Micro-analytical and molecular approaches for understanding the distribution, biochemistry, and molecular biology of selenium in (hyperaccumulator) plants.

MAIN CONCLUSION: Micro-analytical techniques to untangle Se distribution and chemical speciation in plants coupled with molecular biology analysis enable the deciphering of metabolic pathways responsible for Se tolerance and accumulation. Selenium (Se) is not essential for plants and is toxic at high concentrations. However, Se hyperaccumulator plants have evolved strategies to both tolerate and accumulate > 1000 µg Se g-1 DW in their living above-ground tissues. Given the complexity of the biochemistry of Se, various approaches have been adopted to study Se metabolism in plants. These include X-ray-based techniques for assessing distribution and chemical speciation of Se, and molecular biology techniques to identify genes implicated in Se uptake, transport, and assimilation. This review presents these techniques, synthesises the current state of knowledge on Se metabolism in plants, and highlights future directions for research into Se (hyper)accumulation and tolerance. We conclude that powerful insights may be gained from coupling information on the distribution and chemical speciation of Se to genome-scale studies to identify gene functions and molecular mechanisms that underpin Se tolerance and accumulation in these ecologically and biotechnologically important plants species. The study of Se metabolism is challenging and is a useful testbed for developing novel analytical approaches that are potentially more widely applicable to the study of the regulation of a wide range of metal(loid)s in hyperaccumulator plants.

Overexpression of the MYB29 transcription factor affects aliphatic glucosinolate synthesis in Brassica oleracea.

KEY MESSAGE: Overexpression of BoMYB29 gene up-regulates the aliphatic glucosinolate pathway in Brassica oleracea plants increasing the production of the anti-cancer metabolite glucoraphanin, and the toxic and pungent sinigrin. Isothiocyanates, the bio-active hydrolysis products of glucosinolates, naturally produced by several Brassicaceae species, play an important role in human health and agriculture. This study aims at correlating the content of aliphatic glucosinolates to the expression of genes involved in their synthesis in Brassica oleracea, and perform functional analysis of BoMYB29 gene. To this purpose, three genotypes were used: a sprouting broccoli, a cabbage, and a wild genotype (Winspit), a high glucosinolate containing accession. Winspit showed the highest transcript level of BoMYB28, BoMYB29 and BoAOP2 genes, and BoAOP2 expression was positively correlated with that of the two MYB genes. Further analyses of the aliphatic glucosinolates also showed a positive correlation between the expression of BoAOP2 and the production of sinigrin and gluconapin in Winspit. The Winspit BoMYB29 CDS was cloned and overexpressed in Winspit and in the DH AG1012 line. Overexpressing Winspit plants produced higher quantities of alkenyl glucosinolates, such as sinigrin. Conversely, the DH AG1012 transformants showed a higher production of methylsulphinylalkyl glucosinolates, including glucoraphanin, and, despite an up-regulation of the aliphatic glucosinolate genes, no increase in alkenyl glucosinolates. The latter may be explained by the absence of a functional AOP2 gene in DH AG1012. Nevertheless, an extract of DH AG1012 lines overexpressing BoMYB29 provided a chemoprotective effect on human colon cells. This work exemplifies how the genetic diversity of B. oleracea may be used by breeders to select for higher expression of transcription factors for glucosinolate biosynthesis to improve its natural, health-promoting properties.

Functional characterization of NRAMP3 and NRAMP4 from the metal hyperaccumulator Thlaspi caerulescens.

The ability of metal hyperaccumulating plants to tolerate and accumulate heavy metals results from adaptations of metal homeostasis. NRAMP metal transporters were found to be highly expressed in some hyperaccumulating plant species. Here, we identified TcNRAMP3 and TcNRAMP4, the closest homologues to AtNRAMP3 and AtNRAMP4 in Thlaspi caerulescens and characterized them by expression analysis, confocal imaging and heterologous expression in yeast and Arabidopsis thaliana. TcNRAMP3 and TcNRAMP4 are expressed at higher levels than their A. thaliana homologues. When expressed in yeast TcNRAMP3 and TcNRAMP4 transport the same metals as their respective A. thaliana orthologues: iron (Fe), manganese (Mn) and cadmium (Cd) but not zinc (Zn) for NRAMP3; Fe, Mn, Cd and Zn for NRAMP4. They also localize at the vacuolar membrane in A. thaliana protoplasts. Inactivation of AtNRAMP3 and AtNRAMP4 in A. thaliana results in strong Cd and Zn hypersensitivity, which is fully rescued by TcNRAMP3 or TcNRAMP4 expression. However, metal tolerance conferred by TcNRAMP expression in nramp3nramp4 mutant does not exceed that of wild-type A. thaliana. Our data indicate that the difference between TcNRAMP3 and TcNRAMP4 and their A. thaliana orthologues does not lie in a different protein function, but probably resides in a different expression level or expression pattern.