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. 2015 Mar 27;168(1):164–174. doi: 10.1104/pp.114.252882

The Barley Uniculme4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning1,[OPEN]

Elahe Tavakol 1,2,3,4,5,6,7,8, Ron Okagaki 1,2,3,4,5,6,7,8, Gabriele Verderio 1,2,3,4,5,6,7,8, Vahid Shariati J 1,2,3,4,5,6,7,8,2, Ahmed Hussien 1,2,3,4,5,6,7,8, Hatice Bilgic 1,2,3,4,5,6,7,8, Mike J Scanlon 1,2,3,4,5,6,7,8, Natalie R Todt 1,2,3,4,5,6,7,8, Timothy J Close 1,2,3,4,5,6,7,8, Arnis Druka 1,2,3,4,5,6,7,8, Robbie Waugh 1,2,3,4,5,6,7,8, Burkhard Steuernagel 1,2,3,4,5,6,7,8, Ruvini Ariyadasa 1,2,3,4,5,6,7,8, Axel Himmelbach 1,2,3,4,5,6,7,8, Nils Stein 1,2,3,4,5,6,7,8, Gary J Muehlbauer 1,2,3,4,5,6,7,8,*, Laura Rossini 1,2,3,4,5,6,7,8,*
PMCID: PMC4424007  PMID: 25818702

A transcriptional coactivator acts at developmental boundaries to control vegetative branching and leaf patterning.

Abstract

Tillers are vegetative branches that develop from axillary buds located in the leaf axils at the base of many grasses. Genetic manipulation of tillering is a major objective in breeding for improved cereal yields and competition with weeds. Despite this, very little is known about the molecular genetic bases of tiller development in important Triticeae crops such as barley (Hordeum vulgare) and wheat (Triticum aestivum). Recessive mutations at the barley Uniculme4 (Cul4) locus cause reduced tillering, deregulation of the number of axillary buds in an axil, and alterations in leaf proximal-distal patterning. We isolated the Cul4 gene by positional cloning and showed that it encodes a BROAD-COMPLEX, TRAMTRACK, BRIC-À-BRAC-ankyrin protein closely related to Arabidopsis (Arabidopsis thaliana) BLADE-ON-PETIOLE1 (BOP1) and BOP2. Morphological, histological, and in situ RNA expression analyses indicate that Cul4 acts at axil and leaf boundary regions to control axillary bud differentiation as well as the development of the ligule, which separates the distal blade and proximal sheath of the leaf. As, to our knowledge, the first functionally characterized BOP gene in monocots, Cul4 suggests the partial conservation of BOP gene function between dicots and monocots, while phylogenetic analyses highlight distinct evolutionary patterns in the two lineages.

Tillering or vegetative branching is one of the most important components of shoot architecture in cereals because it contributes directly to grain yield (Kebrom et al., 2013; Hussien et al., 2014) and is involved in plant plasticity in response to environmental cues and stresses (Mohapatra et al., 2011; Agusti and Greb, 2013). The shoot apical meristem initiates a series of repetitive units called phytomers, each consisting of a leaf, a node, an internode, and an axillary meristem (AXM) located in the axil between the leaf and the shoot axis (Sussex, 1989). Visually, AXM development and branching can be divided into three stages: (1) establishment of the AXM in the leaf axil; (2) initiation of multiple leaf primordia to form an axillary bud, which may remain dormant; or (3) growth into a branch through expansion of the branch internodes and differentiation of the axillary leaves (Schmitz and Theres, 2005). In grasses, tillers are lateral branches (i.e. culms) that grow from nodes of unelongated internodes at the base of the plant, affecting important agronomical features such as competition with weeds and ease of harvesting (Donald, 1968; Seavers and Wright, 1999). Although sharing some key steps in their development, tillers differ from lateral branches in eudicots in that they can produce adventitious roots and grow independently from the main plant shoot. Primary tillers arising from the main culm initiate new axillary buds that may, in turn, develop into secondary tillers and so on in a reiterative pattern (Hussien et al., 2014).

A complex combination of differential gene expression in conjunction with hormonal signaling and responses and environmental cues determines the number and location of axillary branches (Hussien et al., 2014). A number of evolutionarily conserved genetic pathways control axillary branching in both monocots and eudicots (Kebrom et al., 2013; Janssen et al., 2014). For example, reduced-branching mutant phenotypes are conferred by mutations in the orthologous GRAS (for GIBBERELLIC ACID-INSENSITIVE, REPRESSOR of GA1, and SCARECROW) genes LATERAL SUPPRESSOR in Arabidopsis (Arabidopsis thaliana), LATERAL SUPPRESSOR in tomato (Solanum lycopersicum), and MONOCULM1 in rice (Oryza sativa; Li et al., 2003). However, the molecular mechanisms that control branching in eudicots are not completely conserved with tiller development in grasses (Kebrom et al., 2013; Hussien et al., 2014; Waldie et al., 2014). For example, the reduced-branching mutations in the REGULATOR OF AXILLARY MERISTEMS1, REGULATOR OF AXILLARY MERISTEMS2, REGULATOR OF AXILLARY MERISTEMS3, and BLIND genes of Arabidopsis and tomato (Schmitz et al., 2002; Keller et al., 2006; Müller et al., 2006) are conserved in eudicots but have not been identified in monocot genomes (Keller et al., 2006; Müller et al., 2006).

