Homeotic Conversion Research Articles

B-class floral homeotic gene MapoAPETALA3 may play an important role in the origin and formation of multi-tepals in Magnolia polytepala

In angiosperms, floral architecture diversity reflects its significance in exploring plant evolution. Magnolia polytepala, an endemic and ancient species in China, possesses a unique multi-tepal trait. Notably, the origin and formation of these multi-tepals are poorly understood. In this study, we investigated the origin and formation of multi-tepals from the inner floral whorl and elucidated the underlying molecular regulatory mechanisms by combining phenotypic analysis, sequencing, and molecular experiments. We found that the multi-tepals exhibited morpho-anatomical characteristics similar to normal tepals but differed from petaloid and normal stamens. The temporal dynamics of a large number of differentially expressed genes (DEGs) involved in multiple signaling (transduction) pathways contributed to multi-tepal primordia initiation during early floral differentiation. In particular, the dynamic expression of MpWOX4, MpCLE41, MpULT1, and MpKN1 might be responsible for floral meristem activation and maintenance, while MpTGA1 and MpEJ2 potentially regulated floral organ initiation. Floral homeotic genes, such as MapoAP3, contributed to subsequent organ identity specialization. We further isolated a nucleus-localized APETALA3 homolog from M. polytepala, terming it the MapoAPETALA3 (MapoAP3) gene, which was expressed in almost all vegetative and reproductive tissues. Ectopically expressing MapoAP3 in Arabidopsis resulted in altered phenotypes of rosette leaves, inflorescences, and florets, particularly generating extra petals instead of undergoing homeotic organ conversion. This discovery revealed an additional function of MapoAP3 in regulating organ initiation in addition to its conserved B-function in floral architecture plasticity. In summary, the multi-tepals of M. polytepala originated from the early tepal primordia initiation event rather than stamen petalody. The formation of the multi-tepal trait was attributed to the coordinated regulation of several vital DEGs, with the MapoAP3 gene playing an important role. These results provide additional insight into the regulation underlying the floral architecture formation in ancient Magnolia species and suggest that manipulating the MapoAP3 gene may hold promising potential for genetic breeding in ornamental plants.

Arabidopsis TCP4 transcription factor inhibits high temperature-induced homeotic conversion of ovules

Abnormal high temperature (HT) caused by global warming threatens plant survival and food security, but the effects of HT on plant organ identity are elusive. Here, we show that Class II TEOSINTE BRANCHED 1/CYCLOIDEA/ PCF (TCP) transcription factors redundantly protect ovule identity under HT. The duodecuple tcp2/3/4/5/10/13/17/24/1/12/18/16 (tcpDUO) mutant displays HT-induced ovule conversion into carpelloid structures. Expression of TCP4 in tcpDUO complements the ovule identity conversion. TCP4 interacts with AGAMOUS (AG), SEPALLATA3 (SEP3), and the homeodomain transcription factor BELL1 (BEL1) to strengthen the association of BEL1 with AG-SEP3. The tcpDUO mutant synergistically interacts with bel1 and the ovule identity gene seedstick (STK) mutant stk in tcpDUO bel1 and tcpDUO stk. Our findings reveal the critical roles of Class II TCPs in maintaining ovule identity under HT and shed light on the molecular mechanisms by which ovule identity is determined by the integration of internal factors and environmental temperature.

HvSL1 and HvMADS16 promote stamen identity to restrict multiple ovary formation in barley.

Correct floral development is the result of a sophisticated balance of molecular cues. Floral mutants provide insight into the main genetic determinants that integrate these cues, as well as providing opportunities to assess functional variation across species. In this study, we characterize the barley (Hordeum vulgare) multiovary mutants mov2.g and mov1, and propose causative gene sequences: a C2H2 zinc-finger gene HvSL1 and a B-class gene HvMADS16, respectively. In the absence of HvSL1, florets lack stamens but exhibit functional supernumerary carpels, resulting in multiple grains per floret. Deletion of HvMADS16 in mov1 causes homeotic conversion of lodicules and stamens into bract-like organs and carpels that contain non-functional ovules. Based on developmental, genetic, and molecular data, we propose a model by which stamen specification in barley is defined by HvSL1 acting upstream of HvMADS16. The present work identifies strong conservation of stamen formation pathways with other cereals, but also reveals intriguing species-specific differences. The findings lay the foundation for a better understanding of floral architecture in Triticeae, a key target for crop improvement.

