Dormancy cycling is accompanied by changes in ABA sensitivity in Polygonum aviculare seeds.

Polygonum aviculare seeds show high levels of primary dormancy (PD). Low winter temperatures alleviate dormancy and high spring temperatures induce seeds into secondary dormancy (SD), naturally establishing stable seedbanks cycling through years. The objective of this work was to elucidate the mechanism(s) involved in PD expression and release, and in SD induction in these seeds, and the extent to which abscisic acid (ABA) and gibberellins (GAs) are part of these mechanisms. Quantification of endogenous ABA both prior to and during incubation, and sensitivity to ABA and GAs, were assessed in seeds with contrasting dormancy. Expression analysis was performed for candidate genes involved in hormone metabolism and signaling. It was found that endogenous ABA content does not explain either dormancy release or dormancy induction; moreover, it does not seem to play a role in dormancy maintenance. However, dormancy modifications were commonly accompanied by changes in ABA sensitivity. Concomitantly, induction into SD, but not PD, was characterized by a increased PaABI-5 and PaPYL transcription, and a rise in GA sensitivity as a possible counterbalance effect. These results suggest that dormancy cycling in this species is related to changes in embryo sensitivity to ABA; however, this sensitivity appears to be controlled by different molecular mechanisms in primary and secondary dormant seeds.

Hydrogen sulfide generated by L-cysteine desulfhydrase acts upstream of nitric oxide to modulate abscisic acid-dependent stomatal closure.

Abscisic acid (ABA) is a well-studied regulator of stomatal movement. Hydrogen sulfide (H2S), a small signaling gas molecule involved in key physiological processes in mammals, has been recently reported as a new component of the ABA signaling network in stomatal guard cells. In Arabidopsis (Arabidopsis thaliana), H2S is enzymatically produced in the cytosol through the activity of l-cysteine desulfhydrase (DES1). In this work, we used DES1 knockout Arabidopsis mutant plants (des1) to study the participation of DES1 in the cross talk between H2S and nitric oxide (NO) in the ABA-dependent signaling network in guard cells. The results show that ABA did not close the stomata in isolated epidermal strips of des1 mutants, an effect that was restored by the application of exogenous H2S. Quantitative reverse transcription polymerase chain reaction analysis demonstrated that ABA induces DES1 expression in guard cell-enriched RNA extracts from wild-type Arabidopsis plants. Furthermore, stomata from isolated epidermal strips of Arabidopsis ABA receptor mutant pyrabactin-resistant1 (pyr1)/pyrabactin-like1 (pyl1)/pyl2/pyl4 close in response to exogenous H2S, suggesting that this gasotransmitter is acting downstream, although acting independently of the ABA receptor cannot be ruled out with this data. However, the Arabidopsis clade-A PROTEIN PHOSPHATASE2C mutant abscisic acid-insensitive1 (abi1-1) does not close the stomata when epidermal strips were treated with H2S, suggesting that H2S required a functional ABI1. Further studies to unravel the cross talk between H2S and NO indicate that (1) H2S promotes NO production, (2) DES1 is required for ABA-dependent NO production, and (3) NO is downstream of H2S in ABA-induced stomatal closure. Altogether, data indicate that DES1 is a unique component of ABA signaling in guard cells.

Expression of lorelei-like genes in aposporous and sexual Paspalum notatum plants.