A number of barley (Hordeum vulgare) tillering mutants have been identified, and their characterization has provided insight into the genetic mechanisms of vegetative axillary development and tillering in this important crop plant (Babb and Muehlbauer, 2003; Dabbert et al., 2009, 2010). Despite some recent progress (Dabbert et al., 2010; Mascher et al., 2014), most genes underlying tillering in the Triticeae await identification.

Recessive mutations in the barley Uniculme4 (Cul4) gene result in reduced tillering (Babb and Muehlbauer, 2003). In this study, we show that cul4 mutations affect multiple aspects of branch development and also cause specific defects in leaf patterning. We identified the Cul4 gene by positional cloning and show that it encodes a homolog of the Arabidopsis BLADE-ON-PETIOLE1 (BOP1) and BOP2 genes acting at boundary regions to regulate axillary development and leaf morphogenesis.

RESULTS

Recessive cul4 Mutations Reduce Tiller Number and Disrupt Leaf Patterning

Recessive mutations at the Cul4 gene result in reduced tiller number (Fig. 1, A and B; Dahleen et al., 2007). The cv Bonus and cv Flare typically produced an average of 8.8 ± 1 and 11.9 ± 1.5 tillers, respectively, on fully mature greenhouse-grown plants. In contrast, cul4 mutants showed tillering defects of varying severity, with cul4.5 (Supplemental Table S1) and cul4.16 (both in the cv Bonus background) and cul4.24 (in the cv Flare background) developing an average of 0.4 ± 1, 6.3 ± 1.7, and 4.6 ± 2 tillers, respectively (Fig. 1B). In contrast with the wild type, cul4 tillers were often bent and distorted, possibly as a result of difficulties in emergence from the leaf sheaths that enclosed them (Dahleen et al., 2007); this was frequently observed in cul4.16 plants. In addition, cul4 mutant plants developed some leafy side shoots (Supplemental Fig. S1) that were not considered proper tillers, as they did not elongate to produce lateral culms.

Figure 1.

Figure 1.

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Shoot phenotypes of barley cul4 mutants. A, Whole-plant phenotype of wild-type cv Bowman (left) and the cv Bowman-cul4.5 mutant (right) at the vegetative stage. B, Comparisons between cul4.24 and the wild-type cv Flare and between cul4.16 and cul4.5 and the wild-type cv Bonus for number of tillers in mature plants (n = 9). Error bars represent sd. Asterisks indicate significant differences (P ≤ 0.01) relative to the wild-type background (Student’s t test). C, Wild-type (wt) cv Bowman blade-sheath boundary region. D, cv Bowman-cul4.5 mutant blade-sheath boundary region. The arrow points to the blade-sheath boundary where a ligule would normally develop. E, Scanning electron micrograph of the wild-type (cv Bowman) blade-sheath boundary region. F, Scanning electron micrograph of the cv Bowman-cul4.5 mutant blade-sheath boundary region. The arrow indicates a fringe of tissue at the blade-sheath boundary. The auricle regions that wrap around the culm on both sides of the leaf were removed to take a scanning electron micrograph of the blade-sheath boundary. G, Scanning electron micrograph of wild-type (cv Bowman) auricle tissue from an equivalent position to the asterisk in C. H, Scanning electron micrograph of flap tissue from the cv Bowman-cul4.5 mutant (from an equivalent position to the asterisk in K) exhibits auricle-like cells. I, Wild-type (cv Bowman) leaf. J, cv Bowman-cul4.5 mutant leaf exhibiting a flap of tissue on the margin of the sheath. K, Closeup of flap (from the inset in J) on the sheath margin of a cv Bowman-cul4.5 mutant plant. Bars = 1 mm (E and F) and 100 μm (G and H).

To further examine the defect in tiller development in the cul4 mutant, histological analyses were carried out to compare the number of axillary buds in a near-isogenic line carrying the cul4.5 mutant allele (cv Bowman-cul4.5) and its recurrent parent cv Bowman. At 10 d after planting, shoot apices from cv Bowman-cul4.5 and cv Bowman were very similar (Fig. 2, A and B), except that fewer leaf axils had developed buds in the mutants compared with wild-type plants. Axillary buds were typically observed in two or three leaf axils in wild-type plants (Fig. 2, C and D), whereas in the cul4.5 mutant, only one to two leaf axils contained axillary buds (Fig. 2E). In contrast to the wild type, transverse sections through the cv Bowman-cul4.5 mutant shoot apex revealed two axillary buds in the same leaf axil (compare Fig. 2, F and G). Scanning electron microscopy clearly revealed a single axillary bud developing in leaf axils from cv Bowman seedlings (Fig. 2H), while occasionally leaf axils developing two axillary buds were observed in cv Bowman-cul4.5 seedlings (Fig. 2I). To better dissect the effect of Cul4 on axillary development, we compared the number of active leaf axils (harboring axillary buds, tillers, or leafy side shoots) in different cul4 mutant alleles and the respective backgrounds over the critical period of tiller development (i.e. 2–5 weeks after planting). Our results indicated that the numbers of active axils were significantly lower in all cul4 mutants compared with their wild-type backgrounds (Fig. 2J). Consistent with the reiterative pattern of tiller formation, new axils continued to become active in wild-type plants with secondary buds forming. In contrast, few axillary buds emerged after week 4 in cul4 mutants, and no secondary buds were observed. In addition, most cul4 axillary buds turned into leafy side shoots rather than developed tillers (Supplemental Fig. S1). In agreement with previous histological analyses, the formation of two or multiple axillary buds from a single axil was observed in some cul4 plants (Supplemental Fig. S1). Leafy side shoots were associated with such multiple axillary buds (Supplemental Fig. S1).

