Shun Sakuma

An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice

Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named “eibi1.c,” along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants.

Six-rowed spike4 (Vrs4) controls spikelet determinacy and row-type in barley

Inflorescence architecture of barley (Hordeum vulgare L.) is common among the Triticeae species, which bear one to three single-flowered spikelets at each rachis internode. Triple spikelet meristem is one of the unique features of barley spikes, in which three spikelets (one central and two lateral spikelets) are produced at each rachis internode. Fertility of the lateral spikelets at triple spikelet meristem gives row-type identity to barley spikes. Six-rowed spikes show fertile lateral spikelets and produce increased grain yield per spike, compared with two-rowed spikes with sterile lateral spikelets. Thus, far, two loci governing the row-type phenotype were isolated in barley that include Six-rowed spike1 (Vrs1) and Intermedium-C. In the present study, we isolated Six-rowed spike4 (Vrs4), a barley ortholog of the maize (Zea mays L.) inflorescence architecture gene RAMOSA2 (RA2). Eighteen coding mutations in barley RA2 (HvRA2) were specifically associated with lateral spikelet fertility and loss of spikelet determinacy. Expression analyses through mRNA in situ hybridization and microarray showed that Vrs4 (HvRA2) controls the row-type pathway through Vrs1 (HvHox1), a negative regulator of lateral spikelet fertility in barley. Moreover, Vrs4 may also regulate transcripts of barley SISTER OF RAMOSA3 (HvSRA), a putative trehalose-6-phosphate phosphatase involved in trehalose-6-phosphate homeostasis implicated to control spikelet determinacy. Our expression data illustrated that, although RA2 is conserved among different grass species, its down-stream target genes appear to be modified in barley and possibly other species of tribe Triticeae.

Duplication of a well-conserved homeodomain-leucine zipper transcription factor gene in barley generates a copy with more specific functions

Three spikelets are formed at each rachis node of the cultivated barley (Hordeum vulgare ssp. vulgare) spike. In two-rowed barley, the central one is fertile and the two lateral ones are sterile, whereas in the six-rowed type, all three are fertile. This characteristic is determined by the allelic constitution at the six-rowed spike 1 (vrs1) locus on the long arm of chromosome 2H, with the recessive allele (vrs1) being responsible for the six-rowed phenotype. The Vrs1 (HvHox1) gene encodes a homeodomain-leucine zipper (HD-Zip) transcription factor. Here, we show that the Vrs1 gene evolved in the Poaceae via a duplication, with a second copy of the gene, HvHox2, present on the short arm of chromosome 2H. Micro-collinearity and polypeptide sequences were both well conserved between HvHox2 and its Poaceae orthologs, but Vrs1 is unique to the barley tribe. The Vrs1 gene product lacks a motif which is conserved among the HvHox2 orthologs. A phylogenetic analysis demonstrated that Vrs1 and HvHox2 must have diverged after the separation of Brachypodium distachyon from the Pooideae and suggests that Vrs1 arose following the duplication of HvHox2, and acquired its new function during the evolution of the barley tribe. HvHox2 was expressed in all organs examined but Vrs1 was predominantly expressed in immature inflorescence.

Divergence of expression pattern contributed to neofunctionalization of duplicated HD‐Zip I transcription factor in barley

Barley (Hordeum vulgare) spikes are developmentally switched from two-rowed to six-rowed by a single recessive gene, six-rowed spike 1 (vrs1), which encodes a homeodomain-leucine zipper I class transcription factor. Vrs1 is a paralog of HvHox2 and both were generated by duplication of an ancestral gene. HvHox2 is conserved among cereals, whereas Vrs1 acquired its current function during the evolution of barley. It was unclear whether divergence of expression pattern or protein function accounted for the functionalization of Vrs1. Here, we conducted a comparative analysis of protein functions and gene expression between HvHox2 and Vrs1 to clarify the functionalization mechanism. We revealed that the transcriptional activation activity of HvHOX2 and VRS1 was conserved. In situ hybridization analysis showed that HvHox2 is localized in vascular bundles in developing spikes, whereas Vrs1 is expressed exclusively in the pistil, lemma, palea and lodicule of lateral spikelets. The transcript abundance of Vrs1 was > 10-fold greater than that of HvHox2 during the pistil developmental stage, suggesting that the essential function of Vrs1 is to inhibit gynoecial development. We demonstrated the quantitative function of Vrs1 using RNAi transgenic plants and Vrs1 expression variants. Expression analysis of six-rowed spike mutants that are nonallelic to vrs1 showed that Vrs1 expression was up-regulated by Vrs4, whereas HvHox2 expression was not. These data demonstrate that the divergence of gene expression pattern contributed to the neofunctionalization of Vrs1.

