Zinc is an essential micronutrient in plants. It is taken up from the soil by the plant roots and transported from there throughout the plant tissues, as a final destination ending up in the plant progeny, the seed. In this route from soil to seed, several bottlenecks occur. The export of zinc into apoplastic gaps is required both for xylem loading in the roots and for loading into the apoplastic space in the seeds, the tissue surrounding the next plant generation. The movement of the positively charged zinc ions out of the symplast is against the electrochemical gradient and thus requires active transport. Both in the model plant Arabidopsis thaliana and in the cereals, the P1B2-ATPases have been demonstrated to be involved in the loading of zinc into the xylem, and here we demonstrate that the Arabidopsis P1B2-ATPase AtHMA2 and AtHMA4 are involved in loading of zinc into the seed apoplast. The P1B2-ATPases contain extended C-terminal domains that are believed to serve an autoregulatory function. Studies on the Arabidopsis P1B2-ATPase AtHMA4 have demonstrated that increasing size deletions of the C-terminal domain results in a gradual increase in the pump’s complementation ability of a zinc hypersensitive yeast strain. The emergence of the CRISPR/Cas9 technology enables us to examine the effect of such C-terminal truncations of AtHMA4 in planta by generating premature stop codons in the C-terminal domain by precision mutagenesis. Here, I present the generation of several such C-terminal truncation mutants of AtHMA4. The use of genome editing tools holds the potential to generate precise mutations while obtaining a non-transgenic plant within two generations. The creation of such non-transgenic but improved plants by genome editing techniques is discussed here in a series of reviews, along with the legal, financial and philosophical implications of using these techniques in crops. One of the feasible uses of genome editing techniques is to repair detrimental mutations that may have occurred in crop plants during domestication in genes controlling traits that could not be selected for. One such trait is the grain zinc content of rice, which varies greatly within domestic rice cultivars and is reported to be higher in wild rice relatives. Here, I report the examination of a mutation in OsHMA2 from the domesticated rice Oryza sativa ssp. japonica cv. Nipponbare, in a region which is otherwise conserved within other rice and monocot plant species.
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Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 2017