Analysis and evolution of two functional Y-linked loci in a plant sex chromosome system.

White campion (Silene latifolia) is one of the few examples of plants with separate sexes and with X and Y sex chromosomes. The presence or absence of the Y chromosome determines which type of reproductive organs--male or female--will develop. Recently, we characterized the first active gene located on a plant Y chromosome, SlY1, and its X-linked homolog, SlX1. These genes encode WD-repeat proteins likely to be involved in cell proliferation. Here, we report the characterization of a novel Y-linked gene, SlY4, which also has a homolog on the X chromosome, SlX4. Both SlY4 and SlX4 potentially encode fructose-2,6-bisphosphatases. A comparative molecular analysis of the two sex-linked loci (SlY1/SlX1 and SlY4/SlX4) suggests selective constraint on both X- and Y-linked genes and thus that both X- and Y-linked copies are functional. Divergence between SlY4 and SlX4 is much greater than that between the SlY1 and SlX1 genes. These results suggest that, as for human XY-linked genes, the sex-linked plant loci ceased recombining at different times and reveal distinct events in the evolutionary history of the sex chromosomes.

Molecular and evolutionary analysis of a plant Y chromosome.

Plants have evolved a great diversity of sex determination systems. Among these, the XY system, also found in mammals, is one of the most exciting since it gives the opportunity to compare the evolution of sex chromosomes in two different kingdoms. Whereas genetic and molecular mechanisms controlling sex determination in drosophila and mammals, have been well studied, very little is known about such processes in plants. White campion (Silene latifolia) is an example of plant with X and Y chromosomes. What is the origin of the X and Y chromosomes? How did they evolve from a pair of autosomes? In our laboratory, we have isolated the first active genes located on a plant Y chromosome. We are using them as markers to trace the origin and evolution of sex chromosomes in the Silene genus.

Low variability in a Y-linked plant gene and its implications for Y-chromosome evolution.

Sex chromosomes have evolved independently in several different groups of organisms, but they share common features, including genetic degeneration of the Y chromosome. Suppression of recombination between ancestral proto-X and proto-Y chromosomes is thought to have led to their gradual divergence, and to degeneration of the Y chromosome, but the evolutionary forces responsible are unknown. In non-recombining Y chromosomes, deleterious mutations may be carried to fixation by linked advantageous mutations ("selective sweeps"). Occurrence of deleterious mutations may drive "Muller's ratchet" (stochastic loss of chromosomes with the fewest mutations). Selective elimination of deleterious mutations, causing "background selection" may accelerate stochastic fixation of mildly detrimental mutations. All these processes lower effective population sizes, and therefore reduce variability of genes in evolving Y chromosomes. We have studied DNA diversity and divergence in a recently described X- and Y-linked gene pair (SLX-1 and SLY-1) of the plant Silene latifolia to obtain evidence about the early stages of Y degeneration. Here we show that DNA polymorphism in SLY-1 is 20-fold lower than in SLX-1, but the pattern of polymorphism does not suggest a selective sweep.

SlY1, the first active gene cloned from a plant Y chromosome, encodes a WD-repeat protein.

Unlike the majority of flowering plants, which possess hermaphrodite flowers, white campion (Silene latifolia) is dioecious and has flowers of two different sexes. The sex is determined by the combination of heteromorphic sex chromosomes: XX in females and XY in males. The Y chromosome of S.latifolia was microdissected to generate a Y-specific probe which was used to screen a young male flower cDNA library. We identified five genes which represent the first active genes to be cloned from a plant Y chromosome. Here we report a detailed analysis of one of these genes, SlY1 (S.latifolia Y-gene 1). SlY1 is expressed predominantly in male flowers. A closely related gene, SlX1, is predicted to be located on the X chromosome and is strongly expressed in both male and female flowers. SlY1 and SlX1 encode almost identical proteins containing WD repeats. Immunolocalization experiments showed that these proteins are localized in the nucleus, and that they are most abundant in cells that are actively dividing or beginning to differentiate. Interestingly, they do not accumulate in arrested sexual organs and represent potential targets for sex determination genes. These genes will permit investigation of the origin and evolution of sex chromosomes in plants.

Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant.

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre-meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.

Characterisation and expression of the mitochondrial genome of a new type of cytoplasmic male-sterile sunflower.

A new cytoplasmic male sterile sunflower, CMS3 [44], was characterised in relation to the Petiolaris (PET1) cytoplasmic male-sterile sunflower, CMS89 [25]. Southern blot analysis showed that the mitochondrial genome of CMS3 contains unique rearrangements in at least five loci (atp6, atp9, atpA, nad1 + 5 and coxIII) compared to the PET1 sterile and the fertile cytoplasms. Transcripts of two (coxIII and atp6) of the five rearranged loci differed in CMS3 when compared to the corresponding loci in the PET1 and fertile cytoplasms. In organello protein synthesis experiments showed that the ca. 15 kDa mitochondrial polypeptide, characteristic of PET1, is not present in the CMS3 line. These data suggest that the molecular basis of male sterility in the CMS3 line differs from that of the PET1 cytoplasm. The nucleotide sequences of the coding and the immediate flanking regions of the coxIII and atp6 genes of CMS3 were compared to the corresponding regions from the fertile sunflower. In CMS3 the ORFB-coxIII locus is located immediately 3' to the atpA gene whereas in the fertile cytoplasm these two loci are ca. 60 kb apart. This DNA rearrangement probably involved a 265 bp repeat which may be implicated in the DNA recombination associated with PET1 CMS. The atp6 gene in CMS3 contains a 5'-terminal extention which results in an extended ORF. The potential involvement of the rearrangements associated with the coxIII and atp6 loci in relation to the CMS phenotype is discussed.

