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Upstream signalling, global regulatory control and biochemical function of central components in the zinc homeostasis network: towards the rational design of technologies for bio-fortification

Final Report Summary - CENTZIN (Upstream signalling, global regulatory control and biochemical function of central components in the zinc homeostasis network: ? technologies for bio-fortification)

Publishable Summary

Summary of project objectives
The three objectives of this project are to:
A. Functionally characterise ZIF1, a vacuolar transport protein that is known to be important for Zn tolerance in the model plant Arabidopsis thaliana, particularly with respect to identifying the transported substrate of this protein.
B. Identify, through a forward genetic approach, components of transcriptional regulation of Zn tolerance mechanisms in A. thaliana by screening for mutants with impaired regulation of ZIF1 under Zn stress.
C. Identify components of long-distance or systemic signalling of transcriptional regulation of Zn uptake in A. thaliana by utilising a comparative microarray approach between wild-type plants and hma2hma4 mutants, which are impaired in root-to-shoot transport of Zn.

Description of the work completed
Towards objective A, extensive physiological characterisation of plants with altered expression of ZIF1 has been carried out in order to identify the potential substrate of this transporter as well as progressing towards a better understanding of the physiological function of the protein. Plants over-expressing or mutant in ZIF1 have been analysed for metal and metabolite contents and partitioning in whole tissues and cellular fractions to identify a candidate for ZIF1 transport. In addition, growth response assays and transcriptional phenotyping in these plants grown under a range of micronutrient conditions have revealed novel aspects of ZIF1 function.

Towards objective B, a total of 3 000 mutant seedlings have been screened for altered expression of ZIF1. So far, this has yielded thirteen candidate mutants for further analysis.

Towards objective C, comparative microarray analysis has been carried out in roots and shoots of hma2,hma4 mutants and wild-type controls. This has revealed candidate transcripts implicated in systemic regulation of Zn-deficiency responses and this regulation has been confirmed with quantitative real-time PCR. Promoter-reporter fusions have also been generated to observe spatial regulation of the candidate genes and transformed in hma2,hma4 mutants and wild-type plants.

Main results achieved so far
Towards objective A, ZIF1 has been revealed to be critical for optimal growth of Arabidopsis under newly identified conditions. This is in addition to its previously established role in basal Zn tolerance. ZIF1 is transcriptionally upregulated under this condition and ZIF1 mutants and over-expressors have impaired growth under this condition, consistent with an important role under this condition. In addition, ZIF1 over-expressors have been shown to contain extraordinarily high root concentrations of a metal chelator. This provides evidence concerning the transported substrate of ZIF1.

Towards objective B, 13 candidate mutants have been identified from 3 000 mutant seed screened so far. These have been assessed for regulation of ZIF1 by high-Zn stress and by Fe-deficiency, revealing separable regulation of ZIF1 by these treatments.

Towards objective C, eight candidate transcripts have been selected from the comparative microarray experiment for more detailed analysis. These represent putative targets of either systemic or long-distance regulation of the Zn deficiency response in Arabidopsis and include a putative Zn transporter, several putative transcription factors and transcripts related to genes implicated in regulation of protein abundance via the proteasome.

Expected final results
It is expected that major novel insights will be obtained with respect to the functioning of micronutrient metal homeostasis and its regulation. The comprehensive insights reached in this model organism will allow an application in the biofortification of crops.