Paving the way for genetic improvement of zinc accumulation in Brassica rapa

Brassica rapa L. comprises several vegetable crops, some of which are among the most important vegetables in China, serving as one of the main resources of mineral nutrition for Chinese people. However, the knowledge on the genetics of micronutrient accumulation, including Zn, Fe and Mn, is lacking in B. rapa. By exploring the genetic potential for the improvement of Zn accumulation in B. rapa vegetables I expect to contribute to the reduction of micronutrient malnutrition in China. To characterize the natural variation of Zn accumulation and Zn response upon exposure to different Zn concentrations, I screened a large collection of germplasm representing nine cultivar groups covering the geographic distribution of B. rapa vegetables in China (Chapter 2). The result revealed that there was marked variation in accumulation of Zn (23.2-159.9 µg g-1), Fe (60.3-350.1 µg g-1) and Mn (20.9-53.3 µg g-1) in B. rapa. Zn accumulation correlated with Fe or Mn accumulation both under normal and deficient Zn supply. No significant correlation was detected for the accumulation of these three elements with cultivar groups. A two-fold variation was found for dry-biomass based tolerance to Zn deficiency or excessive Zn. The wide natural variation provides a base for the genetic dissection by quantitative trait locus (QTL) analysis or for developing breeding programs for improved mineral content. QTL analysis is a powerful tool in dissection of complex genetic traits. A doubled haploid (DH) population developed from two Chinese cabbage (B. rapa. ssp. pekinesis) varieties was used for QTL analysis of the accumulation of 11 minerals in leaves and for tolerance to deficient or toxic Zn supplies (Chapter 3). The trait analysis showed significantly positive correlations between the leaf concentrations of the tested minerals, indicating that this must be taken into consideration in breeding programs, especially when toxic minerals (Cd, As) are involved. Seven QTLs were detected for Na, Mg, P, Mn, Zn and Sr leaf concentrations, with an explained variance ranging from 11.1 % to 18.2 % . The tolerance to Zn stress was evaluated by dry shoot biomass of plants grown under different Zn supplies. One common QTL was found affecting Shoot Dry Biomass (SDB) under normal, deficient and excessive Zn supply conditions. An additional QTL was detected for SDB only under Zn excess stress, with an explained variance of 13.0 %. The fact that no major QTL was detected indicates the complexity of the genetic control of the traits, probably involving many loci, each with a limited contribution to mineral accumulation in B. rapa. Furthermore this indicates that the genetic improvement of these traits by classical breeding will be very complicated, as it will require a combination of favourable alleles at many of the genes contributing to the traits. Screening additional populations for larger effect QTLs may provide an alternative. Metal transporters play important roles in maintaining metal homeostasis in plants and are involved in processes of metal uptake, internal transport and storage. Thlaspi caerulescens J&C Presl. is a Zn/Cd/Ni hyperaccumulator, which has been used as a model for plant metal homeostasis research. It has been proposed that the hyperaccumulation is most likely due to the different regulation of the metal homeostasis genes rather than involving genes with novel functions (Van de Mortel et al., 2006). I cloned four metal transporter full-length cDNAs by screening T. caerulescens cDNA libraries, encoding two ZIP transporters (TcZNT5 and TcZNT6, in Chapter 4) and two so-called NRAMP transporters (TcNRAMP3 and TcNRAMP4, in Chapter 5). These four genes showed strongly increased expression in T. caerulescens, as compared to the non-accumulator Arabidopsis thaliana in a wide range of Zn supplies or in the presence of Cd, indicated that these genes were involved in metal hyperaccumulation or tolerance in T. caerulescens. Expression analysis in two T. caerulescens accessions with contrasting Cd accumulation (La Calamine and Ganges) further revealed that their difference in Cd accumulation ability was associated with differences in the regulation of the expression of ZNTs and NRAMPs . Loss of function of the AtZIP5 gene in Arabidopsis increased its tolerance to Cd as measured by reduced root growth, but overexpression of the T. caerulescens orthologue TcZNT5 in Arabidopsis did not lead to any change in phenotype. In contrast, loss of function of AtZIP6 in Arabidopsis did not change its phenotype, but overexpression of the T. caerulescens orthologue TcZNT6 in Arabidopsis increased Cd sensitivity. The Arabidopsis double mutant nramp3 nramp4 showed hypersensitivity to Cd and toxic Zn, in addition to its previously reported hypersensitivity to Fe deficiency. Our study did not reveal functional differences between the T. caerulescens and A. thaliana NRAMPs, which transport the same metals when heterologously expressed in yeast and all four are localized at the vacuolar membrane in plants. Furthermore the phenotype caused by the inactivation of the AtNRAMP3 and AtNRAMP4 genes in Arabidopsis can be rescued by expression of TcNRAMP3. Nevertheless, neither nramp3 nramp4 plants expressing TcNRAMP3, nor wild-type plants expressing TcNRAMP4 at high levels show enhanced Zn or Cd tolerance compared to wild type Arabidopsis. The data presented in this thesis indicate that enhanced Zn accumulation, Zn tolerance or Cd tolerance probably requires elevated and controlled expression of a set of genes, rather than only single metal transporter genes.