Strigolactones (SLs) are a group of newly identified plant hormones that control plant shoot branching. SL signalling requires the hormone-dependent interaction of DWARF 14 (D14), a probable candidate SL receptor, with DWARF 3 (D3), an F-box component of the Skp–Cullin–F-box (SCF) E3 ubiquitin ligase complex. Here we report the characterization of a dominant SL-insensitive rice (Oryza sativa) mutant dwarf 53 (d53) and the cloning of D53, which encodes a substrate of the SCFD3 ubiquitination complex and functions as a repressor of SL signalling. Treatments with GR24, a synthetic SL analogue, cause D53 degradation via the proteasome in a manner that requires D14 and the SCFD3 ubiquitin ligase, whereas the dominant form of D53 is resistant to SL-mediated degradation. Moreover, D53 can interact with transcriptional co-repressors known as TOPLESS-RELATED PROTEINS. Our results suggest a model of SL signalling that involves SL-dependent degradation of the D53 repressor mediated by the D14–D3 complex.

Tillering in rice (Oryza sativa) is one of the most important agronomic traits that determine grain yields. Previous studies on rice tillering mutants have shown that the outgrowth of tiller buds in rice is regulated by a carotenoid-derived MAX/RMS/D (more axillary branching) pathway, which may be conserved in higher plants. Strigolactones, a group of terpenoid lactones, have been recently identified as products of the MAX/RMS/D pathway that inhibits axillary bud outgrowth. We report here the molecular genetic characterization of d27, a classic rice mutant exhibiting increased tillers and reduced plant height. D27 encodes a novel iron-containing protein that localizes in chloroplasts and is expressed mainly in vascular cells of shoots and roots. The phenotype of d27 is correlated with enhanced polar auxin transport. The phenotypes of the d27 d10 double mutant are similar to those of d10, a mutant defective in the ortholog of MAX4/RMS1 in rice. In addition, 29-epi-5-deoxystrigol, an identified strigolactone in root exudates of rice seedlings, was undetectable in d27, and the phenotypes of d27 could be rescued by supplementation with GR24, a synthetic strigolactone analog. Our results demonstrate that D27 is involved in the MAX/RMS/D pathway, in which D27 acts as a new member participating in the biosynthesis of strigolactones.

Strigolactones (SLs) are carotenoid-derived phytohormones that control many aspects of plant development, including shoot branching, leaf shape, stem secondary thickening, and lateral root growth. In rice (Oryza sativa), SL signaling requires the degradation of DWARF53 (D53), mediated by a complex including D14 and D3, but in Arabidopsis thaliana, the components and mechanism of SL signaling involving the D3 ortholog MORE AXILLARY GROWTH2 (MAX2) are unknown. Here, we show that SL-dependent regulation of shoot branching in Arabidopsis requires three D53-like proteins, SUPPRESSOR OF MORE AXILLARY GROWTH2-LIKE6 (SMXL6), SMXL7, and SMXL8. The smxl6 smxl7 smxl8 triple mutant suppresses the highly branched phenotypes of max2 and the SL-deficient mutant max3. Overexpression of a mutant form of SMXL6 that is resistant to SL-induced ubiquitination and degradation enhances shoot branching. Exogenous application of the SL analog rac-GR24 causes ubiquitination and degradation of SMXL6, 7, and 8; this requires D14 and MAX2. D53-like SMXLs form complexes with MAX2 and TOPLESS-RELATED PROTEIN2 (TPR2) and interact with D14 in a GR24-responsive manner. Furthermore, D53-like SMXLs exhibit TPR2-dependent transcriptional repression activity and repress the expression of BRANCHED1. Our findings reveal that in Arabidopsis, D53-like SMXLs act with TPR2 to repress transcription and so allow lateral bud outgrowth but that SL-induced degradation of D53-like proteins activates transcription to inhibit outgrowth.

Copy number variants (CNVs) are associated with changes in gene expression levels and contribute to various adaptive traits. Here we show that a CNV at the Grain Length on Chromosome 7 (GL7) locus contributes to grain size diversity in rice (Oryza sativa L.). GL7 encodes a protein homologous to Arabidopsis thaliana LONGIFOLIA proteins, which regulate longitudinal cell elongation. Tandem duplication of a 17.1-kb segment at the GL7 locus leads to upregulation of GL7 and downregulation of its nearby negative regulator, resulting in an increase in grain length and improvement of grain appearance quality. Sequence analysis indicates that allelic variants of GL7 and its negative regulator are associated with grain size diversity and that the CNV at the GL7 locus was selected for and used in breeding. Our work suggests that pyramiding beneficial alleles of GL7 and other yield- and quality-related genes may improve the breeding of elite rice varieties.

(J.L.).IDEAL PLANT ARCHITECTURE1 (IPA1) is critical in regulating rice (Oryza sativa) plant architecture and substantially enhances grain yield. To elucidate its molecular basis, we first confirmed IPA1 as a functional transcription activator and then identified 1067 and 2185 genes associated with IPA1 binding sites in shoot apices and young panicles, respectively, through chromatin immunoprecipitation sequencing assays. The SQUAMOSA PROMOTER BINDING PROTEIN-box direct binding core motif GTAC was highly enriched in IPA1 binding peaks; interestingly, a previously uncharacterized indirect binding motif TGGGCC/T was found to be significantly enriched through the interaction of IPA1 with proliferating cell nuclear antigen PROMOTER BINDING FACTOR1 or PROMOTER BINDING FACTOR2. Genome-wide expression profiling by RNA sequencing revealed IPA1 roles in diverse pathways. Moreover, our results demonstrated that IPA1 could directly bind to the promoter of rice TEOSINTE BRANCHED1, a negative regulator of tiller bud outgrowth, to suppress rice tillering, and directly and positively regulate DENSE AND ERECT PANICLE1, an important gene regulating panicle architecture, to influence plant height and panicle length. The elucidation of target genes of IPA1 genome-wide will contribute to understanding the molecular mechanisms underlying plant architecture and to facilitating the breeding of elite varieties with ideal plant architecture.

Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice (Oryza sativa) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana. Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security.

Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants(1), but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.

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