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.     

Aneuploid and alloplasmic lines as tools for the study of nuclear and cytoplasmic control of culture ability and regeneration of scutellar calli from common wheat

Summary Twenty four B genome aneuploid lines (di-telosomics, nullisomic-tetrasomics and tetrasomics) of Triticum aestivum cv Chinese Spring were used in an analysis of the culture ability and regeneration capability of scutellar calli. Several correlations were found between the presence or absence of specific chromosomes and chromosomal arms of the B genome of common wheat and the growth and differentiation capabilities of these calli. The rate of callus growth decreased only when the long arm of chromosome 6B was not present. The absence of chromosomes 3B and 7B did not result in an apparent change in morphogenetic capability, while the absence of other B genome chromosomes was significantly correlated to changes in the frequency of calli that regenerated plants. The presence of the short arm of chromosome 1B was negatively correlated with regeneration, whereas its long arm is probably required to counteract this effect and to maintain the normal ratio of regeneration. The presence of the chromosomal arm 2BS seemed to be essential for differentiation to shoots. In the absence of the short arms of chromosomes 4B and 5B, the rate of regeneration was slightly reduced. In the absence of the long arm of chromosome 6B there was a marked reduction of the ability of scutellar calli to regenerate plants. The use of additional aneuploid lines belonging to homoeologous group 6 revealed that only calli derived from lines having chromosome 6D in their complement regenerated plants similarly to the euploid control. Culture ability and regeneration capability were also analysed with alloplasmic lines of T. aestivum cv Chris. The lines were derived from five species, representing plasma-types of different phylogenetic distances from plasma-type B of T. aestivum. The results showed that when the endogenous cytoplasm (B-type) was exchanged with T. timopheevii cytoplasm (G-type) there was a significant increase in the regeneration of shoots from the scutellar calli.     

Morphological alterations by ectopic expression of the rice OsMADS4 gene in tobacco plants

OsMADS4, a rice MADS-box gene, is a member of the GLO/PI family that specifies the identity of petals and stamens in combination with other MADS-box genes. We report here the ectopic expression of OsMADS4 fused to the CaMV 35S promoter in tobacco plants. Transgenic plants carrying the CaMV 35S promoter::OsMADS4 construct generated mutant flowers with a mosaic carpel, in which the tissue around the nectary was elongated and the styles reduced. The fruits were distorted, but viable seeds did develop. These phenotypes mimicked those of transgenic tobacco plants that ectopically express Antirrhinum GLO. However, unlike GLO, OsMADS4 did not cause any homeotic change in the first whorl of the transgenic flowers. These results suggest that the functional role of OsMADS4 in the outer whorls has diverged from that of its dicot counterparts.     

NADP-dependent aromatic alcohol dehydrogenase in polyploid wheats and their diploid relatives. On the origin and phylogeny of polyploid wheats

Summary The three major isoenzymes of the NADP-dependent aromatic alcohol dehydrogenase (ADH-B), distinguished in polyploid wheats by means of polyacrylamide gel electrophoresis, are shown to be coded by homoeoalleles of the locus Adh-2 on short arms of chromosomes of the fifth homoeologous group. Essentially codominant expression of the Adh-2 homoeolleles of composite genomes was observed in young seedlings of hexaploid wheats (T. aestivum s.l.) and tetraploid wheats of the emmer group (T. turgidum s.l.), whereas only the isoenzyme characteristic of the A genome is present in the seedlings of the timopheevii-group tetraploids (T. timopheevii s.str. and T. araraticum).The slowest-moving B³ isoenzyme of polyploid wheats, coded by the homoeoallele of the B genome, is characteristic of the diploid species Aegilops speltoides S.l., including both its awned and awnless forms, but was not encountered in Ae. bicornis, Ae. sharonensis and Ae. longissima. The last two diploids, as well as Ae. tauschii, Ae. caudata, Triticum monococcum s.str., T. boeoticum s.l. (incl. T. thaoudar) and T. urartu all shared a common isoenzyme coinciding electrophoretically with the band B² controlled by the A and D genome homoeoalleles in polyploid wheats. Ae. bicomis is characterized by the slowest isoenzyme, B⁴, not found in wheats and in the other diploid Aegilops species studied.Two electrophoretic variants of ADH-B, B¹ and B², considered to be alloenzymes of the A genome homoeoallele, were observed in T. dicoccoides, T. dicoccon, T. turgidum. s.str. and T. spelta, whereas B² was characteristic of T. timopheevii s.l. and only B¹ was found in the remaining taxa of polyploid wheats. The isoenzyme B¹, not encountered among diploid species, is considered to be a mutational derivative which arose on the tetraploid level from its more ancestral form B² characteristic of diploid wheats.The implication of the ADH-B isoenzyme data to the problems of wheat phylogeny and gene evolution is discussed.     