Roles and evolution of four LEAFY homologs in floral patterning and leaf development in woodland strawberry.

The plant-specific transcription factor LEAFY (LFY), generally maintained as a single-copy gene in most angiosperm species, plays critical roles in flower development. The woodland strawberry (Fragaria vesca) possesses four LFY homologs in the genome; however, their respective functions and evolution remain unknown. Here, we identified and validated that mutations in one of the four LFY homologs, FveLFYa, cause homeotic conversion of floral organs and reiterative outgrowth of ectopic flowers. In contrast to FveLFYa, FveLFYb/c/d appear dispensable under normal growth conditions, as fvelfyc mutants are indistinguishable from wild type and FveLFYb and FveLFYd are barely expressed. Transgenic analysis and yeast one-hybrid assay showed that FveLFYa and FveLFYb, but not FveLFYc and FveLFYd, are functionally conserved with AtLFY in Arabidopsis (Arabidopsis thaliana). Unexpectedly, LFY-binding site prediction and yeast one-hybrid assay revealed that the transcriptional links between LFY and the APETALA1 (AP1) promoter/the large AGAMOUS (AG) intron are missing in F. vesca, which is due to the loss of LFY-binding sites. The data indicate that mutations in cis-regulatory elements could contribute to LFY evolution. Moreover, we showed that FveLFYa is involved in leaf development, as approximately 30% of mature leaves have smaller or fewer leaflets in fvelfya. Phylogenetic analysis indicated that LFY homologs in Fragaria species may arise from recent duplication events in their common ancestor and are undergoing convergent gene loss. Together, these results provide insight into the role of LFY in flower and leaf development in strawberry and have important implications for the evolution of LFY.

Diverse function of the PISTILLATA, APETALA 3, and AGAMOUS-like MADS-box genes involved in the floral development in Alpinia hainanensis (Zingiberaceae)

Zingiberaceae is the vital clue and key node in the decreased process of fertile stamens in Zingiberales, helping to understand the evolution of the ginger families. This study focuses on Alpinia hainanensis to investigate the function of B- and C-class MADS-box genes in floral development. The introns size of two B-class genes AhPI and AhAP3, and one C-class gene AhAG are quite variable. By contrast, the positions of the corresponding introns are conserved, resulting in a similar exon size in homologs. The typical region 70 bp-CCAATCA element was not found in the second intron of AhAG compared to AG homologs. The subcellular localization showed that AhAP3 was in both intranuclear and extranuclear. The heterodimer was formed between APETALA3 and PISTILLATA but not between the B- and C-class proteins using Y2H and BiFC. The 35S::AhAG heterologous transformed Arabidopsis had curly and smaller rosette leaves with early flowering. Floral organs had no homeotic conversion, albeit sepals and petals reduced in size. Siliques development was affected and displayed wrinkled and shorter. By contrast, 35S::AhAP3 and 35S::AhPI did not show any modified phenotype in transgenic Arabidopsis thaliana. We first proposed the model for Alpinia flower development. MADS-box transcription factor binding at particular genomic locations and interaction with partners may be crucial for the development of the floral organ.

Asymmetric expression patterns of B- and C-class MADS-box genes correspond to the asymmetrically specified androecial identities of Canna indica.

