Homeotic Transformation Of Stamens Research Articles

Comparative transcriptome analysis indicates conversion of stamens into pistil-like structures in male sterile wheat (Triticum aestivum L.) with Aegilops crassa cytoplasm

BackgroundAegilops crassa cytoplasm is an important source for investigating cytoplasmic male sterility (CMS). Moreover, the stamens of line C303A exhibit a high degree of pistillody, turning almost white. However, the molecular mechanism that underlies pistillody in C303A remains unclear. Therefore, to obtain a better understanding of pistillody in C303A, the phenotypic and cytological features of C303A were observed to identify the key stage for the homeotic transformation of stamens into pistil-like structures. Transcriptome profiles were determined for stamens using Illumina RNA sequencing.ResultsMorphological observations of the CMS wheat line with Aegilops crassa cytoplasm C303A showed that the pistils developed normally, but the stamens were ultimately aborted and they released no pollen when mature. According to paraffin section observations, the stamens began to transform into pistils or pistil-like structures in the binucleate stage (BNS). Therefore, the stamens were collected from line C303A and its maintainer 303B in the BNS for transcriptome sequencing. In total, 20,444 wheat genes were determined as differentially expressed in C303A and 303B stamens, with 10,283 upregulated and 10,161 downregulated genes. Gene Ontology enrichment analyses showed that most of the differentially expressed genes (DEGs) were annotated with GO terms comprising metabolic process, cell, cellular process, catalytic activity, and cell part. Analysis based on the Kyoto Encyclopedia of Genes and Genomes database showed that the enriched DEGs were mainly associated with energy metabolism. We also found several essential genes that may contribute to pistillody in C303A. These findings suggest that disrupted energy metabolism and reactive oxygen metabolism induce pistillody and eventually lead to abortion in C303A.ConclusionWe determined the complex transcriptome profiles for C303A stamens and demonstrated that disrupted energy metabolism and class B MADS-box genes are related to pistillody. These findings may facilitate future studies of the mechanistic response of the wheat stamen and pollen development in CMS.

Effect of temperature on expression of B- and C-class MADS-box genes in flower buds of sweet cherry

The occurrence of double fruits in sweet cherry (Prunus avium) is due to abnormal differentiation of pistil primordia in the previous growing season. Double pistil formation is caused by high temperature above 30°C during flower differentiation. Furthermore, extremely high temperature induces double pistil formation as well as homeotic transformation of stamens into pistil-like structures. Therefore, the B and C classes of homeotic genes, which control stamen and pistil formations, may be involved in these phenomena. In this study, we investigated the effect of temperature on the expression levels of B- and C-class MADS-box genes in flower buds. On July 20, when the buds had formed sepal and petal primordia, 'Satohnishiki' sweet cherry trees were brought into sunlit growth chambers with controlled temperatures at 25 and 35°C. Shortly after the onset of the treatment (3 and 10 days), spur buds were collected, and the expression levels of the APETALA3 (AP3)-like and PISTILLATA (PI)-like B-class genes and the AGAMOUS (AG)-like and SHATTERPROOF (SHP)-like C-class genes in the buds were analysed using real-time PCR. The transcriptional levels of PaTM6 (TM6 lineage of AP3) and PaAG at 35°C were much higher than those at 25°C. On the other hand, the high temperature considerably reduced the transcriptional levels of PaPI and PaSHP. We expected that increases in the expression level of C-class genes and decreases in that of B-class genes may be involved in double pistil formation and pistil-like stamens due to high temperature. However, only the changes in the expression levels of PaAG and PaPI genes were consistent with our expectations. Next, we plan to compare the expression levels of these genes among cultivars with different frequency of double pistils.