Electrophoretic survey of seedling esterases in wheats in relation to their phylogeny

Summary Evolutionary and ontogenetic variation of six seedling esterases of independent genetic control is studied in polyploid wheats and their diploid relatives by means of polyacrylamide gel electrophoresis. Four of them are shown to be controlled by homoeoallelic genes in chromosomes of third, sixth and seventh homoeologous groups.The isoesterase electrophoretic data are considered supporting a monophyletic origin of both the primitive tetraploid and the primitive hexaploid wheat from which contemporary taxa of polyploid wheats have emerged polyphyletically and polytopically through recurrent introgressive hybridization and accumulation of mutations. Ancestral diploids belonging or closely related to Triticum boeoticum, T. urartu, Aegilops speltoides and Ae. tauschii ssp. strangulata are genetically the most suitable genome donors of polyploid wheats. Diploids of the Emarginata subsection of the section Sitopsis, Aegilops longissima s.str., Ae. sharonensis, Ae. searsii and Ae. bicornis, are unsuitable for the role of the wheat B genome donors, being all fixed for the esterase B and D electromorphs different from those of tetraploid wheats.     

蕙兰CyfaSTK基因的克隆与表达分析

采用同源克隆的方法,从蕙兰（Cymbidium faberi Rolfe）花芽中克隆获得CyfaSTK基因的cDNA序列,并对其进行生物信息学分析及基因表达分析。结果显示,该基因全长843 bp,其中开放阅读框（ORF）长705 bp,共编码234个氨基酸和1个终止密码子。同源蛋白序列比对及分子系统发育分析结果表明,CyfaSTK蛋白属于D类MADS-box转录因子STK-like进化系,含有MADS、I、K和C等4个结构域,其C末端转录激活区含有2个保守的基元：AG motifⅠ和AG motifⅡ,此外,还具有一个在天门冬目植物中相对保守的基元MD motif。基因表达的组织特异性分析结果显示：蕙兰CyfaSTK基因在花萼、花瓣、唇瓣、药帽、子房中均有表达,但在叶片中不表达,其中在子房中的表达量与其他组织相比,差异达到极显著水平;CyfaSTK在花芽经过休眠后的萌动期表达量最高,且在开花当天该基因表达量有上升趋势。研究结果表明CyfaSTK基因不仅参与调控蕙兰花器官的发育过程,且对子房及合蕊柱的正常发育具有重要作用。     

Expression of AODEF,a B-functional MADS-box gene,in stamens and inner tepals of the dioecious species Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is a dioecious species with male and female flowers on separate unisexual individuals. Since B- and C-functional MADS-box genes specify male and female reproductive organs, it is important to characterize these genes to clarify the mechanism of sex determination in monoecious and dioecious species. In this study, we isolated and characterized AODEF gene, a B-functional gene in the development of male and female flowers of A. officinalis. Southern hybridization identified a single copy of AODEF gene in asparagus genome. Northern blot analysis showed that this gene was specifically expressed in flower buds and not in vegetative tissues. In situ hybridization showed that during early hermaphrodite stages, AODEFgene was expressed in the inner tepal and stamen whorls (whorls 2 and 3, respectively), but not in the outer tepals (whorl 1), in both male and female flowers. In late unisexual developmental stages, the expression of AODEF gene was still detected in the inner tepals and stamens of male flowers, but the expression was reduced in whorls 2 and 3 of female flowers. Our results suggest that AODEF gene is probably not involved in tepal development in asparagus and that the expression of AODEF gene is probably controlled directly or indirectly by sex determination gene in the late developmental stages.     

