Homoeologous copy-specific expression patterns of MADS-box genes for floral formation in allopolyploid wheat.

The consensus model for floral organ formation in higher plants, the so-called ABCDE model, proposes that floral whorl-specific combinations of class A, B, C, D, and E genes specify floral organ identity. Class A, B, C, D and E genes encode MADS-box transcription factors; the single exception being the class A gene APETALA2. Bread wheat (Triticum aestivum) is a hexaploid species with a genome constitution AABBDD; the hexaploid originated from a cross between tetraploid T. turgidum (AABB) and diploid Aegilops tauschii (DD). Tetraploid wheat is thought to have originated from a cross between the diploid species T. urartu (AA) and Ae. speltoides (BB). Consequently, the hexaploid wheat genome contains triplicated homoeologous copies (homoeologs) of each gene derived from the different ancestral diploid species. In this study, we examined the expression patterns of homoeologs of class B, C and D MADS-box genes during floral development. For the class B gene wheat PISTILLATA2 (WPI2), the homoeologs from the A and D genomes were expressed, while expression of the B genome homoeolog was suppressed. For the class C gene wheat AGAMOUS1 (WAG1), the homoeologs on the A and B genomes were expressed, while expression of the D genome homoeolog was suppressed. For the class D gene wheat SEEDSTICK (WSTK), the B genome homoeolog was preferentially expressed. These differential patterns of homoeolog expression were consistently observed among different hexaploid wheat varieties and synthetic hexaploid wheat lines developed by artificial crosses between tetraploid wheat and Ae. tauschii. These results suggest that homoeolog-specific regulation of the floral MADS-box genes occurs in allopolyploid wheat.

Identification of a novel homolog for a calmodulin-binding protein that is upregulated in alloplasmic wheat showing pistillody.

Intracellular signaling pathways between the mitochondria and the nucleus are important in both normal and abnormal development in plants. The homeotic transformation of stamens into pistil-like structures (a phenomenon termed pistillody) in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) has been suggested to be induced by mitochondrial retrograde signaling, one of the forms of intracellular communication. We showed previously that the mitochondrial gene orf260 could alter the expression of nuclear class B MADS-box genes to induce pistillody. To elucidate the interactions between orf260 and nuclear homeotic genes, we performed a microarray analysis to compare gene expression patterns in the young spikes of a pistillody line and a normal line. We identified five genes that showed higher expression levels in the pistillody line. Quantitative expression analysis using real-time PCR indicated that among these five genes, Wheat Calmodulin-Binding Protein 1 (WCBP1) was significantly upregulated in young spikes of the pistillody line. The amino acid sequence of WCBP1 was predicted from the full-length cDNA sequence and found to encode a novel plant calmodulin-binding protein. RT-PCR analysis indicated that WCBP1 was preferentially expressed in young spikes at an early stage and decreased during spike maturation, indicating that it was associated with spikelet/floret development. Furthermore, in situ hybridization analysis suggested that WCBP1 was highly expressed in the pistil-like stamens at early to late developmental stages. These results indicate that WCBP1 plays a role in formation and development of pistil-like stamens induced by mitochondrial retrograde signaling.

Dysfunction of mitotic cell division at shoot apices triggered severe growth abortion in interspecific hybrids between tetraploid wheat and Aegilops tauschii.

Common wheat is an allohexaploid species, derived through endoreduplication of an interspecific triploid hybrid produced from a cross between cultivated tetraploid wheat and the wild diploid relative Aegilops tauschii. Hybrid incompatibilities, including hybrid necrosis, have been observed in triploid wheat hybrids. A limited number of A. tauschii accessions show hybrid lethality in triploid hybrids crossed with tetraploid wheat as a result of developmental arrest at the early seedling stage, which is termed severe growth abortion (SGA). Despite the potential severity of this condition, the genetic mechanisms underlying SGA are not well understood. Here, we conducted comparative analyses of gene expression profiles in crown tissues to characterize developmental arrest in triploid hybrids displaying SGA. A number of defense-related genes were highly up-regulated, whereas many transcription factor genes, such as the KNOTTED1-type homeobox gene, which function in shoot apical meristem (SAM) and leaf primordia, were down-regulated in the crown tissues of SGA plants. Transcript accumulation levels of cell cycle-related genes were also markedly reduced in SGA plants, and no histone H4-expressing cells were observed in the SAM of SGA hybrid plants. Our findings demonstrate that SGA shows unique features among other types of abnormal growth phenotypes, such as type II and III necrosis.