Alternative splicing of the AGAMOUS orthologous gene in double flower of Magnolia stellata (Magnoliaceae)

Magnolia stellata is a woody ornamental shrub with more petaloid tepals than related plants from family Magnoliaceae. Recent studies revealed that expression changes in an AGAMOUS (AG) orthologous gene could resulted in double flowers with increased numbers of petals. We isolated three transcripts encoding different isoforms of a single AG orthologous gene, MastAG, mastag_2 and mastag_3, from M. stellata. Sequence alignments and Southern blot analyses suggested that MastAG was a single-copy gene in M. stellata genomes, and that mastag_2 and mastag_3 were abnormally spliced isoforms of MastAG. An 144bp exon skipping in MastAG results in the truncated mastag_2 protein lacking the completely I domain and 18 aa of the K1 subdomain, whereas an 165bp exon skipping of MastAG produces a truncated mastag_3 protein lacking 6 aa of the K3 subdomain and the completely C terminal region. Expression analyses showed that three alternative splicing (AS) isoforms expressed only in developing stamens and carpels. Functional analyses revealed that MastAG could mimic the endogenous AG to specify carpel identity, but failed to regulate stamen development in an Arabidopsis ag-1 mutant. Moreover, the key domain or subdomain deletions represented by mastag_2 and mastag_3 resulted in loss of C-function. However, ectopic expression of mastag_2 in Arabidopsis produced flowers with sepals converted into carpeloid organs, but without petals and stamens, whereas ectopic expression of mastag_3 in Arabidopsis could mimic the flower phenotype of the ag mutant and produced double flowers with homeotic transformation of stamens into petals and carpels into another ag flower. Our results also suggest that mastag_3 holds some potential for biotechnical engineering to create multi-petal phenotypes in commercial ornamental cultivars.

Identification of differentially expressed genes in a pistillody common wheat mutant using an annealing control primer system.

HTS-1 is a new kind of pistillody wheat. All or parts of its stamen are transformed into pistils or pistil-like structures, and it has more seed sets per floret than normal wheat under normal cultivation conditions. To investigate the expression divergence in this mutant, an annealing control primer system was used to identify differentially expressed genes (DEGs) in the young spikelets. As a result, three DEGs, including HDB2, HGF2, and HCG4, were detected, with variable expression in HTS-1 and the control. After further confirmation using real-time reverse transcription polymerase chain reaction analysis, these genes were overexpressed in HTS-1 wheat. NGF2 was identified in the double ridge to floret differentiation stages; HDB2 and HCG4 were identified in the stage of pistil and stamen-differentiating. Therefore, we inferred that the homeotic transformation of stamens into pistil-like structures occurred during the early stage of stamen development. Sequence alignment analysis revealed that HDB2 encodes a putative protein of 189 amino acids, with high homology to the DEAD-box ATP-dependent RNA helicase, and HCG4 was identical to the Chinese spring wheat cDNA clone predicted protein according to GenBank. However, NGF2 was not found to have significant similarity to any reported proteins, suggesting it is a new functional gene in wheat. The results suggest that HDB2, HCG4, and HGF2 are minor genes contributing to pistillody trait formation in HTS-1.

Homeotic Genes and the ABCDE Model for Floral Organ Formation in Wheat.

Floral organ formation has been the subject of intensive study for over 20 years, particularly in the model dicot species Arabidopsis thaliana. These studies have led to the establishment of a general model for the development of floral organs in higher plants, the so-called ABCDE model, in which floral whorl-specific combinations of class A, B, C, D, or E genes specify floral organ identity. In Arabidopsis, class A, B, C, D, E genes encode MADS-box transcription factors except for the class A gene APETALA2. Mutation of these genes induces floral organ homeosis. In this review, I focus on the roles of these homeotic genes in bread wheat (Triticum aestivum), particularly with respect to the ABCDE model. Pistillody, the homeotic transformation of stamens into pistil-like structures, occurs in cytoplasmic substitution (alloplasmic) wheat lines that have the cytoplasm of the related wild species Aegilops crassa. This phenomenon is a valuable tool for analysis of the wheat ABCDE model. Using an alloplasmic line, the wheat ortholog of DROOPING LEAF (TaDL), a member of the YABBY gene family, has been shown to regulate pistil specification. Here, I describe the current understanding of the ABCDE model for floral organ formation in 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.

Class D and Bsister 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.