Figure 2.

Figure 2.

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Axillary development in wild-type and cul4 mutant plants. A, Longitudinal section through the shoot apical meristem (SAM) of a cv Bowman wild-type plant. B, Longitudinal section through the shoot apical meristem of a cv Bowman-cul4.5 plant. C and D, AXMs in two successive leaf axils from the shoot apex shown in A captured in different sectioning planes. E, The only AXM from the same shoot apex shown in B. F, Transverse section through a single axillary shoot (AXS) in wild-type Bowman. G, Transverse section through two single axillary shoots in a single leaf axil in cv Bowman-cul4.5. H, Scanning electron micrograph of an AXM in a leaf axil from a wild-type cv Bowman plant. I, Scanning electron micrograph of two AXMs in a leaf axil from a cv Bowman-cul4.5 mutant. J, Time course (2–5 weeks after planting) of the total number of axils containing axillary buds, side shoots, or tillers in cul4 mutant alleles and the corresponding wild-type backgrounds. Values shown are means ± se of biological replicates. Bars = 200 μm (A–G) and 250 μm (H–I).

Taken together, these results show that the Cul4 gene is required for promoting axillary development in the barley shoot and controlling the number of leaf axils that form AXMs as well as the number of AXMs formed in a single leaf axil. In addition, Cul4 activity is critical for the correct development of existing axillary buds into tillers and the formation of secondary buds on primary tillers.

Mutations at the Cul4 locus also cause specific defects in leaf development. Grass leaves are organized in three distinct regions along the proximal-distal axis: (1) the proximal sheath is offset from (2) the distal blade by (3) a hinge-like structure comprising two wedge-shaped auricles, whereas an epidermal outgrowth called the ligule occurs on the adaxial leaf surface at the base of the auricles (Sylvester et al., 1990; Fig. 1, C and E). All cul4 mutant alleles exhibited a liguleless phenotype, although the boundary between the sheath and blade remained intact with auricles observed at the proper location, and occasionally a fringe of tissue developed in place of the ligule (Fig. 1, D and F; Supplemental Fig. S2). However, ectopic flaps of auricle-like tissue often developed on the margins of cul4 leaf sheaths, altering the proximal-distal development of the mutant leaf (Fig. 1, J and K; Supplemental Fig. S3). Scanning electron microscopy of cul4 flap tissue (Fig. 1H) showed similar cells to those of wild-type auricle tissue (Fig. 1G), confirming that these outgrowths on the sheath margins are ectopic auricles. These phenotypes demonstrate that Cul4 is required for ligule outgrowth and coordinating the proximal-distal patterning of the barley leaf.

Positional Cloning of the Cul4 Gene

Previous mapping positioned Cul4 on the distal end of chromosome 3HL (Pozzi et al., 2003; Druka et al., 2011). High-resolution mapping using 9,898 gametes from the cross cv Bowman-cul4.5 × cv Morex located Cul4 to a 0.55-centimorgan (cM) interval (Fig. 3). To further refine the barley Cul4 region, additional markers were developed based on careful examination of the barley syntenic relationships with Brachypodium distachyon, sorghum (Sorghum bicolor), and rice from the virtual gene order map (genome zipper) of barley (Mayer et al., 2011; Fig. 3B).

Figure 3.

Figure 3.

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Map-based cloning, structure, and molecular characterization of the Cul4 gene. A, Integrated map generated from an analysis of 386 F2 plants from crosses of the cul4.5 mutant allele with six wild-type cultivars (Supplemental Table S2) using 61 polymorphic markers identified in the distal region of chromosome 3HL. Three tightly linked markers, including one cosegregating marker, are boxed. B, High-resolution linkage map of the Cul4 region produced with 4,949 F3 plants from the cv Bowman-cul4.5 × cv Morex cross derived from 72 F2 plants heterozygous in a small interval around Cul4 (black bar in A). The number of recombinants between adjacent markers is indicated above the linkage map. Details of these markers can be found in Supplemental Table S3. At the top, the light-gray bar indicates the bacterial artificial chromosome (BAC) clone HVVMRXALLeA0131P08, and the positions of predicted genes are indicated as black boxes. At the bottom, B. distachyon genes are indicated as black boxes, and anchored genes are connected with dashed lines to the barley genetic map. B. distachyon genes are annotated as follows: 1, Bradi2g60650; 2, Bradi2g60660; 3, Bradi2g60670; 4, Bradi2g60680; 5, Bradi2g60690; 6, Bradi2g60700; 7, Bradi2g60705; 8, Bradi2g60710; 9, Bradi2g60720; 10, Bradi2g60730; 11, Bradi2g60740; and 12, Bradi2g60750. C, Exon-intron structure of the Cul4 gene. Two exons are represented as boxes, with the BTB/POZ domain and ankyrin repeats (ANK) as gray boxes, and the intervening intron is represented as a black line. Mutant alleles of Cul4 show a deletion in cul4.5 and radical amino acid substitutions in cul4.16 and cul4.24 compared with their progenitor backgrounds. D, RT-PCR analysis of Cul4 transcripts in mutant alleles and their corresponding wild-type backgrounds using the primers reported in Supplemental Table S3 (34 PCR cycles). UBIQUITIN (UBQ) was used as an internal control (25 cycles). E, Alignment of predicted amino acid sequences at the ankyrin repeats region of CUL4 with Arabidopsis BOP2 (AT2G41370) and BOP1 (AT3G57130), tobacco NtBOP2 (EF051131), M. truncatula NODULE ROOT (JN180858), and pea COCH (JN180860). Ankyrin repeats are indicated by black lines (Wu et al., 2012). The positions of amino acid substitutions in cul4.16 and cul4.24 are represented by black and white arrowheads, respectively.

