Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha

Background  

Brachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to compare expression profile of target genes and, in order to obtain reliable results, it is important to have suitable reference genes. In this work, we evaluated eight potential reference genes for B. brizantha qRT-PCR experiments, isolated from cDNA ovary libraries. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested to validate the reference genes, including the female gametophyte, where differences in the expression profile between sexual and apomictic plants must occur.     

Activity and localisation of sucrose synthase and invertase in ovules of sexual and apomicticBrachiaria decumbens

Summary Brachiaria decumbens has sexual and apomictic reproduction. Apomixis is facultative and of the aposporic type. In early stages of ovule development, differences in the pattern of callose deposition between sexual and apomictic plants were observed which points to possible differences in carbohydrate metabolism. Therefore, a comparative study on carbohydrate metabolism between a sexual diploid ecotype and an apomictic tetraploidB. decumbens was made. A histochemical determination of two enzymes responsible for sucrose degradation, sucrose synthase and invertase, was performed for all stages of ovule development. In addition, the concentrations of sucrose, glucose, and fructose were measured for each stage of ovule development, both for sexual and apomictic plants. The enzymes were localised by immunohistochemistry with heterologous antibodies. A distinct difference between sexual and apomictic plants was observed in the localisation of sucrose synthase activity as well as in the amount of activity, especially in the early stages of ovular development. Invertase activity localisation was comparable between ovules of the sexual and apomictic plants, but its activity is clearly higher in ovules of sexual plants. The localisation of the enzymes coincided with the place of activity. For both sexual and apomictic plants the amount of sucrose in the ovaries increased with the stage of ovule development. Differences in the amount of sucrose between sexual and apomictic plants in ovaries with ovules in comparable stages of development were detected. A delay in the onset of carbohydrate metabolism during early stages of ovule development characterises the apomictic plant.Abbreviations MMC megaspore mother cell - MC meiocyte - MS megaspore - AI apospore initial - CO coenocyte - MES mature embryo sac - SuSy sucrose synthase - INV invertase - BMM buthylmethyl methacrylate - DTT dithiothreitol - DAPI 4,6-diamidino-2-phenylindole - PBS phosphate-buffered saline     

The MADS domain protein DIANA acts together with AGAMOUS-LIKE80 to specify the central cell in Arabidopsis ovules

MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein–β-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt.     

Dynamics of callose deposition and beta-1,3-glucanase expression during reproductive events in sexual and apomictic Hieracium

Callose accumulates in the walls of cells undergoing megasporogenesis during embryo sac formation in angiosperm ovules. Deficiencies in callose deposition have been observed in apomictic plants and causal linkages between altered callose deposition and apomictic initiation proposed. In apomictic Hieracium, embryo sacs initiate by sexual and apomictic processes within an ovule, but sexual development terminates in successful apomicts. Callose deposition and the events that lead to sexual termination were examined in different Hieracium apomicts that form initials pre- and post-meiosis. In apomictic plants, callose was not detected in initial cell walls and deficiencies in callose deposition were not observed in cells undergoing megasporogenesis. Multiple initial formation pre-meiosis resulted in physical distortion of cells undergoing megasporogenesis, persistence of callose and termination of the sexual pathway. In apomictic plants, callose persistence did not correlate with altered spatial or temporal expression of a β-1,3-glucanase gene (HpGluc) encoding a putative callose-degrading enzyme. Expression analysis indicated HpGluc might function during ovule growth and embryo sac expansion in addition to callose dissolution in sexual and apomictic plants. Initial formation pre-meiosis might therefore limit the access of HpGluc protein to callose substrate while the expansion of aposporous embryo sacs is promoted. Callose deposition and dissolution during megasporogenesis were unaffected when initials formed post-meiosis, indicating other events cause sexual termination. Apomixis in Hieracium is not caused by changes in callose distribution but by events that lead to initial cell formation. The timing of initial formation can in turn influence callose dissolution. Received: 18 April 2000 / Accepted: 10 July 2000     

A DEFICIENS homologue is down-regulated during apomictic initiation in ovules of Hieracium