Diversification of three APETALA1/FRUITFULL-like genes in wheat.

The genomes of grass family species have three paralogs of APETALA1/FRUITFULL (AP1/FUL)-like genes (FUL1, FUL2 and FUL3) that are derived from the FUL lineage. In this study, we focus on the different roles of the wheat AP1/FUL-like genes, WFUL1 (identical to VRN1), WFUL2 and WFUL3, during the transition from vegetative to reproductive growth. Sequence analysis indicated that there was a high level of variability in the amino acid sequence of the C-domain among three WFUL genes. Expression analyses using the spring wheat cultivar Chinese Spring indicated that WFUL1/VRN1 was expressed in leaves as well as spike primordia of non-vernalized plants at the vegetative stage just before phase transition, while WFUL2 and WFUL3 were not expressed in leaves. This result indicates that WFUL1/VRN1 performs a distinct role in leaves before phase transition. In young spikes, WFUL1/VRN1 and WFUL3 were expressed in all developing Xoral organs, whereas WFUL2 expression was restricted in the Xoral organs to the lemma and palea. Furthermore, yeast two-hybrid and three-hybrid analyses revealed that WFUL2, but not WFUL1/VRN1 or WFUL3, interacted with class B and class E proteins. These results suggest that WFUL2 of wheat has class A functions in specifying the identities of Xoral meristems and outer Xoral organs (lemma and palea) through collaboration with class B and class E genes.

Autoimmune response and repression of mitotic cell division occur in inter-specific crosses between tetraploid wheat and Aegilops tauschii Coss. that show low temperature-induced hybrid necrosis.

Common wheat is an allohexaploid species originating from a naturally occurring inter-specific cross between tetraploid wheat and the diploid wild wheat Aegilops tauschii Coss. Artificial allopolyploidization can produce synthetic hexaploid wheat. However, synthetic triploid hybrids show four types of hybrid growth abnormalities: type II and III hybrid necrosis, hybrid chlorosis, and severe growth abortion. Of these hybrid abnormalities, type II necrosis is induced by low temperature. Under low temperature, elongation of stems and expansion of new leaves is repressed in type II necrosis lines, which later exhibit necrotic symptoms. Here, we characterize type II necrosis in detail. Comparative transcriptome analysis showed that a number of defense-related genes were highly up-regulated in seedling leaves that showed type II necrosis. Transmission electron microscopy revealed extensive cell death in the leaves under low-temperature conditions, accompanied by abundant generation of reactive oxygen species. In addition, down-regulation of cell cycle-related genes was observed in shoot apices of type II necrosis lines under low-temperature conditions. Quantitative RT-PCR and in situ hybridization showed repression of accumulation of histone H4 transcripts in the shoot apical meristem of type II necrosis lines. These results strongly suggest that an autoimmune response-like reaction and repression of cell division in the shoot apical meristem are associated with the abnormal growth phenotype in type II necrosis lines.

Duplicate polyphenol oxidase genes on barley chromosome 2H and their functional differentiation in the phenol reaction of spikes and grains.

Polyphenol oxidases (PPOs) are copper-containing metalloenzymes encoded in the nucleus and transported into the plastids. Reportedly, PPOs cause time-dependent discoloration (browning) of end-products of wheat and barley, which impairs their appearance quality. For this study, two barley PPO homologues were amplified using PCR with a primer pair designed in the copper binding domains of the wheat PPO genes. The full-lengths of the respective PPO genes were cloned using a BAC library, inverse-PCR, and 3'-RACE. Linkage analysis showed that the polymorphisms in PPO1 and PPO2 co-segregated with the phenol reaction phenotype of awns. Subsequent RT-PCR experiments showed that PPO1 was expressed in hulls and awns, and that PPO2 was expressed in the caryopses. Allelic variation of PPO1 and PPO2 was analysed in 51 barley accessions with the negative phenol reaction of awns. In PPO1, amino acid substitutions of five types affecting functionally important motif(s) or C-terminal region(s) were identified in 40 of the 51 accessions tested. In PPO2, only one mutant allele with a precocious stop codon resulting from an 8 bp insertion in the first exon was found in three of the 51 accessions tested. These observations demonstrate that PPO1 is the major determinant controlling the phenol reaction of awns. Comparisons of PPO1 single mutants and the PPO1PPO2 double mutant indicate that PPO2 controls the phenol reaction in the crease on the ventral side of caryopses. An insertion of a hAT-family transposon in the promoter region of PPO2 may be responsible for different expression patterns of the duplicate PPO genes in barley.