Canna indica is a common ornamental plant with asymmetric flowers having colourful petaloid staminodes. The only fertile stamen comprises a one-theca anther and a petaloid appendage and represents the lowest stamen number in the order Zingiberales. The molecular mechanism for the asymmetric androecial petaloidy remains poorly understood. Here, we studied the identity specification in Canna stamen. We observed four types of abnormal flower in terms of androecium identity transformation and analysed the corresponding floral symmetry changes. We further tested the expression patterns of B- and C-class MADS-box genes using in situ hybridization in normal Canna stamen. Homeotic conversions in the androecium were accompanied by floral symmetry changes, and the asymmetric stamen is key in contributing to the floral asymmetry. Both B- and C-class genes exhibited higher expression levels in the anther primordium than in other androecial parts. This asymmetric expression pattern precisely corresponded to the asymmetric identities of the Canna androecium. We identified C. indica as a model species for studying androecial organ identity and floral symmetry synthetically in Zingiberales. We hypothesized that homeotic genes specify floral organ identity in a putative dose-dependent manner. The results add to the current understanding of organ identity-related floral symmetry.

Dissection of the Arabidopsis HUA-PEP gene activity reveals that ovule fate specification requires restriction of the floral A-function.

Ovules are essential for sexual plant reproduction and seed formation, and are fundamental for agriculture. However, our understanding of the molecular mechanisms governing ovule development is far from complete. In Arabidopsis, ovule identity is determined by homeotic MADS-domain proteins that define the floral C- (AG) and D- (SHP1/SHP2, STK) functions. Pre-mRNA processing of these genes is critical and mediated by HUA-PEP activity, composed of genes encoding RNA-binding proteins. In strong hua-pep mutants, functional transcripts for C- and D-function genes are reduced, resulting in homeotic transformation of ovules. Thus, hua-pep mutants provide an unique sensitized background to study ovule morphogenesis when C- and D-functions are simultaneously compromised. We found that hua-pep ovules are morphologically sepaloid and show ectopic expression of the homeotic class-A gene AP1. Inactivation of AP1 or AP2 (A-function genes) in hua-pep mutants reduced homeotic conversions, rescuing ovule identity while promoting carpelloid traits in transformed ovules. Interestingly, increased AG dosage led to similar results. Our findings strongly suggest that HUA-PEP activity is required for correct C and D floral functions, which in turn prevents ectopic expression of class-A genes in ovules for their proper morphogenesis, evoking the classic A-C antagonism of the ABC model for floral organ development.

APETALA 2‐like genes AP2L2 and Q specify lemma identity and axillary floral meristem development in wheat

SummaryThe spikelet is the basic unit of the grass inflorescence. In tetraploid (Triticum turgidum) and hexaploid wheat (Triticum aestivum), the spikelet is a short indeterminate branch with two proximal sterile bracts (glumes) followed by a variable number of florets, each including a bract (lemma) with an axillary flower. Varying levels of miR172 and/or its target gene Q (AP2L5) result in gradual transitions of glumes to lemmas, and vice versa. Here, we show that AP2L5 and its related paralog AP2L2 play critical and redundant roles in the specification of axillary floral meristems and lemma identity. AP2L2, also targeted by miR172, displayed similar expression profiles to AP2L5 during spikelet development. Loss‐of‐function mutants in both homeologs of AP2L2 (henceforth ap2l2) developed normal spikelets, but ap2l2 ap2l5 double mutants generated spikelets with multiple empty bracts before transitioning to florets. The coordinated nature of these changes suggest an early role of these genes in floret development. Moreover, the flowers of ap2l2 ap2l5 mutants showed organ defects in paleas and lodicules, including the homeotic conversion of lodicules into carpels. Mutations in the miR172 target site of AP2L2 were associated with reduced plant height, more compact spikes, promotion of lemma‐like characters in glumes and smaller lodicules. Taken together, our results show that the balance in the expression of miR172 and AP2‐like genes is crucial for the correct development of spikelets and florets, and that this balance has been altered during the process of wheat and barley (Hordeum vulgare) domestication. The manipulation of this regulatory module provides an opportunity to modify spikelet architecture and improve grain yield.

