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- Materials and Methods
- Results and Discussion
Noccaea caerulescens (formerly Thlaspi caerulescens) is a highly metal-tolerant plant species which hyperaccumulates nickel (Ni), cadmium (Cd) and zinc (Zn) (reviewed by Broadley et al., 2007; Krämer, 2010). It is a short-lived, self-compatible biennial/perennial species of Brassicaceae which is functionally nonmycorrhizal (Regvar et al., 2003). It occurs on calamine, serpentine and nonmineral soils, with a wide distribution in central, northern and western Europe (Reeves & Brooks, 1983; Baker & Brooks, 1989; Reeves et al., 2001). Current evidence indicates that within the Brassicaceae, hyperaccumulation of Ni has evolved independently at least six times, whereas that of Zn and Cd has occurred only twice (Broadley et al., 2007; Krämer, 2010), once at the base of the Noccaea/Raparia clade, and once in Arabidopsis halleri, a species which is also the focus of intense recent study (e.g. Hanikenne et al., 2008).
Significant advances have been made in understanding the genetics of metal tolerance and accumulation in N. caerulescens. These include numerous studies of natural genetic variation in metal tolerance and accumulation (e.g. Ingrouille & Smirnoff, 1986; Baker et al., 1994; Roosens et al., 2003), expression analysis of metal transporter genes (Assunção et al., 2001), cloning and functional characterization of metal transporter genes in heterologous expression systems (e.g. Papoyan & Kochian, 2004), the development of structured populations used for mapping quantitative trait loci (QTL) (e.g. Assunção et al., 2006), global transcriptome analysis (Hammond et al., 2006; van de Mortel et al., 2006, 2008), and the development of protocols for Agrobacterium tumefaciens-mediated transformation (Peer et al., 2003; Guan et al., 2008). However, dissecting the genetic basis and molecular mechanisms of hyperaccumulation in N. caerulescens is challenging as a result of the length of its life cycle and obligate vernalization requirement. Thus, ecotypes cultivated to date require up to 32 wk to flower, including a 7–12 wk period of short-day vernalization (5°C and 8 h photoperiod), with an additional 4 wk for seed ripening (Peer et al., 2003, 2006). In addition to the length of time per se, growing plants for up to 9 months in controlled environments poses a significant challenge (and cost) in terms of husbandry, and increases the potential for genotype × environment interactions, including those associated with maintaining plants in a disease-free state.
The removal of vernalization requirements to induce flowering has led to the development of rapid-cycling populations in several important model Brassicaceae species, including crop Brassica ssp., and this has facilitated molecular genetic analyses (Williams & Hill, 1986; Iniguez-Luy et al., 2009). In late-flowering ecotypes of Arabidopsis thaliana, the vernalization requirement has been removed through fast neutron-induced mutations in either FLOWERING LOCUS C (FLC) or FRIGIDA (FRI), which interact synergistically to repress flowering (Michaels & Amasino, 1999; Sung & Amasino, 2004). Recent expression analysis has identified conserved roles for FLC homologues in vernalization responses in Brassica rapa (Zhao et al., 2010) and Beta vulgaris (Reeves et al., 2007), as well as in the perennial species Arabis alpina (Wang et al., 2009).
The aim of this study was to produce genetically stable fast cycling lines of N. caerulescens using fast neutron mutagenesis, to support future forward and reverse molecular genetic studies. Mutation breeding using fast neutron bombardment of seeds creates random deletions, ranging from one base to > 100 kb, and is commonly employed in mutating plant genomes, representing a rapid approach to obtain large mutant pools (Kodym & Afza, 2003; Salt et al., 2008; Bruce et al., 2009). This technique is a relatively inexpensive method for producing large mutant populations in species whose genomes are not amenable to T-DNA transformation, generating genome-wide saturation in relatively small populations.
Results and Discussion
- Top of page
- Materials and Methods
- Results and Discussion
We generated genetically stable faster-cycling lines of N. caerulescens which are nonvernal-obligate. From 5500 M0 seeds irradiated at 60 Gy, M1 plants were grown using standard procedures for N. caerulescens, including a 10 wk period of vernalization. Approximately 80% of M1 seeds germinated, with 2% showing signs of leaf colour variegation; 79% of plants survived to maturity. Approximately 80 000 M2 seeds were maintained in a single pool at an average of 25 seeds per M1 plant. M2 seeds were grown initially in modules (Fig. 1a) and transplanted to pots under GC (Fig. 1b). The M2 plants were screened for early-flowering phenotypes with no vernalization requirement (Fig. 1c). A total of 0.49% M2 seedlings demonstrated lethal albinism (Fig. 1a). Floral initials were observed in 35 individuals in the absence of vernalization. Of these, nine individual plants flowered within 12 wk, producing an average of 100 M3 seeds per selfed plant (Fig. 1c). One selfed M2 individual, ‘A2’, produced c. 800 M3 seeds (A2M3). Two of these nine plants (A2M3 and A7M3) were selfed, again without vernalization, to produce A2M4 and A7M4 seeds, respectively. These two lines were compared for flowering and mineral uptake traits with an S2 WT line from the original population.
Figure 1. M2 fast neutron mutagenized Noccaea caerulescens: (a) Seedlings growing in 2 cm2 plug trays, showing lethal albinism; (b) a subset of 80 000 M2 is screened for nonvernal-obligate phenotypes under glasshouse conditions; (c) 12-wk-old, prevernal early-flowering M2 plant in the glasshouse. Bar, 10 cm.