 Hieracium is a member of the Asteraceae family, and contains sexual species in addition to apomictic species that reproduce by apospory and produce seed without fertilization. A homologue of the floral organ-identity gene DEFICIENS (DEF) was isolated from an apomictic line of Hieracium piloselloides (Vill.) following differential display between mature ovules and those initiating autonomous embryogenesis. The gene termed HPDEF has 93% amino acid identity with GDEF2, a DEF homologue isolated from Gerbera hybrida (D. Yu et al., 1999, Plant J. 17: 51–62), another member of the Asteraceae. In-situ analysis showed that early in floral development HPDEF is expressed in stamen and petal primordia, indicating expected B-function activity, according to the ABC model of floral organ identity (J. L. Bowman et al., 1991, Development 112: 1–20; E. S. Coen and E. M. Meyerowitz, 1991, Nature 353: 31–37). However, HPDEF expression was also observed in ovule primordia and expression continued in developing ovules until anthesis, indicating that this gene may have a role in ovule development. Expression of HPDEF was not detected in megaspore mother cells, or in sexual or aposporous embryo sacs. In sexual Hieracium, HPDEF was uniformly expressed throughout the ovule integument until anthesis. In most ovules of the apomict, however, HPDEF expression was transiently down-regulated in a specific zone in the chalazal region where cells initiating aposporous embryo sac formation differentiate. Uniform low-level HPDEF expression was subsequently observed prior to anthesis in ovules from sexual and apomictic plants. HPDEF may be down-regulated as a consequence of apomictic initiation and/or its down-regulation may facilitate progression of apomictic events. Received: 15 September 1999 / Accepted: 12 October 1999     

Direct evidence of pseudogamy in apomictic Brachiaria brizantha (Poaceae)

Brachiaria brizantha is a forage grass that has several apomictic accessions. B. brizantha cv. Marandu is a natural tetraploid aposporous apomict widely cultivated in Brazil. Pseudogamy was detected in this species by observation that seed set is suppressed in plants that have had the stigmas excised from the flowers. The egg cell develops parthenogenetically in the apomictic plants, meaning that fertilisation is necessary for the formation of the endosperm. A thorough knowledge of all the events of seed formation in natural apomictic plants is essential for a complete understanding of this mode of reproduction. In this paper, we show direct evidence of pseudogamy in B. brizantha through the cytological analysis of polar nucleus fertilisation and the determination of triploid level of the endosperm tissue. The development of the male gametophyte gives rise to a reduced tri-celled pollen, the viability of which varies throughout the year, reaching 88% in the peak of the flowering period. Discharge of the male gamete takes place around 10 h after pollination and monospermy is the predominant system observed. Precocious embryony was also observed in these plants; embryos arise from egg cells. Endosperm development followed the free nucleus model and was associated with the presence of an embryo. Cellularisation and reserve uptake occurred 2 days after pollination (DAP) and mature endosperm was observed 8 DAP. The triploid level of the endosperm in the apomictic accession confirmed the 2:1 maternal:paternal ratio of genome contribution in the tissue.     

Characterization of mode of reproduction in Commiphora wightii [(Arnot) Bhandari] reveals novel pollen–pistil interaction and occurrence of obligate sexual female plants

Guggul [Commiphora wightii (Arnot) Bhandari], a polygamous woody tree valued for its medicinal oleoresin gum rich in guggulsterone, is reported to reproduce via sporophytic apomixis. Details about its natural diversity, and mode and extent of sexual reproduction are, however, scanty. Therefore, a comprehensive investigation of guggul reproduction was made employing histology, controlled pollination, flow cytometry and RAPD analyses of progeny to assess the occurrence and extent of sexual reproduction. We report the discovery of obligate sexual female plants of guggul through these studies. Also, we document a unique pollen–pistil incompatibility that prevents all but one pollen tube growth into the style to effect fertilization. Consequently, obligate sexual female plants produced single-seeded fruit although each flower contains four ovules. In apomictic plants bearing more than one seed per fruit, at most only one seed was of sexual origin. Further, flow cytometric analysis conclusively demonstrated that endosperm development occurs either autonomously or following triple fusion. Autonomous endosperm development was invariably associated with endoreduplication, a unique feature of apomixis in guggul. Despite predominance of apomixis, a low frequency of sexual reproduction was found to persist in apomictic plants yielding new genetic variation. RAPD analysis clearly distinguished accessions and was useful in identifying sexual progenies. The implications of the novel pollen–pistil interaction on establishment and spread of apomixis in guggul are discussed. The study has not only revealed novel features of guggul reproduction but also opened new opportunities for molecular genetic analysis of sporophytic apomixis and breeding improvement of guggul.     