The Genetic Basis of Composite Spike Form in Barley and ‘Miracle-Wheat’

Inflorescences of the tribe Triticeae, which includes wheat (Triticum sp. L.) and barley (Hordeum vulgare L.) are characterized by sessile spikelets directly borne on the main axis, thus forming a branchless spike. ‘Compositum-Barley’ and tetraploid ‘Miracle-Wheat’ (T. turgidum convar. compositum (L.f.) Filat.) display noncanonical spike-branching in which spikelets are replaced by lateral branch-like structures resembling small-sized secondary spikes. As a result of this branch formation ‘Miracle-Wheat’ produces significantly more grains per spike, leading to higher spike yield. In this study, we first isolated the gene underlying spike-branching in ‘Compositum-Barley,’ i.e., compositum 2 (com2). Moreover, we found that COM2 is orthologous to the branched headt (bht) locus regulating spike branching in tetraploid ‘Miracle-Wheat.’ Both genes possess orthologs with similar functions in maize BRANCHED SILKLESS 1 (BD1) and rice FRIZZY PANICLE/BRANCHED FLORETLESS 1 (FZP/BFL1) encoding AP2/ERF transcription factors. Sequence analysis of the bht locus in a collection of mutant and wild-type tetraploid wheat accessions revealed that a single amino acid substitution in the DNA-binding domain gave rise to the domestication of ‘Miracle-Wheat.’ mRNA in situ hybridization, microarray experiments, and independent qRT-PCR validation analyses revealed that the branch repression pathway in barley is governed through the spike architecture gene Six-rowed spike 4 regulating COM2 expression, while HvIDS1 (barley ortholog of maize INDETERMINATE SPIKELET 1) is a putative downstream target of COM2. These findings presented here provide new insights into the genetic basis of spike architecture in Triticeae, and have disclosed new targets for genetic manipulations aiming at boosting wheat’s yield potential.

VRS2 regulates hormone-mediated inflorescence patterning in barley

Plant architecture has clear agronomic and economic implications for crops such as wheat and barley, as it is a critical factor for determining grain yield. Despite this, only limited molecular information is available about how grain-bearing inflorescences, called spikes, are formed and maintain their regular, distichous pattern. Here we elucidate the molecular and hormonal role of Six-rowed spike 2 (Vrs2), which encodes a SHORT INTERNODES (SHI) transcriptional regulator during barley inflorescence and shoot development. We show that Vrs2 is specifically involved in floral organ patterning and phase duration by maintaining hormonal homeostasis and gradients during normal spike development and similarly influences plant stature traits. Furthermore, we establish a link between the SHI protein family and sucrose metabolism during organ growth and development that may have implications for deeper molecular insights into inflorescence and plant architecture in crops.

Structure, transcription and post-transcriptional regulation of the bread wheat orthologs of the barley cleistogamy gene Cly1

The majority of genes present in the hexaploid bread wheat genome are present as three homoeologs. Here, we describe the three homoeologous orthologs of the barley cleistogamy gene Cly1, a member of the AP2 gene family. As in barley, the wheat genes (designated TaAP2-A, -B and -D) map to the sub-telomeric region of the long arms of the group 2 chromosomes. The structure and pattern of transcription of the TaAP2 homoeologs were similar to those of Cly1. Transcript abundance was high in the florets, and particularly in the lodicule. The TaAP2 message was cleaved at its miR172 target sites. The set of homoeolog-specific PCR assays developed will be informative for identifying either naturally occurring or induced cleistogamous alleles at each of the three wheat homoeologs. By combining such alleles via conventional crossing, it should be possible to generate a cleistogamous form of bread wheat, which would be advantageous both with respect to improving the level of the crop’s resistance against the causative pathogen of fusarium head blight, and for controlling pollen-mediated gene flow to and from genetically modified cultivars.