Based on the current knowledge of genes involved in shoot development, two candidate genes were identified from annotated genes conserved among B. distachyon, rice, and sorghum. Partial genomic sequences were obtained exploiting EST information and available barley genomic reads (Feuillet et al., 2012; Mayer et al., 2012) and mapped using single-nucleotide polymorphisms (SNPs) identified between cv Bowman-cul4.5 and cv Morex. A GRAS candidate gene (highly related to Bradi2g60750) was mapped 0.38 cM or more from the cul4 locus and was excluded from further analysis. The second candidate gene, encoding a BROAD-COMPLEX, TRAMTRACK, BRIC-À-BRAC (BTB)-ankyrin protein (highly related to Bradi2g60710), showed cosegregation with cul4 in all recombinants identified in the target interval. The two flanking genes in B. distachyon (Bradi2g60705 and Bradi2g60720) defined a 0.22-cM interval flanking cul4 (Fig. 3C). To verify gene content and identify the flanking genes within the corresponding barley genomic region, a sequenced BAC clone (HVVMRXALLeA0131P08; 22× coverage assembled using 454 reads) was identified as matching the cosegregating BTB-ankyrin gene and the ortholog of the proximal B. distachyon gene Bradi2g60705 (Fig. 3B). Physical and genetic mapping yielded new flanking markers 0.02 cM distal to and 0.07 cM proximal from cul4, confirming the genetic position of the locus within this BAC clone (Fig. 3B). Two other predicted genes annotated from this BAC (encoding a pentatricopeptide repeat-containing protein and a hypothetical protein, respectively) showed recombination with the cul4 locus, confirming the correspondence between the BTB-ankyrin candidate gene and the cul4 mutant locus.

Cul4 Encodes a BTB-Ankyrin Protein Related to Arabidopsis BOP1 and BOP2

The Cul4 candidate gene has two exons and one intron, as shown by comparison of genomic and full-length complementary DNA (cDNA) sequences isolated from cv Morex seedlings (and consistent with the published full-length cDNA sequences AK360734.1 and AK355716.1). The Cul4 gene extends 2,632 bp from start to stop codon, with an open reading frame of 1,542 bp encoding a 513-amino acid protein of approximately 54 kD containing a BTB/POX VIRUS AND ZINC FINGER (POZ) domain and ankyrin repeats (Fig. 3C). Sequence comparison of the cul4.5 mutant allele with the cv Bonus background revealed a 3,141-bp deletion spanning most of exon 1 and the 5′ upstream region. The cul4.5 mutant allele showed no expression in reverse transcription (RT)-PCR using a forward primer designed on the exon junction (downstream of the deletion site) and a reverse primer on exon 2 (Fig. 3D). To gain further support for the correspondence between the candidate gene and the cul4 locus, sequences of mutant alleles cul4.16 and cul4.24 were also compared with those from the cv Bonus and cv Flare backgrounds, respectively. One nonsynonymous substitution was uncovered in the cul4.16 allele changing Leu-354 to Gln in the ankyrin repeat region; allele cul4.24 carries a nonconservative substitution of Leu-420 to Gln in a region conserved across highly related genes previously characterized in Arabidopsis, pea (Pisum sativum), Medicago truncatula, and tobacco (Nicotiana tabacum; see below) as well as the substitution of Met-441 to Thr (Fig. 3C). The two amino acid substitutions L354Q and L420Q in cul4.16 and cul4.24, respectively, are located in highly conserved regions among barley and other known genes in dicots (Fig. 3E) and are predicted to have a deleterious impact on the biological function of the protein (SIFT program; P ≤ 0.01). Notably, ankyrin repeats are known to mediate interactions between the BTB-ankyrin protein NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and TGACG sequence-specific binding (TGA) transcription factors to regulate defense responses in Arabidopsis (Zhang et al., 1999; Després et al., 2000, 2003; Zhou et al., 2000), highlighting the functional relevance of these motifs.

Recovery of distinct mutations in three independent cul4 alleles indicates that this gene is responsible for the cul4 phenotype. In addition, we observed that the severity of the cul4 mutations was consistent with the reduction in tiller numbers (Fig. 1B), while all mutant alleles displayed similar leaf phenotypes (Supplemental Figs. S2 and S3).