AGL6-like MADS-box genes are sister to AGL2-like MADS-box genes

AGL6-like genes form one of the major subfamilies of MADS-box genes and are closely related to the AGL2 (Eclass) and SQUA (A-class) subfamilies. In Arabidopsis, AGL6 and AGL13 have been reported from the AGL6 subfamily, and AGL6 controls lateral organ development and flowering time. However, little is known about homologs of these genes in basal angiosperms. We identified new AGL6-like genes from several taxa from gymnosperms, basal angiosperms, monocots, and eudicots. These genes were analyzed together with previously reported AGL6-like genes. Structural analyses showed 1) a one-aa (amino acid) gap in the I-domain in all AGL6-like genes relative to AGL2-like and SQUA-like genes, 2) a seven-aa insertion in the C-domain of genes from asterids, and 3) a one-aa insertion in the C-domain of genes from gymnosperms. Broad phylogenetic analyses strongly showed that AGL6-like genes are sister to AGL2-like genes, and SQUA-like genes are sister to these two groups. The phylogenetic tree of AGL6-like genes generally tracks organismal phylogeny as inferred from multigene data sets; several gene duplications were detected in angiosperms (e.g., within Magnoliales), and one duplication was detected in gymnosperms. We hypothesize that the split between AGL6-like and AGL2-like genes occurred at least 290–309.2 mya based on our phylogenetic tree and the fossil record.     

Sexual reproduction development in apomictic Eulaliopsis binata (Poaceae)

Apomixis is a particular mode of reproduction that allows progeny formation without meiosis and fertilization. Eulaliopsis binata, a tetraploid apomictic species, is widely used for making paper, rope and mats. There is great potential for fixation of heterosis in E. binata due to autonomous endosperm formation in this species. Although most of its embryo sac originates from nucellus cells, termed apospory, we observed sexual reproduction initiation in 86.8 to 96.8% of the ovules, evidenced by callose deposition on the walls of cells undergoing megasporogenesis. However, only 2-3% mature polygonum-type sexual embryo sacs were confirmed by embryological investigation. Callose was not detected on aposporous initial cell walls. The aposporous initial cells differentiated during pre- and post-meiosis of the megaspore mother cell, while the sexual embryo sac degenerated at the megaspore stage. DNA content ratio of embryo and endosperm in some individuals was 2C:3C, based on flow cytometry screening of seed, similar to that of normal sexual seed. These results confirm that apomictic E. binata has conserved sexual reproduction to a certain degree, which may contribute to maintaining genetic diversity. The finding of sexual reproduction in apomictic E. binata could be useful for research on genetic mechanism of apomixis in E. binata.     

Toward in vitro fertilization in Brachiaria spp.

Brachiaria are forage grasses widely cultivated in tropical areas. In vitro pollination was applied to accessions of Brachiaria spp. by placing pollen of non-dehiscent anthers on a solid medium near isolated ovaries. Viability and in vitro germination were tested in order to establish good conditions for pollen development. Comparing sexual to apomictic plants, apomictic pollen has more abortion after meiosis during the microspore stage and a lower viability and, of both types, only some plants have sufficient germination in a high sugar concentration. Using in vitro pollination with the sexual plant, the pollen tube penetrates into the nucellus and micropyle, but the embryo sac degenerates and collapses. In the apomictic B. decumbens, in vitro pollination leads to the transfer of the sperm nuclei into the egg cell and the central cell. The results are discussed according to normal fertilization and barriers in sexual and apomictic plants.     

Sexual and apomictic reproduction in Hieracium subgenus pilosella are closely interrelated developmental pathways

Seed formation in flowering plants requires meiosis of the megaspore mother cell (MMC) inside the ovule, selection of a megaspore that undergoes mitosis to form an embryo sac, and double fertilization to initiate embryo and endosperm formation. During apomixis, or asexual seed formation, in Hieracium ovules, a somatic aposporous initial (AI) cell divides to form a structurally variable aposporous embryo sac and embryo. This entire process, including endosperm development, is fertilization independent. Introduction of reproductive tissue marker genes into sexual and apomictic Hieracium showed that AI cells do not express a MMC marker. Spatial and temporal gene expression patterns of other introduced genes were conserved commencing with the first nuclear division of the AI cell in apomicts and the mitotic initiation of embryo sac formation in sexual plants. Conservation in expression patterns also occurred during embryo and endosperm development, indicating that sexuality and apomixis are interrelated pathways that share regulatory components. The induction of a modified sexual reproduction program in AI cells may enable the manifestation of apomixis in HIERACIUM:     

Paternity and ploidy segregation of progenies derived from tetraploid Malus xiaojinensis