Population-genetic analysis of HvABCG31 promoter sequence in wild barley (Hordeum vulgare ssp. spontaneum)

BackgroundThe cuticle is an important adaptive structure whose origin played a crucial role in the transition of plants from aqueous to terrestrial conditions. HvABCG31/Eibi1 is an ABCG transporter gene, involved in cuticle formation that was recently identified in wild barley (Hordeum vulgare ssp. spontaneum). To study the genetic variation of HvABCG31 in different habitats, its 2 kb promoter region was sequenced from 112 wild barley accessions collected from five natural populations from southern and northern Israel. The sites included three mesic and two xeric habitats, and differed in annual rainfall, soil type, and soil water capacity.ResultsPhylogenetic analysis of the aligned HvABCG31 promoter sequences clustered the majority of accessions (69 out of 71) from the three northern mesic populations into one cluster, while all 21 accessions from the Dead Sea area, a xeric southern population, and two isolated accessions (one from a xeric population at Mitzpe Ramon and one from the xeric ‘African Slope’ of “Evolution Canyon”) formed the second cluster. The southern arid populations included six haplotypes, but they differed from the consensus sequence at a large number of positions, while the northern mesic populations included 15 haplotypes that were, on average, more similar to the consensus sequence. Most of the haplotypes (20 of 22) were unique to a population. Interestingly, higher genetic variation occurred within populations (54.2%) than among populations (45.8%). Analysis of the promoter region detected a large number of transcription factor binding sites: 121–128 and 121–134 sites in the two southern arid populations, and 123–128,125–128, and 123–125 sites in the three northern mesic populations. Three types of TFBSs were significantly enriched: those related to GA (gibberellin), Dof (DNA binding with one finger), and light.ConclusionsDrought stress and adaptive natural selection may have been important determinants in the observed sequence variation of HvABCG31 promoter. Abiotic stresses may be involved in the HvABCG31 gene transcription regulations, generating more protective cuticles in plants under stresses.

Variation in the wheat AP2 homoeologs, the genes underlying lodicule development

The bread wheat genome harbors three homoeologs of the barley gene HvAP2, which determines the cleistogamous/non-cleistogamous flowering. The three homoeologs, TaAP2-A, TaAP2-B and TaAP2-D, are derived from the A, B and D genomes. The importance of lodicule swelling in assuring non-cleistogamous flowering in a range of wild and domesticated wheat accessions of varying ploidy level was established. Re-sequencing of wheat AP2 homoeologous genes was carried out to identify natural variation at both the nucleotide and polypeptide level. The sequences of wheat AP2 homoeologs are highly conserved even across different ploidy levels and no functional variants at the key miR172 targeting site were detected. These results indicate that engineering of cleistogamous wheat will require the presence of a functional TaAP2 modification at each of the three homoeologs.

Extreme suppression of lateral floret development by a single amino acid change in the VRS1 transcription factor

Extreme suppression of lateral floret development in deficiens barley is the result of a single amino acid substitution in the homeodomain-leucine zipper class I transcription factor VRS1. Increasing grain yield is an endless challenge for cereal crop breeding. In barley (Hordeum vulgare), grain number is controlled mainly by Six-rowed spike 1 (Vrs1), which encodes a homeodomain leucine zipper class I transcription factor. However, little is known about the genetic basis of grain size. Here, we show that extreme suppression of lateral florets contributes to enlarged grains in deficiens barley. Through a combination of fine-mapping and resequencing of deficiens mutants, we have identified that a single amino acid substitution at a putative phosphorylation site in VRS1 is responsible for the deficiens phenotype. deficiens mutant alleles confer an increase in grain size, a reduction in plant height, and a significant increase in thousand grain weight in contemporary cultivated germplasm. Haplotype analysis revealed that barley carrying the deficiens allele (Vrs1.t1) originated from two-rowed types carrying the Vrs1.b2 allele, predominantly found in germplasm from northern Africa. In situ hybridization of histone H4, a marker for cell cycle or proliferation, showed weaker expression in the lateral spikelets compared with central spikelets in deficiens. Transcriptome analysis revealed that a number of histone superfamily genes were up-regulated in the deficiens mutant, suggesting that enhanced cell proliferation in the central spikelet may contribute to larger grains. Our data suggest that grain yield can be improved by suppressing the development of specific organs that are not positively involved in sink/source relationships.