NAD-dependent aromatic alcohol dehydrogenase in wheats (Triticum L.) and goatgrasses (Aegilops L.): evolutionary genetics

Summary Evolutionary electrophoretic variation of a NAD-specific aromatic alcohol dehydrogenase, AADH-E, in wheat and goatgrass species is described and discussed in comparison with a NAD-specific alcohol dehydrogenase (ADH-A) and a NADP-dependent AADH-B studied previously. Cultivated tetraploid emmer wheats (T. turgidum s. l.) and hexaploid bread wheats (T. aestivum s. l.) are all fixed for a heterozygous triplet, E^0.58/E^0.64. The slowest isoenzyme, E^0.58, is controlled by a homoeoallelic gene on the chromosome arm 6AL of T. aestivum cv. Chinese Spring and is inherent in all diploid wheats, T. monococcum s. Str., T. boeoticum s. l. and T. urartu. The fastest isoenzyme, E^0.64, is presumably controlled by the B- and D-genome homoeoalleles of the bread wheat and is the commonest alloenzyme of diploid goat-grasses, including Ae. speltaides and Ae. tauschii. The tetraploid T. timopheevii s. str. has a particular heterozygous triplet E^0.56/E^0.71, whereas the hexaploid T. zhukovskyi exhibited polymorphism with electromorphs characteristic of T. timopheevii and T. monococcum. Wild tetraploid wheats, T. dicoccoides and T. araraticum, showed partially homologous intraspecific variation of AADH-E with heterozygous triplets E^0.58/E^0.64 (the commonest), E^0.58/E^0.71, E^0.45/E^0.58, E^0.48/E^0.58 and E^0.56/E^0.58 recorded. Polyploid goatgrasses of the D-genome group, excepting Ae. cylindrica, are fixed for the common triplet E^0.58/E^0.64. Ae. cylindrica and polyploid goatgrasses of the C^u-genome group, excepting Ae. kotschyi, are homozygous for E^0.64. Ae. kotschyi is exceptional, showing fixed heterozygosity for both AADH-E and ADH-A with unique triplets E^0.56/E^0.64 and A^0.49/A^0.56.     

Role of D-genome chromosomes in photosynthesis expression in wheats

Summary The role of D-genome chromosomes in the expression of net photosynthesis in wheats was analysed with the nullitetrasomic and ditelosomic lines of the bread wheat cultivar Chinese Spring. The two arms of chromosome 3 D and the short arm of chromosome 6 D control major mechanisms of photosynthesis. The effect of chromosome 6 D can be thoroughly compensated by that of its homoeologues of genomes A or B, contrary to what can be observed for chromosome 3 D. Chromosome 7 D is responsible for the low photosynthesis of flag leaves developed under high irradiances in genotypes possessing the D-genome, as the likely result of ontogeny or of a loss in adaptability to irradiance.     

Distinct MADS-box gene expression patterns in the reproductive cones of the gymnosperm<Emphasis Type="Italic"> Gnetum gnemon</Emphasis>

Expression patterns from in situ hybridization of four MADS-box genes (GGM7, GGM9, GGM11, and GGM15) from the gymnosperm species Gnetum gnemon are presented. Together with previously published data about putative orthologs of floral homeotic genes from G. gnemon (GGM2, GGM3, GGM13), we describe seven temporally and spatially distinct expression patterns in male, female or both types of reproductive units which very likely reflect the diversity of MADS-box gene function in gymnosperm cones. There is evidence that some aspects of the observed differential expression have been conserved since the last common ancestor of extant angiosperms and gymnosperms about 300 million years ago.Edited by R.J. Sommer     