Ectopic expression of CsMADS24, an AGAMOUS ortholog from cucumber, causes homeotic conversion of sepals into carpels in transgenic arabidopsis plants

The floral homeotic C-function MADS gene AGAMOUS (AG) in Arabidopsis plays crucial roles in specifying stamen and carpel identities as well as determining floral meristem. However, there have been only a few studies of floral homeotic C-function genes in cucumber thus far. In the present study, CsMADS24, a putative AG ortholog from cucumber, was isolated and characterized. Sequence analysis and protein sequence alignment revealed that the deduced CsMADS24 protein contained the typical MIKC structure and the N-terminal extension, as well as two highly conserved AG motifs (I and II). Phylogenetic analysis showed that CsMADS24 fell into the clade of core eudicots, while being distant from the AG orthologs of basal eudicots, monocots and gymnosperms. Expression analysis by RT-PCR showed that CsMADS24 was exclusively expressed in female flower buds. In situ hybridization revealed that CsMADS24 expression was only detected in the carpels. Functional analyses indicated that the sepals were partly converted into carpelloid-like structures in 35S::35S::CsMADS24 transgenic plants. In addition, earlier flowering and delayed floral organ abscission during the development of siliques were also observed in transgenic Arabidopsis. Our findings demonstrate that the AG ortholog plays an exclusive role in carpel specification of cucumber, providing a basis for revealing the mechanisms of reproductive development in cucumber.

The Role of Auxin in the Pattern Formation of the Asteraceae Flower Head (Capitulum).

Nature often creates complex structures by rearranging pre-existing units. One such example is the flower head (capitulum) in daisies, where a group of flowers (florets) and phyllaries (modified bracts) are arranged to superficially mimic a single flower. The capitulum is a key taxonomical innovation that defines the daisy family (Asteraceae), the largest flowering plant group. However, patterning mechanisms underlying its structure remain elusive. Here, we show that auxin, a plant hormone, provides a developmental patterning cue for the capitulum. During capitulum development, a temporal auxin gradient occurs, regulating the successive and centripetal formation of distinct florets and phyllaries. Disruption of the endogenous auxin gradient led to homeotic conversions of florets and phyllaries in the capitulum. Furthermore, auxin regulates floral meristem identity genes, such as Matricaria inodora RAY2 and M inodora LEAFY, which determine floret and phyllary identity. This study reveals the mechanism of capitulum patterning and highlights how common developmental tools, such as hormone gradients, have independently evolved in plants and animals.

An AGAMOUS-like factor is associated with the origin of two domesticated varieties in Cymbidium sinense (Orchidaceae)

Cymbidium has been artificially domesticated for centuries in Asia, which produced numerous cultivated varieties. Flowers with stamenoid tepals or those with multiple tepals have been found in different species of Cymbidium; however, the molecular basis controlling the formation of these phenotypes is still largely unknown. Previous work demonstrated that AGAMOUS/AG lineage MADS genes function in floral meristem determinacy as well as in reproductive organs development in both dicots and monocots, indicating a possible relationship with the origin of two flower varieties in Cymbidium. Here, we characterized and analyzed two AG lineage paralogues, CsAG1 and CsAG2, from Cymbidium sinense, both of which were highly expressed in the gynostemium column of a standard C. sinense. Interestingly, we detected ectopic expression of CsAG1 rather than CsAG2 in all floral organs of a stamenoid-tepal variety and significant down-regulation of CsAG1 in a variety with multiple tepals. Over-expression of CsAG1 in wild type Arabidopsis resulted in petal-to-stamen homeotic conversion, suggesting a conserved C-function of CsAG1 in the development of Cymbidium flower. Altogether, our results supported a hypothesis that disruption of a single AG-like factor would be associated with the formation of two domesticated varieties in C. sinense.

Isolation and Characterization of AGAMOUS-Like Genes Associated With Double-Flower Morphogenesis in Kerria japonica (Rosaceae).