Altered expression of wheat AINTEGUMENTA homolog, WANT-1, in pistil and pistil-like transformed stamen of an alloplasmic line with Aegilops crassa cytoplasm

Homeotic transformation of stamens into pistil-like structures, called pistillody, has been reported in some alloplasmic common wheat lines with Aegilops crassa cytoplasm. An alloplasmic line of Chinese Spring ditelosomic 7BS (CSdt7BS) with Ae. crassa cytoplasm lacking the long arm of the chromosome 7B shows pistillody, and the pistils and transformed stamens are sterile due to abnormal ovule development. To elucidate the molecular mechanism of the ovule abnormality, we compared the expression profiles of floral organs between euplasmic and alloplasmic CSdt7BS lines. Two differential display methods of mRNA profiling demonstrated that Ae. crassa cytoplasm largely affects nuclear gene expression profiles of common wheat. Of the differentially expressed genes, a wheat AINTEGUMENTA (ANT) homolog, WANT-1, was preferentially expressed in pistils but not in stamens, and accumulation of the transcript was limited to ovule primordia at the floral organ development stage. In alloplasmic wheat, WANT-1 expression was patchy and weak at the ovule-development stages. On the other hand, no significant difference in gene expression patterns of wheat AGAMOUS (AG) homologs (WAG-1 and WAG-2) was observed between fertile and sterile pistils. These results indicated that alteration of gene expression after initiation of ovule primordia results in abnormal ovule development, and that the aberrant ovule formation is at least partly associated with the weak expression of WANT-1 around ovule primordia in alloplasmic wheat with Ae. crassa cytoplasm.

Phenotypic and genetic characterization of the pistillate mutation in tomato

Many floral phenotypes have been described in decades of tomato genetics, but for very few of them the underlying genes have been identified so far. Because the increasing availability of genome sequence data will facilitate forward genetics in tomato, novel descriptive and map information will help the attribution of genes to phenotypes. In this contribution, we present our work on pistillate (pi), a genotype that directly recalls mutations affecting class B MADS-box genes, but that has not been further characterized after the first description. Plants homozygous for the pi allele appear with Mendelian proportions and, compared to wild-type, show delayed flowering, a frequently modified sympodial segment, higher occurrence of compound inflorescences, and reversion of the floral meristem to vegetative identity. In pi mutant flowers, the most striking aberration is the homeotic transformation of stamens into carpels. Ultrastructural analysis also reveals more or less subtle sepaloid features in the three inner floral whorls, mainly based on the presence, distribution and amount of glandular and non glandular trichomes. In the ovary, a 'flower within flower' phenotype was seldom observed; in one instance such phenotype was coupled with the setting of a parthenocarpic fruit, that reiterated the differentiation of a new flower. Mapping experiments positioned PI on the distal end of the long arm of chromosome 3. This position was not compatible with any class B or E MADS box gene; differently, the PI genetic window contained the FALSIFLORA (FA) gene, the tomato orthologue of LEAFY (LFY). The pi defects in flowering time and inflorescence development are in agreement with a direct involvement of the floral meristem identity gene. The class B- and E-like phenotypes shown by pi mutant plants are likely an indirect consequence because FA, as LFY, is reported as a positive regulator of homeotic MADS-box genes. Because fa mutant plants do not form complete flowers, the pi mutation deserves a particular interest, producing four-whorled, although modified, flowers useful to study the functional linkage between flower induction and flower organ identity specification.

Orf260cra, a Novel Mitochondrial Gene, is Associated with the Homeotic Transformation of Stamens into Pistil-Like Structures (Pistillody) in Alloplasmic Wheat

