Development of biomarkers for evaluating phosphate stress in Thellungiella salsuginea

Abstract

<p>Phosphorus is a macronutrient required for plant growth and reproduction. Insufficient supplies of phosphate will adversely impact plant growth. In an effort to supply adequate phosphate to crops, large quantities of phosphate-rich fertilizer are applied to fields but much of the phosphate can leach from the soil as run-off, impacting water systems. Therefore, proper management of phosphate and the development of phosphate efficient genotypes of plants are strategies needed for a sustainable agriculture industry.</p> <p>This thesis project focused on the development of biomarkers of phosphate stress in <em>Thellungiella salsuginea, </em>a plant highly tolerant to salt, cold and water deficit. Biomass determinations and real-time quantitative PCR were used to determine the gene expression of several genes selected as known phosphate-responsive genes from studies of phosphate starvation of the related genetic model plant <em>Arabidopsis thaliana.</em></p> <p><em> Thellungiella </em>seedlings were grown on 5 and 500 µM phosphate media. The expression of several genes (<em>RNS1, At4, Pht1;1, Pht1;4, Pht1;5, Siz1, PHR1, WRKY75, </em>and<em> Pht2;1</em>) were assayed for their response to media phosphate content. <em>RNS1</em> and <em>At4 </em>expression was estimated from cDNA prepared from shoot tissues while <em>At4, Pht1;1</em> and <em>Pht1;5</em> expression was determined from root tissues. In all tissue sources, significantly increased expression of <em>RNS1</em>, <em>At4</em>,<em> Pht1;1</em> and <em>Pht1;5</em> was observed under 5 µM phosphate exposure.</p> <p><em> </em>Two natural accessions of <em>Thellungiella</em> were used in this study with one originating from the Yukon Territory, Canada and the second from Shandong Province, China. Seedlings of both ecotypes were grown on defined media plates containing various concentrations of phosphate (0, 25, 125, 250, 500, and 2000 µM). For both accessions, the addition of as little as 25 µM phosphate led to significant increases in root and shoot biomass. Gene expression levels corresponding to <em>RNS1, At4</em> and <em>Pht1;1</em> were the highest in Yukon and Shandong <em>Thellungiella </em>grown on 0 µM phosphate media. The addition of 25 µM phosphate to the media was enough to significantly decrease transcript abundance of <em>RNS1, At4 </em>and <em>Pht1;1. </em>In a test using the transfer of Yukon <em>Thellungiella </em>seedlings from high (500 µM) to low (5 µM) phosphate the expression of <em>At4</em> in roots and shoots increased 30-fold over a five-day period and only <em>Pht1;1</em> expression increased in the roots over the same time period.</p> <p><em>RNS1</em> and <em>At4</em> share attributes that make them suitable biomarkers for phosphate stress in plants. Both genes are expressed in the shoots making it easier to remove tissue for monitoring gene expression, and both genes show readily discernible increases in transcript levels for determination by qPCR. At present, however, the role for their products in phosphate assimilation by plants is uncertain. This lack of knowledge is a deterrent to adopting these genes for widespread use as biomarkers. In particular, more work needs to be done to characterize factors that elicit their expression to test the specificity of their response to phosphate stress in <em>Thellungiella</em>.</p>