Similarity searches show that the Cul4 gene encodes a BTB-ankyrin domain protein sharing high similarity with Arabidopsis BOP1 and BOP2, pea COCHLEATA (COCH), as well as numerous as yet uncharacterized monocot genes (Couzigou et al., 2012), including a paralogous gene in barley (MLOC_61451.6). In a recent survey in a range of species, most plant genomes were found to harbor two or three BOP genes (Khan et al., 2014). Phylogenetic analyses (Supplemental Fig. S4) reveal that the BOP1 and BOP2 genes of Arabidopsis, which mutually share 80% identity at the amino acid level, as well as the soybean (Glycine max) Glyma03g28440 and Glyma19g31180 genes (82% amino acid identity), both derive from independent, recent lineage-specific gene duplications within the dicots. This is consistent with the observed high degree of redundancy of Arabidopsis BOP genes at both the functional and expression pattern levels (Norberg et al., 2005; Ha et al., 2007; Xu et al., 2010). By way of contrast, Cul4 and MLOC_61451.6 share 58% amino acid identity and, as for BOP paralogs in other monocot species, fall into distinct, highly supported clades deriving from a more ancient duplication. These observations are consistent with both the divergent gene expression patterns observed between barley BOP paralogs (see below) and the fact that single cul4 mutated alleles present phenotypic defects in barley. It is unclear whether the inferred gene duplication occurred within monocots after their divergence from dicots, whereby both Cul4 and MLOC_61451.6 should be considered as inparalogs of Arabidopsis BOP1/2, or before the monocot/dicot divergence, implying a loss of one paralog in an ancestor of sampled dicots and orthology of either MLOC_61451.6 or Cul4 with BOP1/2. However, the available data exclude the individual orthology of pairs of barley and Arabidopsis genes.

The best characterized members of this family are Arabidopsis BOP1 and BOP2, which act as complexes with transcription factors (Hepworth et al., 2005) to control leaf development and floral organ determination. Arabidopsis bop1 bop2 double mutants are characterized by the ectopic outgrowth of blade tissue on the petiole (Ha et al., 2003, 2004, 2007; Hepworth et al., 2005). Morphological alterations of the stipules located at the base of the leaf were also observed in loss-of-function mutants of BOP orthologs in pea and M. truncatula (Couzigou et al., 2012). Phenotypic defects in dicot bop mutants and cul4 (Fig. 1, E–G) indicate that the corresponding genes are required for the correct morphogenesis of the proximal region of the leaf. This suggests at least a partial conservation of BOP gene function in leaf development of barley and eudicots. In Arabidopsis, BOP1 and BOP2 have highly redundant functions and near-identical expression patterns (Norberg et al., 2005; Ha et al., 2007; Xu et al., 2010). In contrast, publicly available barley RNA sequencing data show that, while expression patterns partially overlap, Cul4 has significantly higher expression in the embryo of germinating grains (where axillary buds are present) and MLOC_61451.6 exhibits highest expression in the developing inflorescence (Supplemental Fig. S5). In agreement with phylogenetic analyses, these results are consistent with functional divergence between Cul4 and its barley paralog.

Cul4 Expression Is Associated with Axillary Bud and Ligule Formation

The expression of Cul4 in wild-type plants was further analyzed by quantitative RT-PCR and RNA in situ hybridization. Consistent with a role in tiller and leaf development, Cul4 transcripts were detected in 3-d-old seedlings and highly expressed in the crown at the first leaf stage, when axillary buds and leaf primordia develop (Fig. 4A). Transcript accumulation was also detected in leaves at the first leaf stage, while it was lower in roots (Fig. 4A). At the four-leaf stage, Cul4 was strongly expressed in the ligular region of the fully expanded leaf, while it was less expressed in the leaf blade. RNA in situ hybridization showed Cul4 expression in the leaf axil preceding AXM development. Cul4 signal was also observed as a distinct clear pattern in the developing axillary bud, followed by a more diffuse pattern in the more mature bud (Fig. 4B). In addition, Cul4 transcripts were detected in the leaf axil derived from an axillary bud (Fig. 4C). Examination of a cross section of the shoot apex shows that Cul4 is expressed in a crescent of cells on the stem side of the leaf axil (Fig. 4D). Expression was also evident in developing ligules of two successive leaf primordia (Fig. 4B; Supplemental Fig. S6). No signal was detected in the cul4.5 deletion mutant (Fig. 4E). These data indicate that the Cul4 gene is specifically expressed at the leaf axil and at the blade-sheath boundary to guide the development of the axillary bud and the ligule, respectively.

Figure 4.

Figure 4.

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Cul4 expression in cv Bonus wild-type plants using quantitative RT-PCR (A) and in situ RNA hybridization in the shoot apical region of cv Bonus (B–D) and the cul4.5 mutant (E). A, Quantitative RT-PCR was performed using specific primers for cul4 (Supplemental Table S3) on total RNA isolated from seedling (3-d-old seedling, when the first leaf was just emerging through the coleoptile); root, crown, and leaves at the one-leaf stage; and the 1-cm ligular region and the distal half of the blade from the third leaf at the four-leaf stage. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used to normalize the data. The data shown are averages of three biological replicates ± sd. B, Longitudinal section of the shoot apical region exhibiting Cul4 hybridization in the leaf axils (arrowheads), axillary bud (asterisk), and ligule of a leaf primordium (arrow). C, Longitudinal section of an axillary bud; expression in leaf axils (arrowheads) is indicated. D, Cross section of the shoot apical region; Cul4 hybridization on the stem side of the axil (arrowhead) is indicated. The sectioning plane corresponds to the dashed line in B. E, Shoot apical region in the cul4.5 mutant showing no Cul4 expression. p, Leaf primordium; SAM, shoot apical meristem; WT, wild type. Bars = 250 μm (B and E) and 125 μm (C and D).