Homeotic transformation of stamens into pistil-like structures (pistillody) can occur in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) that have the cytoplasm of the related species, Aegilops crassa. Previously we showed that pistillody results from altered patterns of expression of class B MADS-box genes mediated by mitochondrial gene(s) in the Ae. crassa cytoplasm. The wheat cultivar Chinese Spring does not show pistillody when Ae. crassa cytoplasm is introduced. The absence of an effect is due to a single dominant gene (designated Rfd1) located on the long arm of chromosome 7B. To identify the mitochondrial gene involved in pistillody induction, we performed a subtraction analysis using cDNAs derived from young spikes of a pistillody line and a normal line. We found that mitochondrial cDNA clone R04 was abundant in the young spikes of the pistillody line but was down-regulated in the normal line that carried nuclear Rfd1. Sequencing of the full-length cDNA corresponding to clone R04 showed that two genes were present, cox I (cytochrome c oxidase subunit I) and orf260(cra). orf260(cra) shows high sequence similarity to orf256, the T. timopheevii mitochondrial gene responsible for cytoplasmic male sterility (CMS). orf260(cra) was also present in the cytoplasms of Ae. juvenalis and Ae. vavilovii, which induce pistillody, but not in the cytoplasms of other species not associated with pistillody. Furthermore, Western blot analysis revealed that the ORF260cra protein was more abundant in the pistillody line than in the normal line. We suggest therefore that orf260(cra) is associated with pistillody induction.

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.

細胞質置換コムギ系統で誘発する pistillody（雄ずいの雌ずい化）には花器官決定遺伝子の発現パターンの変化が関与する

細胞質置換コムギ系統で誘発する pistillody（雄ずいの雌ずい化）には花器官決定遺伝子の発現パターンの変化が関与する

Preferential expression of a HLP homolog encoding a mitochondrial L14 ribosomal protein in stamens of common wheat

Interaction between nucleus and cytoplasm has essential roles in plant development, including that of floral organs. We isolated a wheat homolog Whlp of Arabidopsis HUELLENLOS PARALOG ( HLP) gene encoding a mitochondrial (mt) ribosomal protein L14. Transient expression analysis using the green fluorescent protein (GFP) fusion protein showed that 50 amino residues located on the N-terminal of the wheat HLP homolog (WHLP) protein acted as a mt-targeting signal (MTS). Expression patterns of the Whlp gene were compared among floral organs of alloplasmic lines of wheat, in which intrinsic cytoplasms were replaced by the cytoplasm of a wild relative Aegilops crassa. In these alloplasmic lines, pistillody (homeotic transformation of stamens into pistil-like organs) is induced by the alien cytoplasm in the absence of nuclear restorer genes. The Whlp transcripts preferentially accumulated in stamens compared with pistils, leaves, and roots. The expression level of Whlp in the pistillate stamens of the alloplasmic lines was similar to that in genuine pistils of both euplasmic lines and fertile alloplasmic lines. The result suggested that the elevated expression of the Whlp gene plays a role in aiding the development of male reproductive organ but not in the determination of its whorl identity. A comparable expression pattern was observed in another nuclear-encoded mt ribosomal protein gene but not in a mt-encoded gene. The different expression patterns of different mt ribosomal protein genes suggest that the abundance of mt ribosomal proteins is differentially regulated in the organ/tissue development in wheat.

Pistillody is caused by alterations to the class-B MADS-box gene expression pattern in alloplasmic wheats.

Class-B floral homeotic genes are involved in specifying petal and stamen identity during flower development in plant species. Homeotic transformation of stamens into pistil-like structures (called pistillody) has been observed in alloplasmic lines of bread wheat ( Triticum aestivum L.) having the cytoplasm of a wild relative species, Aegilops crassa Boiss. To obtain information about the molecular mechanism underlying pistillody induction, we isolated two PISTILLATA ( PI)-type class-B MADS-box genes, WPI1 ( wheat PI STILLATA # 1) and WPI2, from wheat. Phylogenetic reconstruction indicated that WPI1 is orthologous to OsMADS4 and that WPI2 is probably an ortholog of OsMADS2. Both OsMADS4 and OsMADS2 genes were suggested to be PI orthologs in rice, and the function of OsMADS4 as a class-B gene was proven by the transgenic study. An in situ hybridization study demonstrated that the WPI1 gene is expressed in primordia of lodicules and stamens in developing florets in wheat. In the alloplasmic wheat line exhibiting pistillody, the WPI1 transcripts were not detected in the primordia of pistil-like stamens, whereas WPI1 was expressed in the lodicules. The wheat APETALA3 ( AP3)-type class-B MADS-box gene WAP3 ( wheat AP3) /TaMADS#82 showed an expression pattern similar to that of WPI1. These results suggest that pistillody in alloplasmic wheats is caused by alterations to the expression pattern of class-B MADS-box genes.