Class D and B(sister) MADS-box genes are associated with ectopic ovule formation in the pistil-like stamens of alloplasmic wheat (Triticum aestivum L.).

Homeotic transformation of stamens into pistil-like structures (pistillody) has been reported in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum L.) that have the cytoplasm of a related wild species, Aegilops crassa. An ectopic ovule differentiates in the pistil-like stamen in the alloplasmic wheat. The SEEDSTICK (STK)-like class D MADS-box gene, wheat STK (WSTK), was expressed in the primordia of ectopic ovules in the pistil-like stamens as well as in the true pistil, suggesting that ectopic ovule formation results from WSTK expression in the pistil-like stamens of alloplasmic wheat. The ectopic ovule is abnormal as it fails to form complete integuments. Based on the expression pattern of WSTK and B(sister) MADS-box gene, WBsis (wheat B ( sister )), we conclude that WSTK plays a role in determination of ovule identity in the pistil-like stamen, but complete ovule development fails due to aberrant expression of WBsis.

Heterochronic development of the floret meristem determines grain number per spikelet in diploid, tetraploid and hexaploid wheats.

BACKGROUND AND AIMS: The inflorescence of grass species such as wheat, rice and maize consists of a unique reproductive structure called the spikelet, which is comprised of one, a few, or several florets (individual flowers). When reproductive growth is initiated, the inflorescence meristem differentiates a spikelet meristem as a lateral branch; the spikelet meristem then produces a floret meristem as a lateral branch. Interestingly, in wheat, the number of fertile florets per spikelet is associated with ploidy level: one or two florets in diploid, two or three in tetraploid, and more than three in hexaploid wheats. The objective of this study was to identify the mechanisms that regulate the architecture of the inflorescence in wheat and its relationship to ploidy level. METHODS: The floral anatomy of diploid (Triticum monococcum), tetraploid (T. turgidum ssp. durum) and hexaploid (T. aestivum) wheat species were investigated by light and scanning electron microscopy to describe floret development and to clarify the timing of the initiation of the floret primordia. In situ hybridization analysis using Wknox1, a wheat knotted1 orthologue, was performed to determine the patterning of meristem formation in the inflorescence. KEY RESULTS: The recessive natural mutation of tetraploid (T. turgidum ssp. turgidum) wheat, branching head (bh), which produces branched inflorescences, was used to demonstrate the utility of Wknox1 as a molecular marker for meristematic tissue. Then an analysis of Wknox1 expression was performed in diploid, tetraploid and hexaploid wheats and heterochronic development of the floret meristems was found among these wheat species. CONCLUSIONS: It is shown that the difference in the number of floret primordia in diploid, tetraploid and hexaploid wheats is caused by the heterochronic initiation of floret meristem development from the spikelet meristem.

A genetic network of flowering-time genes in wheat leaves, in which an APETALA1/FRUITFULL-like gene, VRN1, is upstream of FLOWERING LOCUS T.

To elucidate the genetic mechanism of flowering in wheat, we performed expression, mutant and transgenic studies of flowering-time genes. A diurnal expression analysis revealed that a flowering activator VRN1, an APETALA1/FRUITFULL homolog in wheat, was expressed in a rhythmic manner in leaves under both long-day (LD) and short-day (SD) conditions. Under LD conditions, the upregulation of VRN1 during the light period was followed by the accumulation of FLOWERING LOCUS T (FT) transcripts. Furthermore, FT was not expressed in a maintained vegetative phase (mvp) mutant of einkorn wheat (Triticum monococcum), which has null alleles of VRN1, and never transits from the vegetative to the reproductive phase. These results suggest that VRN1 is upstream of FT and upregulates the FT expression under LD conditions. The overexpression of FT in a transgenic bread wheat (Triticum aestivum) caused extremely early heading with the upregulation of VRN1 and the downregulation of VRN2, a putative repressor gene of VRN1. These results suggest that in the transgenic plant, FT suppresses VRN2 expression, leading to an increase in VRN1 expression. Based on these results, we present a model for a genetic network of flowering-time genes in wheat leaves, in which VRN1 is upstream of FT with a positive feedback loop through VRN2. The mvp mutant has a null allele of VRN2, as well as of VRN1, because it was obtained from a spring einkorn wheat strain lacking VRN2. The fact that FT is not expressed in the mvp mutant supports the present model.