DISCUSSION

In this study, we have shown that Cul4 is required for tiller development and leaf patterning in barley. Multiple lines of evidence provide conclusive proof that Cul4 encodes a BTB-ankyrin protein highly related to Arabidopsis BOP1 and BOP2 (Norberg et al., 2005), including (1) cosegregation with the phenotype in 9,898 gametes; (2) physical mapping and recombination with adjacent genes identified within the BAC clone spanning the Cul4 locus; (3) identification of three independent mutant alleles confirming that Cul4 mutations account for recessive cul4 phenotypes of different severity; and (4) gene expression in the boundary regions at the leaf axil and ligule coincident with the alterations in morphology in cul4 mutants. To our knowledge, Cul4 is the first BOP gene functionally characterized in monocots.

Our results revealed the involvement of the Cul4 gene in the control of tiller development, ligule formation, and proximal-distal leaf patterning. The related Arabidopsis genes BOP1 and BOP2 function redundantly to regulate the growth and development of lateral organs and are expressed at the leaf/meristem boundary governing leaf proximal-distal and adaxial-abaxial patterning (Ha et al., 2003, 2004; Hepworth et al., 2005; Norberg et al., 2005; Barton, 2010; Jun et al., 2010; Xu et al., 2010; Khan et al., 2012). In addition, Arabidopsis bop1 bop2 double mutants showed partial reduction in the number of rosette paraclades (Khan et al., 2012), although this phenotype was not well characterized. Here, we observed that cul4 mutants develop fewer axillary buds than the wild type, although a single axil can sometimes produce two to three axillary buds. Compared with the wild type, the overall reduction in cul4 tiller numbers is linked to the formation of fewer active axils (i.e. axils that initiate axillary buds), the lack of secondary buds, and the formation of leafy side shoots instead of normal tillers. These results indicate that Cul4 controls the number of leaf axils that develop an axillary bud and the number of axillary buds that develop in a single axil. The location of Cul4 expression at the leaf axil preceding axillary bud development indicates that Cul4 is involved in defining a boundary between an existing developmental axis (i.e. the main culm) and a new axis of lateral growth. In addition, Cul4 function is required for tiller outgrowth, as some buds formed in cul4 mutants developed into leafy side shoots rather than elongated lateral culms. Together, these results show that Cul4 acts to control tiller development at multiple levels.

Cul4 is also responsible for proper proximal sheath cell fate and the location of distal auricle cells, as shown by the development of ectopic auricle-like tissue on the sheath margins in cul4 mutants: this is reminiscent of the laminar outgrowths that form on petiolar edges of Arabidopsis bop1 bop2 double mutants (Hepworth et al., 2005; Norberg et al., 2005). Although the homology of the leaf sheath in grasses, and its relationship to the petiole of eudicot plants, are controversial (Arber, 1918; Kaplan, 1973), it is intriguing that these ectopic outgrowths occur following the loss of function of homologous genes in both barley and Arabidopsis. In pea, loss-of-function mutations in the BOP-like COCH gene result in the reduction and absence of stipules in basal leaves (Couzigou et al., 2012), suggesting a conserved role of BOP-like genes in proximal-distal leaf patterning. In addition, in cul4 mutants, the boundary between the sheath and blade is preserved, but the ligule does not develop, indicating that Cul4 is required for outgrowth of the ligule but does not play a role in demarcating the separation between blade and sheath. Gene expression in the developing ligule is consistent with a specific role for Cul4 in the differentiation of this structure. Other genes required for ligule development have been identified from genetic analyses in maize (Zea mays; Bolduc et al., 2012). Among them, LIGULELESS2 (LG2) encodes a TGA basic Leu zipper transcription factor (Walsh et al., 1998), which was proposed to link proximal-distal leaf patterning signals and the induction of ligule development (Bolduc et al., 2012). Interestingly, BOP and NPR1 proteins have been shown to bind TGA transcription factors to regulate different processes (Khan et al., 2014). In particular, BOP1 and BOP2 interact with the TGA factor PERIANTHIA to pattern Arabidopsis floral meristems (Hepworth et al., 2005), suggesting the possibility that CUL4 controls leaf patterning in barley through interaction with yet unknown TGA factors possibly related to maize LG2.

Taken together, cul4 mutants exhibit a range of phenotypes including proximal-distal leaf patterning defects, lack of ligule outgrowth, and defective tiller development that, combined with the expression patterns in the developing ligule and leaf axil, suggest that Cul4 functions to define developmental boundaries. Comparison with Arabidopsis and pea suggests that BOP genes share conserved functions in the patterning of monocot and dicot leaves. In addition, Cul4 plays a major role in the control of tillering in barley through the regulation of AXM formation and outgrowth.

In contrast to the largely redundant activities of BOP1 and BOP2 in Arabidopsis, phenotypic defects of cul4 single mutants and phylogenetic analysis indicate that Cul4 plays a specific and distinct function from its barley paralog MLOC_61451.6. While functional characterization of MLOC 61451.6 will tell if some level of redundancy with Cul4 is maintained, expression data show that Cul4 is more active in germinating embryos where axillary buds are developing, while the paralogous gene is highly expressed in developing inflorescences and might play a function in reproductive development, similar to the role of Arabidopsis BOPs in the specification and patterning of inflorescence architecture (for review, see Khan et al., 2014).

