Limits...
Overexpression of the transporters AtZIP1 and AtMTP1 in cassava changes zinc accumulation and partitioning.

Gaitán-Solís E, Taylor NJ, Siritunga D, Stevens W, Schachtman DP - Front Plant Sci (2015)

Bottom Line: Eighteen transgenic events from four constructs, out of a total of 73 events generated, showed significantly higher zinc concentrations in the edible portion of the storage root compared to the non-transgenic controls.The zinc content in the transgenic lines ranged from 4 to 73 mg/kg dry weight (DW) as compared to the non-transgenic control which contained 8 mg/kg.Striking changes in whole plant phenotype such as smaller plant size and chlorotic leaves were observed in transgenic lines that over accumulated zinc.

View Article: PubMed Central - PubMed

Affiliation: Donald Danforth Plant Science Center, St Louis, MO USA.

ABSTRACT
Zinc deficiency in humans is a serious problem worldwide with an estimated one third of populations at risk for insufficient zinc in diet, which leads to impairment of cognitive abilities and immune system function. The goal of this research was to increase the bioavailable zinc in the edible portion of cassava roots to improve the overall zinc nutrition of populations that rely on cassava as a dietary staple. To increase zinc concentrations, two Arabidopsis thaliana genes coding for ZIP1 and MTP1 were overexpressed with a tuber-specific or constitutive promoter. Eighteen transgenic events from four constructs, out of a total of 73 events generated, showed significantly higher zinc concentrations in the edible portion of the storage root compared to the non-transgenic controls. The zinc content in the transgenic lines ranged from 4 to 73 mg/kg dry weight (DW) as compared to the non-transgenic control which contained 8 mg/kg. Striking changes in whole plant phenotype such as smaller plant size and chlorotic leaves were observed in transgenic lines that over accumulated zinc. In a confined field trial five transgenic events grown for 12 months showed a range of zinc concentrations from 18 to 217 mg/kg DW. Although the overexpression of zinc transporters was successful in increasing the zinc concentrations in 25% of the transgenic lines generated, it also resulted in a decrease in plant and tuber size and overall yield due to what appears to be zinc deficiency in the aerial parts of the plant.

No MeSH data available.


Related in: MedlinePlus

Copy number determination using Southern blot hybridization of genomic DNA samples isolated from leaves of wild type and transgenic lines disgested with HindIII (A) and BamHI (B). WT, wild type.
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Figure 2: Copy number determination using Southern blot hybridization of genomic DNA samples isolated from leaves of wild type and transgenic lines disgested with HindIII (A) and BamHI (B). WT, wild type.

Mentions: Confirmation of transgene integration was confirmed by Southern blot analysis. Transgenic lines mostly had one to two copies of integrated T-DNA with only one line showing integration of three copies (Figure 2). Northern blot analysis was performed on all 18 Zn accumulating plant lines shown in Figure 3 to confirm expression of AtMTP1 and AtZIP1 transcripts in different plant parts. RNA from 4-months-old plants was loaded across the blot from lines containing high zinc concentrations to those containing lower concentrations in the starchy roots (Figure 3) to visualize any relationship between zinc concentration and transgenic RNA expression. All zinc accumulating events showed strong transgene expression (Figure 3C, upper panel). Levels of expression were similar for transgenic plants containing cassettes driven by the patatin and FMV promoters, with strongest expression seen in the storage root tissue, followed by the leaves and then fibrous roots. Data indicate that the FMV promoter was driving expression of AtZIP1 to higher levels than the patatin promoter. This correlates with the higher levels of zinc accumulation seen in storage roots for FMV:AtZIP1 events compared to PAT:AtZIP1 (Figure 1). While transgene expression across all tissue types is expected in the case of the constitutive FMV promoter, the analysis of RNA transcripts showed that the patatin promoter is effective for driving transgene expression in the storage roots but expression was not restricted to this organ in plants 4 months after transfer to soil in the greenhouse.