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Lines of A2M4, A7M4 and the S2 WT were transplanted to pots under CE conditions at 7 DAS; germination in module trays was > 98% for all lines by 7 DAS. By 66 DAS, all A2M4 plants had developed floral initials, by 71 DAS all A2M4 plants had unopened flower buds, and by 79 DAS all A2M4 plants had fully opened flowers (Fig. 2). The A7M4 flowering was c. 3 wk slower than A2M4. Thus, by 87 DAS, all A7M4 plants developed floral initials, by 97 DAS all had unopened flower buds, and by 104 DAS all had fully open flowers. Silicle development was well established for all A2M4 and the majority of A7M4 individuals by 92 and 123 DAS, respectively. No floral or silicle development was observed in any of the S2 WT plants at these dates.
Figure 2. Noccaea caerulescens lines. S2 wild-type (WT), A2M4 (A2) and A7M4 (A7) growing under controlled-environment conditions (16 h photoperiod, 19°C (± 2°C), and relative humidity 83% (± 10%)). Photographs taken at 70 (a), 96 (b) and 112 (c) d after sowing. Bars, 15 cm.
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Wild-type N. caerulescens produced 32 and 23% more leaf biomass than the A2M4 and A7M4 lines, respectively. However, there was no significant difference in leaf tissue DW between the two faster-cycling lines (data not shown). Mineral analysis of dried leaf tissue demonstrated that both A2M4 and A7M4 rapid-cycling mutant lines contained similar leaf Zn concentrations to the WT, which were in the range > 0.3% Zn on a DW basis. This indicates that the hyperaccumulation phenotype (Reeves & Brooks, 1983; Broadley et al., 2007) was retained. Thus, when grown with the addition of 455 mg Zn kg−1 to the compost, WT, A2M4 and A7M4 plants accumulated 0.34, 0.33 and 0.35% Zn on a DW basis, respectively (Fig. 3a). However, WT, A2M4 and A7M4 plants differed in leaf concentrations of other minerals, including the macronutrients Mg, Ca and K, the micronutrient Fe and Cd, which were all typically higher in the WT. Leaf Mg concentrations (in WT, A2M4 and A7M4 plants, respectively) were 0.42, 0.31 and 0.37%, leaf Ca concentrations were 0.80, 0.61 and 0.69%, leaf K concentrations were 2.86, 2.19 and 2.86% on a DW basis, leaf Fe concentrations were 140, 48 and 103 mg kg−1 DW, and leaf Cd concentrations were 1.1, 0.5 and 0.8 mg kg−1 DW (Fig. 3b–f). These variations might be the result of phenological differences between lines (Nord & Lynch, 2009). In this study, no exogenous Cd was supplied to soil and therefore it is not known if the Cd-hyperaccumulating phenotype has been retained. This requires further study.
Figure 3. Mean leaf concentrations of zinc (Zn; a), magnesium (Mg; b), calcium (Ca; c), potassium (K; d), iron (Fe; e) and cadmium (Cd; f) in rosette leaves of Noccaea caerulescens lines M4A2 (A2), M4A7 (A7) and S2 wild-type (WT) growing under controlled-environment conditions for 123 d. Bars sharing a lower case letter are statistically indistinguishable based on ANOVA and least-significant-difference test (P = 0.05).
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Phenotypically, both mutant lines were stable and there was no evidence of significant intraline variability in flowering, growth or Zn accumulation. Neither A2 nor A7 displayed seed dormancy or altered germination, and all seeds germinated within 7 d. However, there is a significant reproductive cost of accelerated life cycle in terms of decreased fertility. Wild-type plants can typically produce between 500 and 3000 seeds, whereas the mean number of seeds per plant for A2 was 109, and for A7 was 19.
The A2 and A7 lines had clearly lost the requirement for vernalization to initiate flowering whilst remaining self-fertile. From a preliminary backcross experiment to WT Noccaea lines, there is no evidence to date that this nonvernalization trait is dominant. Both mutant lines exhibited much more rapid flowering and seed maturation phenotypes than any WT grown under our conditions. It is likely that these lines will significantly reduce the period currently required to cultivate vernal-obligate WT N. caerulescens (Peer et al., 2003, 2006), enabling production of up to four generations of seed in a single year. Both lines appear to have retained the Zn hyperaccumulator phenotype, and so these lines have potential for establishing further molecular genetic insights, especially when efficient transformation systems and full genome sequence become available. If faster-cycling lines of other N. caerulescens ecotypes can be similarly developed, there is scope for establishing additional mapping populations and introgression lines to facilitate locus resolution. As we have found with rapid-cycling Brassica, the elimination of a vernalization requirement greatly accelerates the ability to resolve traits introgressed from a wide range of germplasm, including subsequent selection for reduced time to flowering and seed maturation. It may also be possible to further mutate lines A2 and A7 to produce even faster-cycling lines in the future. Lines A2 and A7 are available as a community resource from the European Arabidopsis Stock Centre (NASC; http://arabidopsis.info).
We have not yet investigated the molecular basis for rapid cycling in the N. caerulescens A2 and A7 lines. In the first instance, it will be interesting to test if functional homologues of FLOWERING LOCUS C (FLC) and FRIGIDA (FRI) (Michaels & Amasino, 1999; Sung & Amasino, 2004; Reeves et al., 2007; Wang et al., 2009; Zhao et al., 2010) have been affected. It may be possible to test this hypothesis using high-throughput transcriptome sequencing or DNA hybridizations to tiling or exon arrays designed for Arabidopsis (Mockler et al., 2005) or Brassica (Love et al., 2010) using heterologous- (cross-) species-based approaches (Broadley et al., 2008). However, further selfing, backcrossing and complementation will most likely still be required since the mutational load is not yet known.
Our results demonstrate that fast neutron mutagenesis is a viable approach to develop nonvernal-obligate, faster-cycling N. caerulescens lines. It is anticipated that these lines will become a valuable community resource for future molecular genetic investigations into metal tolerance and hyperaccumulation.