In conclusion, identification of the Cul4 gene opens new opportunities for the genetic dissection and manipulation of shoot branching in Triticeae species. As tillers contribute directly to grain yield, competition with weeds, and plant plasticity in response to environmental conditions and stress, such knowledge can be applied in breeding for more adaptable and productive crops.

MATERIALS AND METHODS

Plant Materials

The cul4.5 allele was derived from x-ray mutagenesis of barley (Hordeum vulgare ‘Bonus’), and the cul4.16 and cul4.24 alleles were derived from fast-neutron mutagenesis of cv Bonus and cv Flare, respectively (Supplemental Table S1). The cul4.5 mutant allele was backcrossed five times into cv Bowman, a two-rowed spring feed barley (Franckowiak et al., 1985), to obtain the cul4.5 near-isogenic line (cv Bowman-cul4.5). Detailed information about cul4 mutant stocks, the corresponding wild-type backgrounds, and the segregating populations used in this work can be found in Supplemental Tables S1 and S2.

Morphological Analysis

For quantitative phenotyping of mutant stocks cul4.5, cul4.16, and cul4.24 and their corresponding wild-type backgrounds, cv Bonus and cv Flare, single plants were grown in 1.5-L pots in a greenhouse in a completely randomized design with nine replicates under natural photoperiod and temperature in Lodi, Italy, from December 2011 to June 2012. Phenotyping data were analyzed in SAS version 9.1.3.

Histological analysis of the shoot apex from 10-d-old seedlings of cv Bowman and the cv Bowman-cul4.5 mutant were performed to examine tiller development. Tissue from six to 10 plants was fixed, passed through an ethanol dehydration series, and embedded in paraffin wax. Ten-micrometer-thick longitudinal and transverse sections through the apical meristem region were obtained and stained with Toluidine Blue following the protocols described by Ruzin (1999).

For scanning electron microscopy, a minimum of five shoot apices were dissected from seedlings of cv Bowman and cv Bowman-cul4.5. Tissue samples were attached to aluminum stubs using double-sided carbon tape and/or carbon paint and immediately frozen in liquid nitrogen. Frozen tissue samples were viewed on a cold stage of the scanning electron microscope, and images were taken at 1.8 to 2.3 kV (Ahlstrand, 1996).

To further examine the effects of cul4 mutations on tiller development, the numbers of leaf axils harboring axillary buds, tillers, or leafy side shoots were recorded in seven to nine seedlings of the cv Bowman-cul4.5, cul4.16, and cul4.24 mutant stocks and the corresponding wild-type backgrounds, cv Bowman, cv Bonus, and cv Flare, respectively, from 2 to 5 weeks after planting (Fig. 2J; Supplemental Fig. S1).

Linkage Mapping and Positional Cloning

To identify SNPs tightly linked to the cul4 locus and select the most appropriate cross for high-resolution mapping, initially, 386 F2 plants from six segregating populations (Supplemental Table S2) were genotyped using the Illumina Goldengate assay (Fan et al., 2003). Starting from eight SNPs identified previously as linked to cul4 by comparison of the cv Bowman-cul4.5 and recurrent parent cv Bowman (data not shown), a total of 96 EST-derived SNP markers covering the interval 120.6 to 173.2 cM in the 3HL telomeric region (Close et al., 2009; Barley HarvEST database; http://harvest.ucr.edu/) were examined. An integrated genetic linkage map was constructed from the six initial mapping populations using JoinMap 4.1 (Stam, 1993).

A population of 4,949 F3 plants from the cv Bowman-cul4.5 × cv Morex cross was generated by selfing 72 F2 plants heterozygous for the cul4 region; KASPar genotyping (KBioscience) with SNPs 8919-758 and 2825-1609, which flank cul4, identified 174 recombinants. Phenotyping was conducted in the same conditions as above from December 2009 to June 2012. Plants exhibiting defective tillering and liguleless phenotype were classified as homozygous cul4. Wild-type F3 individuals harboring recombination events in the vicinity of the locus were propagated, and F4 progeny were phenotyped for discrimination of homozygous Cul4 and heterozygotes. The identified recombinants were then genotyped with SNP U35_6520_551 (cosegregating with cul4 in the initial 266 F2 plants of the cv Bowman-cul4.5 × cv Morex mapping population).

To refine the location of the recombination events nearest to cul4 and evaluate colinearity with Brachypodium distachyon chromosome 2 and rice (Oryza sativa) chromosome 1 genomic regions, markers were developed on the basis of revised genome zipper information (Mayer et al., 2011). Gene-based markers were developed exploring EST information and the available assembly of barley genomic reads (Mayer et al., 2012): Specific primers were designed (Supplemental Table S3), and genomic PCR amplicons were sequenced in parents cv Morex and cv Bowman-cul4.5 using the Sanger method at the Genomics Platform, Parco Tecnologico Padano. The resulting polymorphic markers were mapped using the same method mainly on 55 recombinants between the cul4 flanking markers U34_6520_551 and 2825-1609 (selected from 4,949 F3 plants); in the case of markers that were not mapped within this region, additional F3 plants were genotyped, allowing us to determine their positions and better resolve colinearity with reference genomes.