WAG, a wheat AGAMOUS homolog, is associated with development of pistil-like stamens in alloplasmic wheats

Pistillody, homeotic transformation of stamens into pistil-like structures, has been reported in cytoplasmic substitution (alloplasmic) lines of common wheat (Triticum aestivum) with Aegilops crassa cytoplasm. The induction of pistillody is suppressed by the Rfd1 gene detected on the long arm of chromosome 7B in wheat cultivar 'Chinese Spring' (CS). Because of the absence of Rfd1, the alloplasmic line CS ditelosomic 7BS [(cr)-CSdt7BS] lacking the long arm of chromosome 7B exhibits pistillody in all florets, whereas the euplasmic CS ditelosomic 7BS (CSdt7BS) with normal cytoplasm forms normal stamens. To study the molecular mechanism of pistillody caused by nuclear-cytoplasm interaction in alloplasmic wheat, we cloned and characterized a wheat AGAMOUS homolog, WAG. The WAG gene copy number in the wheat genome was estimated at three, located on the homoeologous group 1 chromosomes. Northern blot analysis with a normal wheat line revealed that the transcription level of WAG was lower in young spikes and increased during spike development. The highest expression was observed in spikes at the booting to heading stage. The 1.1 kb WAG transcript accumulated in both the reproductive (pistil and stamen) and non-reproductive (palea and lemma) portions of spikes at the heading stage, whereas an extra transcript of different molecular size (1.3 kb) was observed in pistil-like stamens of line (cr)-CSdt7BS as well as in pistils of CSdt7BS and (cr)-CSdt7BS. These results suggest that the product of the 1.3 kb WAG transcript is involved in pistil development and is associated with pistillody caused by a nuclear-cytoplasm interaction in alloplasmic wheat.

Pistillody, homeotic transformation of stamens into pistil-like structures, caused by nuclear-cytoplasm interaction in wheat.

Homeotic transformation of stamens into pistil-like structures (pistillody) has been observed in a cytoplasmic substitution (alloplasmic) line of wheat (Triticum aestivum L.) cv. Norin 26, which has the cytoplasm of a wild relative species, Aegilops crassa L. On the other hand, an alloplasmic line of wheat cv. Chinese Spring (CS) with Ae. crassa cytoplasm has normal flowers. This is due to the presence in the CS nucleus of a fertility-restoring gene, Rfd1. Deletion mapping analysis revealed that Rfd1 is located on the middle part of the long arm of chromosome 7B. To investigate the function of the Rfd1 gene by a loss-of-function strategy, we produced alloplasmic lines of CS ditelosomic 7BS [(cr)-CSdt7BS] and CS monotelodisomic 7BS [(cr)-CSmd7BS] with the Ae. crassa cytoplasm, and characterized their phenotypes. The line (cr)-CSdt7BS without Rfd1 exhibited pistillody in all florets, and also female sterility. Scanning electron microscopy of the young spikes revealed that the pistillody was induced at an early stage of stamen development. The pistillate stamens often developed incomplete ovule-like structures with integuments instead of tapetum and pollen grains. It is possible that MADS box genes are associated with the induction of pistillody, because the expression of wheat APETALA3 homologue (WAP3) was reduced in the young spikes of (cr)-CSdt7BS. In addition, a histological study indicated that the female sterility in (cr)-CSdt7BS is due to the abnormality of the ovule, which fails to form an inner epidermis and integuments in the chalaza region. The line (cr)-CSmd7BS, hemizygous for Rfd1, showed partial pistillody (51%) and restored female fertility up to 72%. These results suggest that the induction of both pistillody and ovule deficiency caused by the Ae. crassa cytoplasm is inhibited by the Rfd1 gene in a dose-dependent manner.