Overexpression of the transporters AtZIP1 and AtMTP1 in cassava changes zinc accumulation and partitioning.

Gaitán-Solís E, Taylor NJ, Siritunga D, Stevens W, Schachtman DP - Front Plant Sci (2015)

Copy number determination using Southern blot hybridization of genomic DNA samples isolated from leaves of wild type and transgenic lines disgested with HindIII (A) and BamHI (B). WT, wild type.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496839&req=5

Figure 2: Copy number determination using Southern blot hybridization of genomic DNA samples isolated from leaves of wild type and transgenic lines disgested with HindIII (A) and BamHI (B). WT, wild type.
Mentions: Confirmation of transgene integration was confirmed by Southern blot analysis. Transgenic lines mostly had one to two copies of integrated T-DNA with only one line showing integration of three copies (Figure 2). Northern blot analysis was performed on all 18 Zn accumulating plant lines shown in Figure 3 to confirm expression of AtMTP1 and AtZIP1 transcripts in different plant parts. RNA from 4-months-old plants was loaded across the blot from lines containing high zinc concentrations to those containing lower concentrations in the starchy roots (Figure 3) to visualize any relationship between zinc concentration and transgenic RNA expression. All zinc accumulating events showed strong transgene expression (Figure 3C, upper panel). Levels of expression were similar for transgenic plants containing cassettes driven by the patatin and FMV promoters, with strongest expression seen in the storage root tissue, followed by the leaves and then fibrous roots. Data indicate that the FMV promoter was driving expression of AtZIP1 to higher levels than the patatin promoter. This correlates with the higher levels of zinc accumulation seen in storage roots for FMV:AtZIP1 events compared to PAT:AtZIP1 (Figure 1). While transgene expression across all tissue types is expected in the case of the constitutive FMV promoter, the analysis of RNA transcripts showed that the patatin promoter is effective for driving transgene expression in the storage roots but expression was not restricted to this organ in plants 4 months after transfer to soil in the greenhouse.

Bottom Line: Eighteen transgenic events from four constructs, out of a total of 73 events generated, showed significantly higher zinc concentrations in the edible portion of the storage root compared to the non-transgenic controls.The zinc content in the transgenic lines ranged from 4 to 73 mg/kg dry weight (DW) as compared to the non-transgenic control which contained 8 mg/kg.Striking changes in whole plant phenotype such as smaller plant size and chlorotic leaves were observed in transgenic lines that over accumulated zinc.

View Article: PubMed Central - PubMed

Affiliation: Donald Danforth Plant Science Center, St Louis, MO USA.

ABSTRACT
Zinc deficiency in humans is a serious problem worldwide with an estimated one third of populations at risk for insufficient zinc in diet, which leads to impairment of cognitive abilities and immune system function. The goal of this research was to increase the bioavailable zinc in the edible portion of cassava roots to improve the overall zinc nutrition of populations that rely on cassava as a dietary staple. To increase zinc concentrations, two Arabidopsis thaliana genes coding for ZIP1 and MTP1 were overexpressed with a tuber-specific or constitutive promoter. Eighteen transgenic events from four constructs, out of a total of 73 events generated, showed significantly higher zinc concentrations in the edible portion of the storage root compared to the non-transgenic controls. The zinc content in the transgenic lines ranged from 4 to 73 mg/kg dry weight (DW) as compared to the non-transgenic control which contained 8 mg/kg. Striking changes in whole plant phenotype such as smaller plant size and chlorotic leaves were observed in transgenic lines that over accumulated zinc. In a confined field trial five transgenic events grown for 12 months showed a range of zinc concentrations from 18 to 217 mg/kg DW. Although the overexpression of zinc transporters was successful in increasing the zinc concentrations in 25% of the transgenic lines generated, it also resulted in a decrease in plant and tuber size and overall yield due to what appears to be zinc deficiency in the aerial parts of the plant.

No MeSH data available.


Related in: MedlinePlus