Progress 07/15/02 to 07/14/05
Outputs
White lupin (Lupinus albus) can acquire soil phosphorus (P) effectively through the development of proteoid roots and a number of other coordinated root system responses, including induction of organic acid synthesis and excretion. The objectives of this grant were to: 1) develop and use ESTs to analyze global gene expression in P deficiency induced proteoid roots; and 2) isolate the promoter for a minus P-induced MATE (multi-drug and toxin efflux) gene and evaluate MATE expression and function. Nylon filter arrays spotted with about 3200 ESTs derived from cDNA libraries isolated at different stages of proteoid root development were performed to evaluate global aspects of white lupin gene expression in response to minus P stress. Some 30 plus genes were consistently upregulated in all experiments by at least 2-fold under P-deficiency conditions. We have developed transgenic proteoid roots via Agrobacterium rhizogenes, which coupled to RNAi technology, will allow us to
perform functional analysis of white lupin genes involved in proteoid root development. We successfully silenced an endogenous P-deficiency induced MATE gene in white lupin proteoid roots. We have also pursued the identification of gene promoters controlling P-signaling in white lupin through the isolation and functional characterization of 5'-upstream promoter regions for P-deficiency induced phosphate transporter 1 (PT1), secreted acid phosphatase (SAP), and multi-drug-toxin extrusion (MATE).
Impacts
Increasing our understanding of the gene regulation of phosphorus deficiency in white lupin may lead to crop improvements that allow more efficient acquisition of this increasingly expensive, non-renewable agricultural input.
Publications
- Liu, J.Q., D.A. Samac, B. Bucciarelli, D.L. Allan, and C.P. Vance. (2005) Signaling of phosphorus deficiency-induced gene expression in white lupin requires sugar and phloem transport. Plant Journal 41:257-268
- Uhde-Stone, C., J. Liu, K. Zinn, D.Allan, and C. Vance. (2005) Transgenic proteoid roots of white lupin: A vehicle for characterizing and silencing root genes involved in adaptation to P stress. Plant Journal (accepted 6 Sept, 2005)
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Progress 01/01/04 to 12/31/04
Outputs
The specific objectives of our work on white lupin were to: 1) develop and use ESTs to analyze global gene expression in -P induced proteoid roots; and 2) isolate the promoter for a -P induced MATE (multidrug toxin extrusion) gene and evaluate MATE expression and function. In addition to the 2,100 ESTs derived from proteoid root cDNA libraries we have previously reported, we have sequenced an additional 1,100 ESTs derived from a cDNA library prepared with RNA isolated from the root zone containing pre-emergent newly initiated cluster root meristems. These sequences contain an abundance of highly redundant ESTs encoding putative transcription factors, membrane channel/transporters, and hormone metabolism related proteins. In efforts to better understand the mechanisms underpinning the signaling of P-deficiency, we evaluated the effect of light, exogenous sugars, and phloem transport on the expression of P-deficiency induced genes in seedlings and cluster roots of white
lupin (84). Exogenous sucrose, glucose, and fructose stimulated phosphate transporter 1 (PT1), secreted acid phosphatase (SAP), and multidrug-toxin extrusion (MATE) transcript accumulation in dark-grown, P-sufficient seedlings. Furthermore, in intact P-deficient white lupin PT1, SAP, and MATE expression in cluster roots was strikingly reduced in dark-adapted plants with expression rapidly restored upon re-exposure to light. Likewise, interruption of phloem supply to P-deficient cluster roots resulted in a rapid decline in PT1, SAP, and MATE transcript accumulation. Our most recent experiments have shown that hexokinase transcripts are upregulated in -P-stressed cluster roots and are modulated by light and sugars similar to PT1, SAP, and MATE. We have also pursued the identification of gene promoters controlling P-signaling in white lupin through the isolation and functional characterization of 5'-upstream promoter regions for P-deficiency induced PT1, SAP, and MATE. Recently, we have
isolated the promoter for a P-deficiency cluster root associated MYB transcription factor gene and shown its functional regulation of a reporter gene in transgenic lupin roots. We have recently achieved success in transforming white lupin with A. rhizogenes engineered to carry several promoter::GUS chimeric reporter genes and RNAi constructs. Upon transformation of white lupin with A. rhizogenes containing a GUS::reporter gene driven by the promoters of genes induced in response to P-deficiency, we found reporter gene activity greatest in proteoid roots of transformed P-stressed plants. In addition, we have been successful in silencing an endogenous P-deficiency induced MATE gene in white lupin proteoid roots after transformation with an appropriate RNAi construct. The plants that were silenced in MATE gene expression were less vigorous than control plants (average dry weight reduced 20-25%). Furthermore, H+ extrusion and P and Al accumulation appeared to be reduced in the RNAi
plants. It is important to note that proteoid root growth and development of plants transformed with A. rhizogenes appears normal (visually comparable to untransformed proteoid roots).
