The role of gibberellin in the reproductive development of Arabidopsis thaliana

The plant hormone gibberellin (GA) promotes several processes during Arabidopsis reproductive development, including the transition to flowering, floral organ growth and fertility. GA functions during stamen development to promote degradation of the tapetum cell layer through programmed cell death (PCD) and in post-anthesis pollen development. Bioactive GA is synthesised through a multi-step pathway, in which the last two biosynthetic steps are expressed as conserved multigene families. One of these, the GA 20-oxidases (GA20ox) consists of five paralogues in Arabidopsis, though physiological functions have only been ascribed to two (AtGA20ox1 and -2). Through a reverse genetics approach, this project demonstrates that AtGA20ox1, -2 and -3 account for almost all GA20ox activity in Arabidopsis, with very little evidence of any functions for AtGA20ox4 or -5. Unlike AtGA20ox1, -2, -3 and -4, AtGA20ox5 possesses only partial GA20ox activity, performing the first two out of three sequential catalytic conversions in vitro. Partial functional redundancy occurs between AtGA20ox1, -2 and -3 across Arabidopsis development, although AtGA20ox1 and -2 dominate. Mapping of floral AtGA20ox expression through qPCR and the creation of transgenic GUS reporter lines found that the relationship between these three paralogues is complex, and not explicable through the simple hypothesis of co-expression in the same tissues. During anther development, the reported expression of AtGA20ox1, -2, -3 and -4 is mainly restricted to the tapetum cell layer, and loss of AtGA20ox1, -2 and -3 results in an anther developmental arrest in which the tapetum does not degrade. This project demonstrates that stamen development is dependent on an optimum level of GA, with GA-deficiency restricting filament elongation to prevent pollination and GA-overdose negatively affecting anther development. DELLA repression of GA signalling is necessary for successful pollen development, with two of the five DELLA paralogues, RGA and GAI, critical to this process in the Columbia ecotype

DELLA activity is required for successful pollen development in the Columbia ecotype of Arabidopsis.

Excessive gibberellin (GA) signalling, mediated through the DELLA proteins, has a negative impact on plant fertility. Loss of DELLA activity in the monocot rice (Oryza sativa) causes complete male sterility, but not in the dicot model Arabidopsis (Arabidopsis thaliana) ecotype Landsberg erecta (Ler), in which DELLA function has been studied most extensively, leading to the assumption that DELLA activity is not essential for Arabidopsis pollen development. A novel DELLA fertility phenotype was identified in the Columbia (Col-0) ecotype that necessitates re-evaluation of the general conclusions drawn from Ler. Fertility phenotypes were compared between the Col-0 and Ler ecotypes under conditions of chemical and genetic GA overdose, including mutants in both ecotypes lacking the DELLA paralogues REPRESSOR OF ga1-3 (RGA) and GA INSENSITIVE (GAI). Ler displays a less severe fertility phenotype than Col-0 under GA treatment. Col-0 rga gai mutants, in contrast with the equivalent Ler phenotype, were entirely male sterile, caused by post-meiotic defects in pollen development, which were rescued by the reintroduction of DELLA into either the tapetum or developing pollen. We conclude that DELLA activity is essential for Arabidopsis pollen development. Differences between the fertility responses of Col-0 and Ler might be caused by differences in downstream signalling pathways or altered DELLA expression

Protocol: genetic transformation of the fern Ceratopteris richardii through microparticle bombardment

BACKGROUND: The inability to genetically transform any fern species has been a major technical barrier to unlocking fern biology. Initial attempts to overcome this limitation were based on transient transformation approaches or achieved very low efficiencies. A highly efficient method of stable transformation was recently reported using the fern Ceratopteris richardii, in which particle bombardment of callus tissue achieved transformation efficiencies of up to 72%. As such, this transformation method represents a highly desirable research tool for groups wishing to undertake fern genetic analysis. RESULTS: We detail an updated and optimized protocol for transformation of C. richardii by particle bombardment, including all necessary ancillary protocols for successful growth and propagation of this species in a laboratory environment. The C. richardii lifecycle comprises separate, free-living gametophyte and sporophyte stages. Callus is induced from the sporophyte apex through growth on cytokinin-containing tissue culture medium and can be maintained indefinitely by sub-culturing. Transgene DNA is introduced into callus cells through particle bombardment, and stable genomic integration events are selected by regeneration and growth of T(0) sporophytes for a period of 8 weeks on medium containing antibiotics. Selection of T(1) transgenic progeny is accomplished through screening T(1) gametophytes for antibiotic resistance. In many cases sexual reproduction and development of transgenic embryos requires growth and fertilization of gametophytes in the absence of antibiotics, followed by a separate screen for antibiotic resistance in the resultant sporophyte generation. CONCLUSIONS: Genetic transformation of C. richardii using this protocol was found to be robust under a broad range of bombardment and recovery conditions. The successful expansion of the selection toolkit to include a second antibiotic for resistance screening (G-418) and different resistance marker promoters increases the scope of transformations possible using this technique and offers the prospect of more complex analysis, for example the creation of lines carrying more than one transgene. The introduction of a robust and practicable transformation technique is a very important milestone in the field of fern biology, and its successful implementation in C. richardii paves the way for adoption of this species as the first fern genetic model. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-015-0080-8) contains supplementary material, which is available to authorized users