ramosa2 encodes a LATERAL ORGAN BOUNDARY domain protein that determines the fate of stem cells in branch meristems of maize

Abstract

Genetic control of grass inflorescence architecture is critical given that cereal seeds provide most of the world's food. Seeds are borne on axillary branches, which arise from groups of stem cells in axils of leaves and whose branching patterns dictate most of the variation in plant form. Normal maize (Zea mays) ears are unbranched, and tassels have long branches only at their base. The ramosa2 (ra2) mutant of maize has increased branching with short branches replaced by long, indeterminate ones. ra2 was cloned by chromosome walking and shown to encode a LATERAL ORGAN BOUNDARY domain transcription factor. ra2 is transiently expressed in a group of cells that predicts the position of axillary meristem formation in inflorescences. Expression in different mutant backgrounds places ra2 upstream of other genes that regulate branch formation. The early expression of ra2 suggests that it functions in the patterning of stem cells in axillary meristems. Alignment of ra2-like sequences reveals a grass-specific domain in the C terminus that is not found in Arabidopsis thaliana. The ra2-dm allele suggests this domain is required for transcriptional activation of ra1. The ra2 expression pattern is conserved in rice (Oryza sativa), barley (Hordeum vulgare), sorghum (Sorghum bicolor), and maize, suggesting that ra2 is critical for shaping the initial steps of grass inflorescence architecture.

Figure 1.

The ra2 Mutant Phenotype. (A) Wild-type tassel; inset, a spikelet pair. (B) ra2-mum4 tassel. (C) The transition of branch lengths in a ra2 mutant. Mixed branches are formed after main branches (data not shown) and bear single spikelets and spikelet pairs. Spikelet multimers are formed after the mixed branches and consist of more than two single spikelets. mb, mixed branch; sm, spikelet multimer; sp, spikelet pair; ss, single spikelet. (D) Wild-type ear with well-defined rows of kernels. (E) ra2-mum4 ear with disorganized rows. (F) Open-pollinated ra2-dm ear with numerous branches. (G) to (J) Scanning electron microscopy of developing ears. (G) Wild-type ear. im, inflorescence meristem; sm, spikelet meristem; spm, spikelet pair meristem. (H) Scanning electron micrograph of a ra2-R ear showing meristems initiating branches (arrows) and some well-developed branches at the base. (I) Detail of a wild-type ear row consisting of a pair of spikelet meristems. (J) Detail of the formation of three spikelet meristems in a ra2-R ear. Bars = 0.5 mm in (G) and (H) and 50 μm in (I) and (J).

Figure 2.

Tassel Branching Patterns in A188, B73, and W22 Maize Inbred Lines and ra2 Mutants. (A) The distribution of axillary meristem fates in tassels of ra2 alleles and inbred lines. The area of each class of branch represents its percentage of all tassel branches. Single spikelets were only found in W22 and W22-introgressed families. See Table 1 for error values and significance of a t test. (B) and (C) A simplified schematic showing the change in branch determinacy imposed on branches of ra2 mutant tassels. (B) A wild-type tassel, consisting mainly of spikelet pair–bearing branches at the bottom and spikelet pairs directly on the main axis above. (C) A typical ra2 tassel showing mixed branches and spikelet multimers that gradually become spikelet pairs toward the apex. Branch angle difference is not illustrated.

Figure 3.

Cloning and Characterization of ra2. (A) The genetic map of a portion of maize chromosome 3 (top) is compared with the syntenous physical region of rice chromosome 1 (bottom). Black circles are maize markers/genes. White circles are rice genes and ORFs. The 175-kb segment indicated for the rice physical map corresponds to rice clone AP003339. The rice homolog to maize RFLP marker asg48 is located in an unlinked location of rice chromosome 1, and it is the only nonsyntenic gene of the region shown here between maize and rice. There are four ORFs (white squares) in a 17-kb region between the Osmyb and Osra2 genes for which there are no significantly similar maize sequences. The number and order of rice genes follow their annotation in GenBank but are not drawn to proportion. HP, hypothetical protein. (B) Structure of ra2 with the LOB domain in gray; diagram begins and ends at transcription initiation and polyadenylation site, respectively. The alleles of ra2 are shown with the ra2-dm deletion indicated by the zigzag marks in intron 1 and exon 2. eb3, the fragment used as a probe for DNA and RNA gel blot hybridizations; ra2full, the probe used for in situ hybridizations. (C) Alignment of the deduced full-length amino acid sequence of ra2 from maize, sorghum (Sbra2), rice (Osra2), barley (Hvra2), and ASL4 of Arabidopsis. The LOB domain is located near the N terminus and consists of a C-rich domain, a GAS block, and a Leu-zipper domain. Grass ra2 orthologs share a conserved C terminus domain of unknown function. The asterisk indicates the mutation (Arg to His) in the ra2-mum4 allele.

Figure 4.

Tissue Localization of ra2 and ra1 Expression. RNA gel blot of inbred line B73 seedling (lane 1), B73 root (lane 2), B73 tassel (lane 3), B73 ear (lane 4), ra2-R tassel (lane 5), ra2-dm ear (lane 6), ra2-mum4 tassel (lane 7), and ra1-R ear (lane 8). Top panel is ra2 hybridization, middle is ra1, and bottom is ubiquitin. Note the smaller size transcript in ra2-dm due to the deletion of the LOB domain and that ra1 is expressed in this allele but not the other ra2 alleles.

Figure 5.

ra2 Expression Marks the Position of Inflorescence Axillary Meristems. (A) to (C) and (E) to (I) ra2 expression in wild-type tissue. (A) Tassel. (B) Ear. (C) Longitudinal section of a tassel tip showing ra2 expression predicting the position of spikelet pair meristems. (D) Detail of ra1 expression in spikelet pair meristems of a tassel. (E) Detail of ra2 expression in the anlagen of spikelet meristem. (F) Front view of a row of spikelet pair meristems in an ear. (G) Expression in a tassel primordium that is just initiating branch meristems. (H) Hybridization of a young tassel tip showing expression of ra2 in axillary meristem anlagen above the bract primordia (red arrows). (I) Tip of a tassel of similar stage of development as in (A) showing hybridization with a gene-specific probe. (J) and (K) ra2 expression in ra mutants: ra3 tassel (J); ra1 tassel branch (K). Bars = 0.5 mm (A), (B), and (G) to (J) and 50 μm in (C) to (F) and (K).

Figure 6.

ra2 Transiently Marks the Position of Axillary Inflorescence Meristems in Sorghum, Rice, and Barley. (A) to (C) Sorghum. (A) Expression in first-order branch primordia. (B) Expression in second-order branch primordia. (C) Expression in spikelet pair meristem primordia. (D) and (E) Rice. (D) Expression in primary branch primordia. (E) Expression in spikelet meristem primordia. (F) Expression in the anlagen of a spikelet meristem of a barley inflorescence. Notice that the expression is above the bracts (red arrows). Bars = 250 μm.