TRANSCRIPT ANALYSIS OF PSFRO AND RIT1 IN WILD-TYPE AND MUTANT PEA PLANTS USING QUANTITATIVE PCR

Authors: LI, CHEE-MING - BAYLOR COLLEGE MED; Grusak, Michael

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 7/15/2002
Publication Date: 7/15/2002
Citation: LI, C., GRUSAK, M.A. TRANSCRIPT ANALYSIS OF PSFRO AND RIT1 IN WILD-TYPE AND MUTANT PEA PLANTS USING QUANTITATIVE PCR. ABSTRACTS OF THE XI INTERNATIONAL SYMPOSIUM ON IRON NUTRITION AND INTERACTIONS IN PLANTS. 2002. p. 131.

Interpretive Summary:

Technical Abstract: In Strategy I plants, such as pea, iron-deficiency stress leads to an enhancement in root Fe(III) reductase activity, along with an apparent increase in Fe2+ transport capacity. In concert with these functional changes, previous studies with Arabidopsis, pea, and tomato have shown similar iron-deficiency induced increases in mRNA levels for the reductase and/or the Fe2+ transporter genes. These expression studies generally have utilized Northern analyses to make gross, qualitative comparisons in plants grown with or without iron. However, no detailed, quantitative expression data have been reported for these important iron acquisition genes, and thus we have no clear understanding of the relationship between expressed levels of mRNA and observed functional activity. For several years, we have used pea as a model plant to study root iron nutritional physiology and have made much use of the iron-hyperaccumulating mutants, dgl and brz, which exhibit constitutively elevated levels of root reductase activity. We are fortunate that the root Fe(III) reductase gene, PsFRO, and the Fe2+ transporter gene, RIT1, recently have been cloned in pea. With these sequences available, we designed primers to measure relative mRNA levels using real-time, or quantitative PCR techniques (Quant-PCR), in roots of wild-type (cv. Sparkle) and mutant (brz and dgl) plants. All PsFRO and RIT1 transcript levels were calculated in terms of their abundance relative to 18S ribosomal RNA. Plants were grown with or without Fe and harvested roots were immediately frozen in liquid nitrogen prior to mRNA isolation. Quant-PCR analysis revealed that PsFRO transcript levels were 35- to 90-fold higher in -Fe WT and +/-Fe brz and dgl roots, relative to +Fe-grown WT roots. This is interesting, in that previous studies of root Fe(III) reductase activity showed only a 3- to 4-fold increase in -Fe WT or mutant roots, relative to +Fe WT controls. This discrepancy in the induction of transcripts, relative to functional activity, suggests that either there is a problem with the translational efficiency of PsFRO, or that other factors limit the measurable activity. Availability/turn-over of internal reductant is one possibility. For RIT1, Fe deficiency resulted in only a 4- to 6-fold increase in the level of transcripts, relative to +Fe-grown plants; this is an induction much more in line with relative changes in Fe2+ transport rates. However, these results also suggest the possibility of differential transcriptional regulation of PsFRO relative to RIT1. We will discuss these findings in terms of their relevance to whole-plant iron acquisition and homeostasis.