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Title: TRANSGENIC PROTEOID ROOTS OF WHITE LUPIN: A VEHICLE FOR CHARACTERIZING AND SILENCING ROOT GENES INVOLVED IN ADAPTATION TO P STRESS

Author
item UHDE-STONE, CLAUDIA - UNIVERSITY OF MINNESOTA
item LIU, JUNQI - UNIVERSITY OF MINNESOTA
item ZINN, KELLY - UNIVERSITY OF MINNESOTA
item ALLAN, DEBORAH - UNIVERSITY OF MINNESOTA
item Vance, Carroll

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/14/2005
Publication Date: 10/1/2005
Citation: Uhde-Stone, C., Liu, J., Zinn, K.E., Allan, D.L., Vance, C.P. 2005. Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress. Plant Journal. 44(5):840-853.

Interpretive Summary: Plant roots are critical organs for acquiring the nutrients needed for plant growth. Under growth conditions in which nutrients are limiting, roots undergo striking changes in development and physiology. Low amounts of phosphorus (P) limit the growth of crop plants on 33% of Earth’s farm land. Research in this report focuses on biochemical and molecular changes that occur in the roots of white lupin plants in response to insufficient P. When white lupin plants are grown under low P conditions, they develop very unusual root systems. Under insufficient P, white lupin forms large numbers of roots that look very much like bottle brushes. These roots are called cluster roots. Formation of cluster roots increases the root surface area allowing more soil to be explored for acquiring P. Cluster roots also have many biochemical modifications for acquiring P including secretion of enzymes that solubilize P, increase in synthesis of proteins that help in taking P from the soil solution, and release of small organic compounds that help release soil P. The objective of this research was to characterize a unique gene (MATE) that is highly expressed and encodes a protein found in cluster roots only under conditions of P deficiency. The unique MATE protein appears to be involved in cluster root cell membrane function. Analysis of gene expression, protein localization, and gene sequence showed that MATE was synthesized early in P-deficiency stress as cluster roots form. The protein has 11 membrane domains and is located only in cluster root cells. When expression of MATE is blocked, the protein is not detectable in P-deficient cluster roots and plant shoots are reduced in growth. These data indicate that MATE is critical to lupin cluster root adaptation to P-deficiency. Discovery of this gene is useful because it may be used by plant breeders and biotechnologists to improve crop adaptation to low P soils.

Technical Abstract: White lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to -P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system we have developed an A. rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNAi based gene silencing. We used this system to characterize a lupin multi-drug and toxin efflux (LaMATE) gene previously shown to have enhanced expression under -P stress. Here we show that LaMATE had high expression in proteoid roots not only under -P but also under -Fe, -N, -Mn, and +Al stress. A portion containing the putative LaMATE promoter was fused to GUS and EGFP reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis points to the plasma membrane as the likely location of the LaMATE protein. LaMATE displayed homology to FRD3 in Arabidopsis but did not complement an Arabidopsis FRD3 mutant. RNAi based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.