Gametophytic apomictic plants form non-reduced embryo sacs that generate clonal embryos by parthenogenesis, in the absence of both meiosis and egg-cell fertilization. Here we report the sequence and expression analysis of a lorelei-like Paspalum notatum gene, n20gap-1, which encodes a GPI-anchored protein previously associated with apomixis in this species. Phylogeny trees showed that n20gap-1 was evolutionary related to the Arabidopsis thaliana lorelei genes At4g26466 and At5g56170. The lorelei At4g26466 disruption was shown to be detrimental to sperm cell release in arabidopsis. RFLP (Restriction Fragment Length Polymorphism) analysis revealed the occurrence of several homologous sequences in the Paspalum notatum genome, exhibiting polymorphisms genetically linked to apomixis. Real-time PCR showed that lorelei-family genes present a minor activity peak at pre-meiosis and a major one at anthesis. The apomictic genotype analyzed showed a significantly increased activity at pre-meiosis, post-meiosis and anthesis with respect to a sexual genotype. In situ hybridization assays revealed expression in integuments, nucellus and the egg-cell apparatus. Several n20gap-1 alleles differing mainly at the 3' UTR sequence were identified. Allele-specific real-time PCR experiments showed that allele 28 was significantly induced in reproductive tissues of the apomictic genotype with respect to the sexual genotype at anthesis. Our results indicate that P. notatum lorelei-like genes are differentially expressed in representative sexual (Q4188) and apomictic (Q4117) genotypes, and might play a role in the final stages of the apomixis developmental cascade. However, the association of n20gap-1 expression with the trait should be confirmed in significant number of sexual and apomictic genotypes.

Gene expression analysis at the onset of aposporous apomixis in Paspalum notatum.

Apomixis is a route of asexual reproduction through seeds, that progresses in the absence of meiosis and fertilization to generate maternal clonal progenies. Gametophytic apomicts are usually polyploid and probably arose from sexual ancestors through a limited number of mutations in the female reproductive pathway. A differential display analysis was carried out on immature inflorescences of sexual and apomictic tetraploid genotypes of Paspalum notatum, in order to identify genes associated with the emergence of apospory. Analysis of approximately 10,000 transcripts led to the identification of 94 high-quality differentially expressed sequences. Assembling analysis, plus validation, rendered 65 candidate unigenes, organized as 14 contigs and 51 singletons. Thirty-four unigenes were isolated from apomictic plants and 31 from sexual ones. A total of 45 (69.2%) unigenes were functionally categorized. While several of the differentially expressed sequences appeared to be components of an extracellular receptor kinase (ERK) signal transduction cascade, others seemed to participate in a variety of central cellular processes like cell-cycle control, protein turnover, intercellular signalling, transposon activity, transcriptional regulation and endoplasmic reticulum-mediated biosynthesis. In silico mapping revealed that a particular group of five genes silenced in apomictic plants clustered in a rice genomic area syntenic with the region governing apospory in Paspalum notatum and Brachiaria brizantha. Two of these genes mapped within the set of apo-homologues in P. notatum. Four genes previously reported to be controlled by ploidy were identified among those expressed differentially between apomictic and sexual plants. In situ hybridization experiments were performed for selected clones.

Genome polymorphisms and gene differential expression in a 'back-and-forth' ploidy-altered series of weeping lovegrass (Eragrostis curvula).

Molecular markers were used to analyze the genomic structure of an euploid series of Eragrostis curvula, obtained after a tetraploid dihaploidization procedure followed by chromosome re-doubling with colchicine. Considerable levels of genome polymorphisms were detected between lines. Curiously, a significant number of molecular markers showed a revertant behavior following the successive changes of ploidy, suggesting that genome alterations were specific and conferred genetic structures characteristic of a given ploidy level. Genuine reversion was confirmed by sequencing. Cluster analysis demonstrated grouping of tetraploids while the diploid was more distantly related with respect to the rest of the plants. Polymorphic revertant sequences involved mostly non-coding regions, although some of them displayed sequence homology to known genes. A revertant sequence corresponding to a P-type adenosine triphosphatase was found to be differentially represented in cDNA libraries obtained from the diploid and a colchiploid, but was not found expressed in the original tetraploid. Transcriptome profiling of inflorescence followed by real-time polymerase chain reaction validation showed 0.34% polymorphic bands between apomictic tetraploid and sexual diploid plants. Several of the polymorphic sequences corresponded to known genes. Possible correlation between the results observed here and a recently reported genome-wide non-Mendelian inheritance mechanism in Arabidopsis thaliana are discussed.