numbers than lax1, lax2, and moc1, suggesting that DES4 may work upstream of the three genes.

Keywords: DNA repair; LRR (leucine-rich repeat); rice (Oryza sativa L.); spikelet number; tetratricopeptide.

Figure 1

Phenotypical characterization of the des4 and wild type (WT). (A) Plant architecture, (B) panicle structure, and (C) leaf shape of des4 and WT, bar = 10 cm. (D) Quantification of the plant height, tiller number, primary branches, secondary branches, and spikelet numbers per panicle. Numerical values are expressed as the mean; error bars denote one standard deviation of the mean; asterisk denotes significant difference between mutant and WT within each treatment (** p < 0.01; Student’s t-test).

Figure 2

Genetic mapping and complementation analysis of DES4. (A) Map-based cloning of DES4. The markers for PCR-based mapping are listed in Table S1. “Recombinant” indicates recombination between maker and phenotype; the numbers indicate the numbers of recombinant lines out of 1002 mutant type lines analyzed. Red shape indicates Os02g0782800 candidate gene; blue shapes indicate the 16 sequenced candidate genes; and grey shape indicates the transposon genes. (B) Schematic presentation of the gene structure of DES4. Boxes represent the exons and the line indicates the intron. One base pair deletion occurred on the last exon of DES4. The mutated sequence is presented in Data S1. (C) Panicle architectures of negative control (empty vector) line, genetically complemented line, and WT.

Figure 3

Protein structure, phylogenetic, and expression analysis of DES4. (A) Conserved domain analysis of the DES4 amino acid sequence. (B) Phylogenetic tree of DES4 and orthologs from other species. Numbers are the bootstrap values. Red line indicates the clade of grasses. (C–E) mRNA in situ hybridization analysis of DES4 in differentiating panicles meristems (C) and tiller meristems (D). Boxes highlight the tissue parts with strong expression signals. Bar = 10 μm. (E) Negative control using sense probes. (F) Subcellular localization of DES4 in tobacco leaf epidermal cells.

Figure 4

Genotoxic stresses on des4 and WT seedlings. (A) Growth of des4 and WT seedlings under different methyl methanesulfonate (MMS) concentrations. (B) Quantification of the seedling heights under different concentrations of MMS and zeocin. ** p < 0.01.

Figure 5

qRT-PCR analysis of double-strand break (DSB) repair genes and spikelet number genes. qRT-PCR analysis of transcript levels of DSB repair genes and spikelet number genes in WT and des4. Rice actin (LOC_Os03g61970) was used as an internal control. The locus ID for each gene can be found in Table S1. Values are means ± SE. Asterisks indicate significant differences between WT and des4 (* p < 0.05; Student’s t-test).