向日葵MADS-Box基因HAM23-like克隆和表达分析

为了解MADS-box基因在向日葵(Helianthus annuus)花发育过程中的作用,采用RT-PCR技术克隆了1个MADS-box基因新成员HAM23-like,开放阅读框为831bp,编码276个氨基酸,相对分子量为30.52k D,理论等电点为9.42。系统发育分析表明,HAM23-like与拟南芥的AGL18聚于同一分支,具有较近的亲缘关系。qRT-PCR分析表明,HAM23-like基因在花和成熟果实(籽粒饱满期)中的表达量较高;HAM23-like在开花当天的雄蕊中的表达量最高;随着花的发育,HAM 23-like表达量逐渐升高,在开花后5 d (果实形成早期)达到最高表达水平。因此,推断HAM23-like基因可能与向日葵花器官后期发育和瘦果早期发育相关。     

Species relationships and genetic variation in the diploid wheats (Triticum,Aegilops) as revealed by starch gel electrophoresis

Twenty enzyme loci were examined in the diploid species ofTriticum andAegilops for allelic variation by starch gel electrophoresis. SectionSitopsis, including the five species,Ae. speltoides, Ae. lingissima, Ae. sharonensis, Ae. bicornis andAe. searsii form a close subgroup withAe. speltoides slightly removed from the others.T. monococcum s. lat., was found to be closest to the species of theSitopsis group.Ae. comosa, Ae. umbellulata andAe. uniaristata form a second subgroup withAe. caudata most closely related to these species.Ae. squarrosa appears almost equally related to all of the species, showing no special affinity for any one species group. Nineteen out of twenty loci examined were polymorphic with a mean of 6.7 alleles per locus. Species could be, for most loci, characterized by the presence of predominant alleles. A conspicious genetic characteristic ofTriticum-Aegilops is the sharing of these predominant alleles between species. Within species variation is characterized by a diffuse distribution of secondary alleles.     

The pleiotropic effects induced by therolC gene in transgenic plants are caused by expression restricted to protophloem and companion cells

Expression of therolC gene fromAgrobacterium rhizogenes causes morphological and developmental alterations in transgenic plants. The histological alterations underlying the macroscopic changes and the cellular localization of the site of expression of therolC gene have shown that: (i) the expression of therolC gene is developmentally regulated, (ii) in vegetative transgenic plants, the expression of therolC gene under the control of its own promoter is restricted to companion and protophloem cells, (iii) the site of action of the product(s) of the activity of the rolC enzyme is distinct from its site of expression, (iv) precise localization of the rolC peptide has been achieved by immunocytochemistry but not by the histochemical GUS assay. These results imply that the sites of action and expression of therolC gene in trangenic plants are physically separated. Thus the product(s) of the activity of the rolC enzyme must be a factor capable of being transported. Current models forrolC gene action are discussed taking into account the reported results.     

<Emphasis Type="Italic">AVAG2</Emphasis> is a putative D-class gene from an ornamental asparagus

Members of the AGAMOUS (AG) family of MADS-box genes play important roles in regulating the development of reproductive organs in flowering plants. To elucidate the molecular mechanisms of floral development in Asparagus virgatus, we isolated and characterized an Asparagus AG-homologue, AVAG2. AVAG2 contains an open reading frame that encodes a deduced protein with 234 amino acid residues. Phylogenetic analysis indicated that AVAG2 belongs to the D-lineage of the AG gene family. AVAG2 mRNA was detected in the flower, but not in vegetative organs. Moreover, in in situ hybridization experiments, AVAG2 signals were observed in the stamens and carpels during early flower development, and appeared in the ovule only at later developmental stages. This suggests that the AVAG2 gene is involved in ovule formation. Thus, our expression data support the phylogenetic analysis indicating that AVAG2 belongs to the D-class gene family.