Cell-specific regulation of gene expression in mitochondria during anther development in sunflower.

Mitochondrial gene expression was characterized during meiosis in sunflower anthers. In situ hybridization experiments showed that there was a marked accumulation of four mitochondrial gene transcripts (atpA, atp9, cob, and rrn26) in young meiotic cells. This pattern of transcript accumulation was only detected for mitochondrial genes and not for transcripts of two nuclear genes (atpB and ANT) encoding mitochondrial proteins or another nuclear gene transcript (25S rRNA). Immunolocalization studies showed that the pattern of accumulation of the protein product of the atpA gene, the F1-ATP synthase alpha subunit, reflects that of the transcript. The expression of the novel mitochondrial orf522, which is associated with the cytoplasmic male-sterile (CMS) phenotype, was also studied by in situ hybridization. The orf522 transcripts were reduced in abundance in meiotic cells in the presence of fertility restorer genes. These results suggest that mitochondrial gene expression is regulated in a cell-specific fashion in developing anthers and that the restorer gene(s) may act cell specifically.

Nuclear restoration of cytoplasmic male sterility in sunflower is associated with the tissue-specific regulation of a novel mitochondrial gene.

We have previously shown that cytoplasmic male sterility in sunflower is associated with the insertion into the mitochondrial DNA of a novel open reading frame (ORF) located 3' to the atpA gene. Here, we show that in mitochondria from the sterile line, this novel ORF (ORF522) is cotranscribed with atpA. We have identified the product of the ORF522 as being a 15 kDa protein previously observed in sterile plant mitochondria by in organello translation. Both Western blot analysis and in organello translation assays show reduced levels of the 15 kDa polypeptide upon restoration of fertility. Interestingly, this reduction is tissue specific since it is only observed in the male florets from restored hybrid plants. These results suggest that the 15 kDa novel polypeptide is probably responsible for the CMS phenotype. Northern blot analysis using RNA from both seedlings and male florets shows a flower-specific reduction in the level of the ORF522 transcript in the restored hybrid line. The reduction is not due to a reduced transcription rate as demonstrated by 'run-on' experiments using mitochondria isolated from male florets. This suggests that the product of the nuclear restorer gene acts at the post-transcriptional level to destabilize the novel mitochondrial transcript in a tissue-specific manner and restore male fertility.

Expression of Chloroplast and Mitochondrial Genes during Microsporogenesis in Maize.

Mitochondrial and plastid gene expression has been examined during maize (Zea mays) microsporogenesis. Accumulation of transcripts was found for three mitochondrial genes studied (cob, atp6, and atp9) at the mid-term of pollen development. In contrast, these mitochondrial transcripts were undetectable in mature pollen. Southern and DNA gel blot experiments showed that the copy number of mitochondrial genes was amplified in microspores at stages preceding the accumulation of these transcripts. Plastid transcripts of the photosynthetic psbA and rbcL genes could not be detected after the two mitoses, whereas precursors of the 16S rRNA are detected at low levels.

Mitochondrial genome organization and expression associated with cytoplasmic male sterility in sunflower (Helianthus annuus).

A comparative investigation of the organization and expression of the mitochondrial genome in fertile and cytoplasmic male sterile (CMS) sunflower (Helianthus annuus) has been undertaken. A region of mitochondrial genome variation between the two phenotypes has been located in the 3' flanking region of the gene encoding the alpha subunit of the F1 ATPase (atpA). Physical mapping and sequence analysis have been used to show that a rearrangement involving an inversion and an insertion has occurred immediately downstream of the atpA coding region in the mitochondrial DNA from sterile sunflower. This rearrangement has resulted in the creation of a new open reading frame (ORFc) which is co-transcribed with atpA in sterile sunflower. In organello labelling of mitochondrial translation products from the two types of sunflower shows that a 15 kDa protein is synthesized by the mitochondria from sterile sunflower but not by those from fertile plants. The ORFc sequence could encode this 15 kDa protein which may be causally related to the CMS phenotype.

In vitro protein synthesis in isolated microspores of Zea mays at several stages of development.

A new procedure has been used providing large and homogenous populations of pollen from maize at different stages of their development. In order to label proteins synthesized during the course of microsporogenesis, a method has been developed that allows an efficient uptake of amino acids in the microspores. Results are presented showing that during pollen development three specific steps are involved: an early period active in protein synthesis, followed by a rest period when starch is accumulated, and a third period preceding the sorting out of mature pollen grains and during which protein synthesis starts again at a relatively low level. New polypeptides, some of which are very basic, appear at the time of starch deposition and accumulate up to the mature stage.

Characterization of a large inversion in the spinach chloroplast genome relative to Marchantia: a possible transposon-mediated origin.

A 7,022 bp BamHI-EcoRI fragment, located in the inverted repeat of spinach chloroplast, has been sequenced. It contains a 2131 codon open reading frame (ORF) homologous to both tobacco ORFs 581 and 1708, and to Marchantia ORF 2136. Relative to the Marchantia chloroplast genome, spinach ORF 2131 is located at the end of a large inversion; the other end point is close to trnL, the position of which is the same in Marchantia, tobacco and spinach. In Marchantia, two 8 bp direct repeats flanking two 10 bp indirect repeats are present near the end points of the inversion. These repeats may result from a transposon-mediated insertion which would have facilitated the subsequent inversion. From a comparison of the gene organization of the spinach, tobacco, and Marchantia genomes in this region, we propose a step-wise process to explain the expansion of the inverted repeat from a Marchantia-like genome to the spinach/tobacco genome.