Impacts
The information obtained from these studies will be useful for developing strategies to improve nutrient uptake efficiency in other plant systems. Such improvements have ecological and environmental implications by reducing the need for P fertilizer and extending the potential for growing crops in P deficient soils.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
White lupin can effectively acquire P even though it does not form a mycorrhizal symbiosis. Instead, its adaptation to P stress is a highly coordinated modification of root development and biochemistry resulting in cluster (proteoid) roots that exude copious amounts of organic acids and acid phosphatase. The specific objectives of the current project are to: 1) develop and use ESTs to analyze global gene expression in -P stress induced proteoid roots; and 2) determine whether a -P induced EST with striking similarity to a MATE (multi-drug and toxin efflux) protein encodes an organic acid transporter. Over the past 2.5 years we have isolated, sequenced, contiged, deposited in GenBank, and analyzed on nylon filter arrays 2,100 ESTs derived from proteoid root cDNA libraries. One of the 35 P-responsive contigs identified displayed homology to a MATE gene. Several approaches have been utilized to define the possible function of the corresponding protein, which was
predicted to be an integral membrane protein. Due to inherent difficulties in functional analysis of membrane proteins using heterologous systems, we are currently assessing the function of MATE directly in white lupin through RNAi technology. This approach was made possible by our recent success with A. rhizogenes-mediated transformation of white lupin roots. Another approach of our current work is the isolation of P-responsive genes. In the current granting period we have isolated and sequenced the genomic region of LaMATE and LaMYB, the latter encoding a MYB transcription factor. The 5'-upstream putative promoter region of LaMATE was fused to the GUS and GFP reporter genes. In addition, a translation fusion was made between the coding region of LaMATE and GFP under the control of the LaMATE promoter. All three constructs were used for A. rhizogenes-mediated transformation of white lupin. The LaMATE promoter directed -P-dependent GUS and GFP expression to proteoid roots, and
expression varied according to the stage of proteoid root development. Arabidopsis transformed with the MATE:GUS and MATE::GFP also showed root specific reporter gene activity. Most recently we have made a series of reporter gene promoter deletions (via PCR) at 300 bp intervals for 1.5 kb of the 5'-upstream region of two formerly isolated P-responsive genes, LaAPase and LaPt. The reporter deletions were transformed into Arabidopsis and are currently being analyzed.
Impacts
The information obtained from these studies will be useful for developing strategies to improve nutrient uptake efficiency in other plant systems. Such improvements have ecological and environmental implications by reducing the need for P fertilizer and extending the potential for growing crops in P deficient soils.
Publications
- Uhde-Stone, C., G. Gilbert, J. M-F Johnson, R. Litjens, K.E. Zinn, S.J. Temple, C.P. Vance, and D.L. Allan. 2003. Acclimation of white lupin to phosphorus deficiency involves enhanced expression of genes related to organic acid metabolism. Plant Soil 248:99-116.
- Uhde-Stone, C., K.E. Zinn, M. Ramirez-Yanez, A. Li, C.P. Vance, and D.L. Allan. 2003. Nylon filter arrays reveal differential gene expression in proteoid roots of white lupin in response to phosphorus deficiency. Plant Physiol. 131:1064-1079.
- Vance, C.P., C. Uhde-Stone, and D.L. Allan. 2003. Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol. 157:423-447.
- Uhde-Stone, C., K. Zinn, J. Liu, S. Miller, A. Reinders, J. Ward, D. Allan, and C. Vance. 2003. A MATE transporter gene from phosphorus deficient white lupin: possible involvement in proteoid root function. 2nd International Symposium on Phosphorus Dynamics in the Soil-Plant Continuum, Perth, W. Australia, September, 2003.
- Uhde-Stone, C., J. Liu, M. Ramirez, S. Miller, D. Samac, D. Allan, and C. Vance. 2003. Modification of the rhizosphere by cluster roots of phosphorus stressed white lupin. Midwest Rhizosphere Research Symposium, Danforth Plant Science Center, St. Louis, MO, September, 2003.
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Progress 01/01/02 to 12/31/02
Outputs
White lupin (Lupinus albus) can acquire soil phosphorus (P) effectively through the development of proteoid roots and a number of other coordinated root system responses, including induction of organic acid synthesis and excretion. The objectives of the current project are to: 1) develop and use ESTs to analyze global gene expression for white lupin roots under P stress as well as other nutrient stresses; 2) determine whether a -P stress induced EST with striking similarity to a MATE (multi-drug and toxin extrusion) protein encodes an organic acid efflux protein; and 3) define the 5' upstream region for several -P induced genes and characterize whether they share common regulatory elements. To further characterize the MATE EST we have identified, we will combine RNA blot analysis, in situ hybridization, immunolocalization, and functional analysis in yeast and/or Xenopus oocytes. Two strains of yeast have already been transformed with the MATE gene or expression vector
only; the culture medium was then purified and analyzed for organic acids by HPLC. MATE transformation resulted in a large increase in size of an unidentified peak for both strains. Samples are currently being submitted for mass spec analysis to confirm the identity of the peak. To understand how the expression of genes induced in -P proteoid roots is controlled, three types of experimental approaches will be utilized. We have further characterized the temporal and spatial patterns of a phosphate transporter gene (LaPT1) and an acid phosphatase gene (LaSAP1) by introducing promoter::GUS reporter gene fusions into another legume species, alfalfa. Both LaPT1::GUS and LaSAP1::GUS reporter genes showed significant induction by P deficiency in transgenic alfalfa. Another approach underway is the isolation of genes induced in -P proteoid roots and computational analysis of the 5'-upstream region, which has already revealed the presence of common cis elements. Finally, we hope to identify
the trans-acting DNA binding factors that interact with specific elements within promoters by using a yeast one-hybrid system.
Impacts
The ultimate goal of this project is to develop strategies that may improve nutrient uptake efficiency in other plant systems. Improvement of P uptake efficiency has both ecological and economic implications by reducing the need for P fertilizer and extending the potential for growing crops in P-deficient soils.
Publications
- No publications reported this period
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