Candidate genes were considered based on annotated genes conserved among the three reference genomes of B. distachyon, rice, and sorghum (Sorghum bicolor) and current knowledge of genes involved in shoot development. Candidate genes were amplified from genomic DNA using the primers listed in Supplemental Table S3, and genomic sequences were compared in cv Bowman-cul4.5 and cv Morex. Identified polymorphisms were used to map them as described for newly developed markers.

A BAC contig (FPcontig_460) of the barley physical map (Schulte et al., 2011) was identified by sequence homology search with cul4 flanking markers to barley genomic sequence information. The BAC clone HVVMRXALLeA0131P08 was sequenced using Roche/454 Genome Sequencer FLX technology and assembled after the removal of short sequences, adapter and vector trimming, and assembly using a previously described procedure (Steuernagel et al., 2009). The Triannot pipeline gene prediction program (Leroy et al., 2012; http://urgi.versailles.inra.fr/Species/Wheat/Triannot-Pipeline) was used to annotate potential genes. Genomic markers were developed using the same method as above or insertion site-base polymorphism markers (Paux et al., 2010) using the primers listed in Supplemental Table S3.

For allelic comparisons, genomic PCR and resequencing of the Cul4 gene were carried out in the three available allelic mutant stocks cul4.5 (GenBank accession no. KF151193), cul4.16 (KF151195), and cul4.24 (KF151196) and the backgrounds cv Bonus (KF151192) and cv Flare (KF151194) using the primers described in Supplemental Table S3. The amino acid substitution’s impact on protein function was evaluated using http://sift.bii.a-star.edu.sg/www/SIFT_seq_submit2.html (Ng and Henikoff, 2001).

RNA Extraction, Expression Analysis, and Quantitative RT-PCR

Relative Cul4 expression was measured in 3-d-old seedlings when the first leaf was just emerging through the coleoptile (GRO:0007059; www.gramene.org); root, crown, and leaves at the one-leaf stage (GRO:0007060); and the 1-cm ligular region and the distal half of the blade from the third leaf at the four-leaf stage (GRO:0007063). Total RNA was isolated using TRI-Reagent (Sigma-Aldrich) and treated with RNase-free DNase I (Invitrogen) according to the manufacturers’ instructions. The concentration of RNA was determined using Agilent Bioanalyser 2100 (Agilent Technologies). First strand cDNA was synthesized from 1.5 μg of total RNA using SuperScript III Reverse Transcriptase (Invitrogen). After RT, cDNA samples were diluted 4-fold, and 2 μL was used for further analysis. Quantitative analyses were carried out with three biological and technical replications on the 7300 Real-time PCR System (Applied Biosystems) using the primers reported in Supplemental Table S3 and SYBR Green Master Mix according to the manufacturer’s instructions. Normalization was carried out using the GAPDH (accession no. EF409629) and UBQ (Osnato et al., 2010) genes and the ΔΔCT method where ΔΔCT = (CT,Cul4 − CT,GAPDH)tissue2 − (CT,Cul4 − CT,GAPDH)tissue1 (Livak and Schmittgen, 2001).

For expression analyses of Cul4 in various tissues/stages, three biological replications were carried out, with each replication including samples from five cv Bonus plants grown in a growth chamber under a 16-h-light/8-h-dark photoperiod with day/night temperatures of 20°C/17°C, respectively.

For comparison of the expression of Cul4 and MLOC_61451.6, publicly available RNA sequencing data (Mayer et al., 2012) were queried at the MorexGenes Barley RNA-seq database (http://ics.hutton.ac.uk/morexGenes/index.html) to obtain fragments per kilobase of exon per million fragments mapped data for three biological replicates.

RNA in Situ Hybridization

Two gene-specific fragments of 306 and 362 bp from the 5′ untranslated region and the 3′ end of the Cul4 cDNA were PCR amplified with the primers shown in Supplemental Table S3. Samples from 14-d-old seedlings were fixed, processed, sectioned, and hybridized to both probes as described (Juarez et al., 2004).

Cul4 gene sequences reported in this article have been deposited in the National Center for Biotechnology Information database with accession numbers KF151192, KF151193, KF151194, KF151195, and KF151196. Sequences for BAC HVVMRXALLeA0131P08 have been deposited at the National Center for Biotechnology (accession no. AC256289; assembled contigs) and at the European Bioinformatics Institute′s European Nucleotide Archive (accession no. PRJEB4166; raw data).

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_168_1_164__index.html (816B, html)

Acknowledgments

We thank Francesco Salamini and Carlo Pozzi for providing cul4 crosses with cv Proctor and cv Nudinka; Etienne Paux for help in designing insertion site-base polymorphism markers; Kevin Smith for providing field space; Tiziana Fusca for helping with the development of the initial mapping populations; and Bruna Bucciarelli and Gail Celio for assistance with histology and for scanning electron microscopy.

Glossary

AXM

axillary meristem

cM

centimorgan

SNP

single-nucleotide polymorphism

BAC

bacterial artificial chromosome

cDNA

complementary DNA

RT

reverse transcription

TGA

TGACG sequence-specific binding

Footnotes

1

This work was supported by the European Community’s Seventh Framework Programme (FP7/2007–2013; grant no. FP7–212019 to L.R. and N.S.); the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service-National Research Initiative Plant Growth and Development Program (grant no. 2004–03440 to G.J.M.); and the GABI-FUTURE program of the German Ministry of Education and Research (grant no. Barlex–0314000A to N.S.).

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