Double-flower phenotype is more popular and attractive in garden and ornamental plants. There is great interest in exploring the molecular mechanisms underlying the double-flower formation for further breeding and selection. Kerria japonica, a commercial ornamental shrub of the Rosaceae family, is considered an excellent system to determine the mechanisms of morphological alterations, because it naturally has a single-flower form and double-flower variant with homeotic conversion of stamens into petals and carpels into leaf-like carpels. In this study, Sf-KjAG (AGAMOUS homolog of single-flower K. japonica) and Df-KjAG (AGAMOUS homolog of double-flower K. japonica) were isolated and characterized as two AGAMOUS (AG) homologs that occur strictly in single- and double-flower K. japonica, respectively. Our sequence comparison showed that Df-KjAG is derived from ectopic splicing with the insertion of a 2411 bp transposon-like fragment, which might disrupt mRNA accumulation and protein function, into intron 1. Ectopic expression analysis in Arabidopsis revealed that Sf-KjAG is highly conserved in specifying carpel and stamen identities. However, Df-KjAG did not show any putative C-class function in floral development. Moreover, yeast-two-hybrid assays showed that Sf-KjAG can interact with KjAGL2, KjAGL9, and KjAP1, whereas Df-KjAG has lost interactions with these floral identity genes. In addition, loss-of-function of Df-KjAG affected not only its own expression, but also that of other putative floral organ identity genes such as KjAGL2, KjAGL9, KjAP1, KjAP2, KjAP3, and KjPI. In conclusion, our findings suggest that double-flower formation in K. japonica can be attributed to Df-KjAG, which appears to be a mutant produced by the insertion of a transposon-like fragment in the normal AG homolog (Sf-KjAG) of single-flower K. japonica.Highlights: Sf-KjAG and Df-KjAG are different variations only distinguished by a transposon-like fragment insertion which lead to the evolutionary transformation from single-flower to double-flowers morphogenesis in Kerria japonica.

Brassinosteroids Modulate Meristem Fate and Differentiation of Unique Inflorescence Morphology in Setaria viridis.

Inflorescence architecture is a key determinant of yield potential in many crops and is patterned by the organization and developmental fate of axillary meristems. In cereals, flowers and grain are borne from spikelets, which differentiate in the final iteration of axillary meristem branching. In Setaria spp, inflorescence branches terminate in either a spikelet or a sterile bristle, and these structures appear to be paired. In this work, we leverage Setaria viridis to investigate a role for the phytohormones brassinosteroids (BRs) in specifying bristle identity and maintaining spikelet meristem determinacy. We report the molecular identification and characterization of the Bristleless1 (Bsl1) locus in S. viridis, which encodes a rate-limiting enzyme in BR biosynthesis. Loss-of-function bsl1 mutants fail to initiate a bristle identity program, resulting in homeotic conversion of bristles to spikelets. In addition, spikelet meristem determinacy is altered in the mutants, which produce two florets per spikelet instead of one. Both of these phenotypes provide avenues for enhanced grain production in cereal crops. Our results indicate that the spatiotemporal restriction of BR biosynthesis at boundary domains influences meristem fate decisions during inflorescence development. The bsl1 mutants provide insight into the molecular basis underlying morphological variation in inflorescence architecture.

Divergence of the Floral A-Function between an Asterid and a Rosid Species.

The ABC model is widely used as a genetic framework for understanding floral development and evolution. In this model, the A-function is required for the development of sepals and petals and to antagonize the C-function in the outer floral whorls. In the rosid species Arabidopsis thaliana, the AP2-type AP2 transcription factor represents a major A-function protein, but how the A-function is encoded in other species is not well understood. Here, we show that in the asterid species petunia (Petunia hybrida), AP2B/BLIND ENHANCER (BEN) confines the C-function to the inner petunia floral whorls, in parallel with the microRNA BLINDBEN belongs to the TOE-type AP2 gene family, members of which control flowering time in Arabidopsis. In turn, we demonstrate that the petunia AP2-type REPRESSOR OF B-FUNCTION (ROB) genes repress the B-function (but not the C-function) in the first floral whorl, together with BEN We propose a combinatorial model for patterning the B- and C-functions, leading to the homeotic conversion of sepals into petals, carpels, or stamens, depending on the genetic context. Combined with earlier results, our findings suggest that the molecular mechanisms controlling the spatial restriction of the floral organ identity genes are more diverse than the well-conserved B and C floral organ identity functions.

