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Grass inflorescences support floral structures that each bear a single grain, where variation in branch architecture directly impacts yield. The maize (Zea mays) RAMOSA1 (ZmRA1) transcription factor acts as a key regulator of inflorescence development by imposing branch meristem determinacy. Here, we show RA1 transcripts accumulate in boundary domains adjacent to spikelet meristems in sorghum (Sorghum bicolor, Sb) and green millet (Setaria viridis, Sv) inflorescences similar as in the developing maize tassel and ear. To evaluate the functional conservation of syntenic RA1 orthologs and promoter cis sequences in maize, sorghum, and setaria, we utilized interspecies gene transfer and assayed genetic complementation in a common inbred background by quantifying recovery of normal branching in highly ramified ra1-R mutants. A ZmRA1 transgene that includes endogenous upstream and downstream flanking sequences recovered normal tassel and ear branching in ra1-R. Interspecies expression of two transgene variants of the SbRA1 locus, modeled as the entire endogenous tandem duplication or just the nonframeshifted downstream copy, complemented ra1-R branching defects and induced unusual fasciation and branch patterns. The SvRA1 locus lacks conserved, upstream noncoding cis sequences found in maize and sorghum; interspecies expression of a SvRA1 transgene did not or only partially recovered normal inflorescence forms. Driving expression of the SvRA1 coding region by the ZmRA1 upstream region, however, recovered normal inflorescence morphology in ra1-R. These data leveraging interspecies gene transfer suggest that cis-encoded temporal regulation of RA1 expression is a key factor in modulating branch meristem determinacy that ultimately impacts grass inflorescence architecture. [ABSTRACT FROM AUTHOR]

Axillary branch suppression is a favorable trait bred into many domesticated crop plants including maize compared with its highly branched wild ancestor teosinte. Branch suppression in maize was achieved through selection of a gain of function allele of the teosinte branched1 (tb1) transcription factor that acts as a repressor of axillary bud growth. Previous work indicated that other loci may function epistatically with tb1 and may be responsible for some of its phenotypic effects. Here, we show that tb1 mediates axillary branch suppression through direct activation of the tassels replace upper ears1 (tru1) gene that encodes an ankyrin repeat domain protein containing a BTB/POZ motif necessary for protein-protein interactions. The expression of TRU1 and TB1 overlap in axillary buds, and TB1 binds to two locations in the tru1 gene as shown by chromatin immunoprecipitation and gel shifts. In addition, nucleotide diversity surveys indicate that tru1, like tb1, was a target of selection. In modern maize, TRU1 is highly expressed in the leaf trace vasculature of axillary internodes, while in teosinte, this expression is highly reduced or absent. This increase in TRU1 expression levels in modern maize is supported by comparisons of relative protein levels with teosinte as well as by quantitativemeasurements of mRNA levels. Hence, a major innovation in creating ideal maize plant architecture originated from ectopic overexpression of tru1 in axillary branches, a critical step in mediating the effects of domestication by tb1. [ABSTRACT FROM AUTHOR]

Genetic control of branching is a primary determinant of yield, regulating seed number and harvesting ability, yet little is known about the molecular networks that shape grain-bearing inflorescences of cereal crops. Here, we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. Developmental dynamics of genes targeted in vivo by the transcription factor RAMOSA1, a key regulator of determinacy, revealed potential mechanisms for repressing branches in distinct stem cell populations, including interactions with KNOTTED1, a master regulator of stem cell maintenance. Our results uncover discrete developmental modules that function in determining grass-specific morphology and provide a basis for targeted crop improvement and translation to other cereal crops with comparable inflorescence architectures.

Advances in plant biology have been frustrated by the lack of an efficient means to create targeted mutations. Zinc finger nucleases (ZFNs) hold much promise for overcoming this limitation: they can be used to generate targeted gene knockouts through imprecise repair of broken chromosomes by non-homologous end joining (NHEJ), or they can stimulate the introduction of specific DNA sequence changes through homologous recombination. Critical to the function of ZFNs is their ability to access and cleave chromosomal target sites. Numerous factors may obscure cleavage, including packaging of DNA into chromatin, DNA methylation, or the presence of other proteins at the target site. Here we describe a transient assay that rapidly assesses ZFN function at chromosomal targets in plant cells. The assay monitors the ability of a ZFN to introduce mutations by imprecise repair through NHEJ, resulting in the loss of a restriction endonuclease recognition sequence. The requirement for the restriction endonuclease recognition sequence coincident with the ZFN spacer region has thus far not been a limiting factor in identifying ZFN target sites in genes of interest suitable for this assay.