Identification of a protein kinase gene associated with pistillody, homeotic transformation of stamens into pistil-like structures, in alloplasmic wheat.

Homeotic transformation of stamens into pistil-like structures (called pistillody) has been reported in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) having the cytoplasm of a wild relative species, Aegilops crassa. Our previous studies indicated that pistillody is caused by alterations of the class B MADS-box gene expression pattern associated with mitochondrial gene(s) in the Ae. crassa cytoplasm. To elucidate the nuclear gene involved in the cross-talk between pistillody-related mitochondrial gene(s) and nuclear homeotic genes, we performed cDNA subtraction analysis using cDNAs derived from young spikes of a pistillody line and a normal line. As a result, we identified a protein kinase gene, WPPK1 (wheat pistillody-related protein kinase 1), which is upregulated in the young spikes of the pistillody line. RT-PCR analysis indicated that WPPK1 is strongly expressed in pistils and pistil-like stamens in the pistillody line, suggesting that it is involved in the formation of pistil-like stamens as well as pistils. The full-length cDNA sequence for WPPK1 showed high similarity with a flowering plant PVPK-1 protein kinase, and phylogenetic analysis indicated that it is a member of AGC group protein kinases. Furthermore, a phosphorylation assay indicated that it has protein kinase activity. In situ hybridization analysis revealed that WPPK1 is expressed in developing pistils and pistil-like stamens as well as in their primordia. These indicate that in the alloplasmic line, WPPK1 plays a role in formation and development of pistil-like stamens.

Genetic and epigenetic alteration among three homoeologous genes of a class E MADS box gene in hexaploid wheat.

Bread wheat (Triticum aestivum) is a hexaploid species with A, B, and D ancestral genomes. Most bread wheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the ancestral species. Here, we report that both genetic and epigenetic alterations have occurred in the homoeologs of a wheat class E MADS box gene. Two class E genes are identified in wheat, wheat SEPALLATA (WSEP) and wheat LEAFY HULL STERILE1 (WLHS1), which are homologs of Os MADS45 and Os MADS1 in rice (Oryza sativa), respectively. The three wheat homoeologs of WSEP showed similar genomic structures and expression profiles. By contrast, the three homoeologs of WLHS1 showed genetic and epigenetic alterations. The A genome WLHS1 homoeolog (WLHS1-A) had a structural alteration that contained a large novel sequence in place of the K domain sequence. A yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein had no apparent function. The B and D genome homoeologs, WLHS1-B and WLHS1-D, respectively, had an intact MADS box gene structure, but WLHS1-B was predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat. This is a situation where three homoeologs are differentially regulated by genetic and epigenetic mechanisms.

The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase, is caused by a deletion in the VRN1 gene.

The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase (mvp), was induced by nitrogen ion-beam treatment and was identified by its inability to transit from the vegetative to reproductive phase. In our previous study, we showed that WAP1 (wheat APETALA1) is a key gene in the regulatory pathway that controls phase transition from vegetative to reproductive growth in common wheat. WAP1 is an ortholog of the VRN1 gene that is responsible for vernalization insensitivity in einkorn wheat. The mvp mutation resulted from deletion of the VRN1 coding and promoter regions, demonstrating that WAP1/VRN1 is an indispensable gene for phase transition in wheat. Expression analysis of flowering-related genes in mvp plants indicated that wheat GIGANTIA (GI), CONSTANS (CO) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) genes either act upstream of or in a different pathway to WAP1/VRN1.

WFL, a wheat FLORICAULA/LEAFY ortholog, is associated with spikelet formation as lateral branch of the inflorescence meristem.

FLORICAULA (FLO) of Antirrhinum and LEAFY (LFY) of Arabidopsis encode plant-specific transcription factors, which are necessary and sufficient to specify floral meristem identity. We isolated WFL, a wheat FLO/LFY ortholog, and analyzed its expression pattern. RT-PCR analysis indicated that WFL is expressed predominantly in young spike. The WFL expression pattern during reproductive development was analyzed in more detail by using in situ hybridization technique. WFL transcripts were observed in all layers of the young spike excepting spikelet initiation sites as axillary meristem. In the double-ridge stage, WFL transcripts were localized in the lower ridge but were absent in the upper ridge, where spikelet meristem initiates. The WFL expression pattern indicated that WFL is associated with spikelet formation rather than floral meristem identity in wheat. As development of floret proceeds, the WFL transcripts were detectable in the developing palea, but not in other floral organs, suggesting that WFL may play a novel role in developing palea in the wheat floret.