Isolation and characterization of a floral homeotic gene in Fraxinus nigra causing earlier flowering and homeotic alterations in transgenic Arabidopsis

Reproductive sterility, which can be obtained by manipulating floral organ identity genes, is an important tool for gene containment of genetically engineered trees. In Arabidopsis, AGAMOUS (AG) is the only C-class gene responsible for both floral meristem determinacy and floral organ identity, and its mutations produce sterility. As a first step in an effort to develop transgenic sterile black ash (Fraxinus nigra), an AG ortholog in black ash (FnAG) was isolated using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. Analysis of the deduced amino acid sequence showed a typical MIKC structure of type II plant MADS-box protein with a highly conserved MADS-domain. Phylogenetic analysis revealed that FnAG had a close relationship with AG orthologs from other woody species. FnAG transcript was mainly expressed in reproductive tissues, but rarely detected in the vegetative tissues, consistent with the ABC model for floral development. A functional analysis was performed by ectopic expression of FnAG driven by the CaMV 35S promoter in transgenic Arabidopsis. Transformed plants showed homeotic conversions of carpeloid sepals and stamenoid petals. Curled leaves, reduced plant size, and earlier flowering were also observed in transgenic Arabidopsis. These data indicated that the FnAG functions in the same way as AG in Arabidopsis. These results provide the framework for targeted genome editing of black ash, an ecologically and economically important wetland species.

Conservation vs divergence in LEAFY and APETALA1 functions between Arabidopsis thaliana and Cardamine hirsuta

A conserved genetic toolkit underlies the development of diverse floral forms among angiosperms. However, the degree of conservation vs divergence in the configuration of these gene regulatory networks is less clear. We addressed this question in a parallel genetic study between the closely related species Arabidopsis thaliana and Cardamine hirsuta. We identified leafy (lfy) and apetala1 (ap1) alleles in a mutant screen for floral regulators in C.hirsuta. C. hirsuta lfy mutants showed a complete homeotic conversion of flowers to leafy shoots, mimicking lfy ap1 double mutants in A.thaliana. Through genetic and molecular experiments, we showed that AP1 activation is fully dependent on LFY in C.hirsuta, by contrast to A.thaliana. Additionally, we found that LFY influences heteroblasty in C.hirsuta, such that loss or gain of LFY function affects its progression. Overexpression of UNUSUAL FLORAL ORGANS also alters C.hirsuta leaf shape in an LFY-dependent manner. We found that LFY and AP1 are conserved floral regulators that act nonredundantly in C.hirsuta, such that LFY has more obvious roles in floral and leaf development in C.hirsuta than in A.thaliana.

Partial redundancy and functional specialization of E-class SEPALLATA genes in an early-diverging eudicot

Plant MADS-box genes have duplicated extensively, allegedly contributing to the immense diversity of floral form in angiosperms. In Arabidopsis thaliana (a core eudicot model plant), four SEPALLATA (SEP) genes comprise the E-class from the extended ABCE model of flower development. They are redundantly involved in the development of the four types of floral organs (sepals, petals, stamens and carpels) and in floral meristem determinacy. E-class genes have been examined in other core eudicots and monocots, but have been less investigated in non-core eudicots. Our goal was to functionally characterize the E-class genes in the early-diverging eudicot Thalictrum thalictroides (Ranunculaceae), whose flowers are apetalous. We identified four SEP orthologs, which when placed in a phylogenetic context, resulted from a major gene duplication event before the origin of angiosperms and a subsequent duplication at the origin of the Ranunculales. We used Virus-Induced Gene Silencing (VIGS) to down-regulate the three expressed paralogs individually and in combination to investigate their function and to determine the degree of conservation versus divergence of this important plant transcription factor. All loci were partially redundant in sepal and stamen identity and in promoting petaloidy of sepals, yet the SEP3 ortholog had a more pronounced role in carpel identity and development. The two other paralogs appear to have subfunctionalized in their cadastral roles to keep the boundaries between either sepal and stamen zones or stamen and carpel zones. Double knockdowns had enhanced phenotypes and the triple knockdown had an even more severe phenotype that included partial to complete homeotic conversion of stamens and carpels to sepaloid organs and green sepals, highlighting a role of E-class genes in petaloidy of sepals in this species. While no floral meristem determinacy defects were observed, this could be due to residual amounts of gene expression in the VIGS experiments being sufficient to perform this function or to the masking role of a redundant gene.