Apomixis is usually subdivided into parthenogenesis, apogamy, and apospory on the basis of different mechanisms of development and cytogenetic effects. Although most plants and animals undergo sexual reproduction to multiply, it is important to understand the role of apomixis in plant reproduction because of its useful applications in the breeding and propagation of some crops. Tetraploid Malus xiaojinensis Cheng et Jiang is a typical facultative apomictic plant species. Paternity and ploidy analyses of M. xiaojinensis seedlings were performed with flow cytometry in conjunction with microsatellite and single nucleotide polymorphism markers. The proportion of apomictic derived progeny of M. xiaojinensis × Malus baccata was 54.05, 53.96, and 43.55 % from 2008 to 2010, respectively. The progeny of M. xiaojinensis × M. baccata comprised hybrids of 2x, 3x, 4x, or 5x ploidy, whereas apomictic offspring were 2x, 3x, or 4x. Among the apomictic derived progeny, the ploidy of restituted apomictic progeny was 2x, 3x, or 4x, whereas that of nonrestituted apomictic progeny was 4x only. The proportions of the different types of apomictic derived progeny differed among the years 2008 to 2010. The ploidy of progeny from emasculated flowers also segregated into 2x, 3x, and 4x individuals. These results indicate that both parthenogenesis and unreduced meiotic diplospory play important roles in apomictic reproduction in M. xiaojinensis.     

An <Emphasis Type="Italic">Hieracium</Emphasis> mutant, <Emphasis Type="Italic">loss of</Emphasis><Emphasis Type="Italic">apomeiosis 1</Emphasis> (<Emphasis Type="Italic">loa1</Emphasis>) is defective in the initiation of apomixis

Asexual seed formation (apomixis) in Hieracium aurantiacum occurs by mitotic embryo sac formation without prior meiosis in ovules (apomeiosis), followed by fertilization-independent embryo and endosperm development. Sexual reproduction begins first in Hieracium ovules with megaspore mother cell (MMC) formation. Apomixis initiates with the enlargement of somatic cells, termed aposporous initial (AI) cells, near sexual cells. AI cells grow towards sexually programmed cells undergoing meiosis, which degrade as the dividing nuclei of AIs obscure and displace them. Following Agrobacterium-mediated transformation of an aneuploid Hieracium aurantiacum apomict, a somaclonal mutant designated “loss of apomeiosis 1” (loa1) was recovered, which had significantly lost the ability to form apomictic seed. Maternal apomictic progeny were rare and low levels of germinable seedlings were primarily derived from meiotically derived eggs. Cytological analysis revealed defects in AI formation and function in loa1. Somatic cells enlarged some distance away from sexual cells and unlike AI cells, these expanded away from sexual cells without nuclear division. Surprisingly, many accumulated callose in the walls, a marker associated with meiotically specified cells. These defective AI (DAI) cells only had partial sexual identity as they failed to express a marker reflecting entry to meiosis that was easily detected in MMCs and they ultimately degraded. DAI cell formation did not lead to a compensatory increase in functional sexual embryo sacs, as collapse of meiotic embryo sacs was prevalent in the aneuploid somaclonal mutant. Positional cues that are important for AI cell differentiation, growth and fate may have been disrupted in the loa1 mutant and this is discussed. The authors Takashi Okada, Andrew S. Catanach and Susan D. Johnson made equal contributions to the data.     

Anatomy of ovary and ovule in dandelions (Taraxacum, Asteraceae)

The genus Taraxacum Wigg. (Asteraceae) forms a polyploid complex within which there are strong links between the ploidy level and the mode of reproduction. Diploids are obligate sexual, whereas polyploids are usually apomictic. The paper reports on a comparative study of the ovary and especially the ovule anatomy in the diploid dandelion T. linearisquameum and the triploid T. gentile. Observations with light and electron microscopy revealed no essential differences in the anatomy of both the ovary and ovule in the examined species. Dandelion ovules are anatropous, unitegmic and tenuinucellate. In both sexual and apomictic species, a zonal differentiation of the integument is characteristic of the ovule. In the integumentary layers situated next to the endothelium, the cell walls are extremely thick and PAS positive. Data obtained from TEM indicate that these special walls have an open spongy structure and their cytoplasm shows evidence of gradual degeneration. Increased deposition of wall material in the integumentary cells surrounding the endothelium takes place especially around the chalazal pole of the embryo sac as well as around the central cell. In contrast, the integumentary cells surrounding the micropylar region have thin walls and exhibit a high metabolic activity. The role of the thick-walled integumentary layers in the dandelion ovule is discussed. We also consider whether this may be a feature of taxonomic importance.