A Homolog of Blade-On-Petiole 1 and 2 (BOP1/2) Controls Internode Length and Homeotic Changes of the Barley Inflorescence.

Inflorescence architecture in small-grain cereals has a direct effect on yield and is an important selection target in breeding for yield improvement. We analyzed the recessive mutation laxatum-a (lax-a) in barley (Hordeum vulgare), which causes pleiotropic changes in spike development, resulting in (1) extended rachis internodes conferring a more relaxed inflorescence, (2) broadened base of the lemma awns, (3) thinner grains that are largely exposed due to reduced marginal growth of the palea and lemma, and (4) and homeotic conversion of lodicules into two stamenoid structures. Map-based cloning enforced by mapping-by-sequencing of the mutant lax-a locus enabled the identification of a homolog of BLADE-ON-PETIOLE1 (BOP1) and BOP2 as the causal gene. Interestingly, the recently identified barley uniculme4 gene also is a BOP1/2 homolog and has been shown to regulate tillering and leaf sheath development. While the Arabidopsis (Arabidopsis thaliana) BOP1 and BOP2 genes act redundantly, the barley genes contribute independent effects in specifying the developmental growth of vegetative and reproductive organs, respectively. Analysis of natural genetic diversity revealed strikingly different haplotype diversity for the two paralogous barley genes, likely affected by the respective genomic environments, since no indication for an active selection process was detected.

Cell-to-cell movement of mitochondria in plants

We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer from Nicotiana sylvestris into Nicotiana tabacum cells. The alloplasmic N. tabacum line we used carries Nicotiana undulata cytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer from N. sylvestris to N. tabacum could be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility to orf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation.

Transcriptomic Analysis of Differentially Expressed Genes during Flower Organ Development in Genetic Male Sterile and Male Fertile Tagetes erecta by Digital Gene-Expression Profiling.

Tagetes erecta is an important commercial plant of Asteraceae family. The male sterile (MS) and male fertile (MF) two-type lines of T. erecta have been utilized in F1 hybrid production for many years, but no report has been made to identify the genes that specify its male sterility that is caused by homeotic conversion of floral organs. In this study, transcriptome assembly and digital gene expression profiling were performed to generate expression profiles of MS and MF plants. A cDNA library was generated from an equal mixture of RNA isolated from MS and MF flower buds (1 mm and 4 mm in diameter). Totally, 87,473,431 clean tags were obtained and assembled into 128,937 transcripts among which 65,857 unigenes were identified with an average length of 1,188 bp. About 52% of unigenes (34,176) were annotated in Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO (KEGG Ortholog database) and/or GO. Taking the above transcriptome as reference, 125 differentially expressed genes were detected in both developmental stages of MS and MF flower buds. MADS-box genes were presumed to be highly related to male sterility in T. erecta based on histological and cytological observations. Twelve MADS-box genes showed significantly different expression levels in flower buds 4 mm in diameter, whereas only one gene expressed significantly different in flower buds 1 mm in diameter between MS and MF plants. This is the first transcriptome analysis in T. erecta and will provide a valuable resource for future genomic studies, especially in flower organ development and/or differentiation.