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. 2010 Mar;3(2):347-60.
doi: 10.1093/mp/ssq007. Epub 2010 Feb 10.

A Contribution to Identification of Novel Regulators of Plant Response to Sulfur Deficiency: Characteristics of a Tobacco Gene UP9C, Its Protein Product and the Effects of UP9C Silencing

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A Contribution to Identification of Novel Regulators of Plant Response to Sulfur Deficiency: Characteristics of a Tobacco Gene UP9C, Its Protein Product and the Effects of UP9C Silencing

Malgorzata Lewandowska et al. Mol Plant. .
Free PMC article

Abstract

Extensive changes in plant transcriptome and metabolome have been observed by numerous research groups after transferring plants from optimal conditions to sulfur (S) deficiency. Despite intensive studies and recent important achievements, like identification of SLIM1/EIL3 as a major transcriptional regulator of the response to S-deficiency, many questions concerning other elements of the regulatory network remain unanswered. Investigations of genes with expression regulated by S-deficiency stress encoding proteins of unknown function might help to clarify these problems. This study is focused on the UP9C gene and the UP9-like family in tobacco. Homologs of these genes exist in other plant species, including a family of four genes of unknown function in Arabidopsis thaliana (LSU1-4), of which two were reported as strongly induced by S-deficit and to a lesser extent by salt stress and nitrate limitation. Conservation of the predicted structural features, such as coiled coil region or nuclear localization signal, suggests that these proteins might have important functions possibly mediated by interactions with other proteins. Analysis of transgenic tobacco plants with silenced expression of UP9-like genes strongly argues for their significant role in regulation of plant response to S-deficit. Although our study shows that the UP9-like proteins are important components of such response and they might be also required during other stresses, their molecular functions remain a mystery.

Figures

Figure 1.
Figure 1.
Amino Acid Sequence Alignment of UP9-Like Proteins from Tobacco and Arabidopsis (A) and Circular Dichroism Spectrum of Recombinant UP9C (B). (A) All tobacco UP9-like ESTs identified after a database similarity search with a coding region of UP9C were grouped into six clusters (arbitrary denoted UP9A-F) according to the sequence of their coding regions: UP9A (GB# DQ444223, DV999485, FG623584, BP535499), UP9B (GB# BP533592, FG638291, FG637038), UP9C (GB# AY547446, FG641107, EB445189), UP9D (GB# FG636997), UP9E (GB# BP531586, EB430507), UP9F (GB# BY31337). The alignment of six open reading frames deduced from each of the tobacco EST group and of four homologs from A. thaliana (NP_190527, NP_190526, NP_197854, and NP_56450) was performed using the MAFFT. The asterisks above the sequences indicate residues identical in six tobacco proteins, while the asterisks below the sequences indicate the residues identical in all aligned proteins. The stripped bar above alignment shows the alpha-helical region predicted by GOR4 and its filled part indicates a two-stranded coiled coil detected by MULTICOIL. (B) The mean values of three independent measurements with SD-indicated molar ellipticity (deg cm2 dmol−1) of 64 μM UP9C protein in a light spectrum between 190 and 250 nm are shown.
Figure 2.
Figure 2.
Sub-Cellular Localization of the UP9C–EYFP Fusion Protein in the Root of 3-Week-Old Transgenic Tobacco. (A) Light microscopy image showing the cell nucleus marked by an arrow. (B) Fluorescence microscopy image showing the nuclear localization of the UP9C–EYFP fusion protein. (C) Fluorescence microscopy image showing the nucleus stained with DAPI. (D) Overlay of (B) and (C). (E) Overlay of (A), (B), and (C). Scale bar  =  10 μm.
Figure 3.
Figure 3.
Evaluation of Expression Levels of UP9-Like Transcripts in Mature Leaves of the Analyzed Plant Lines. The primers used in semiquantitative RT–PCR (sqRT–PCR) were designated to the non-coding sequences of the indicated EST clones and they are listed in Table 2. The GB#, the name of the protein cluster, and the number of cycles are marked next to the corresponding expression profile; wt, the parental line (LA Burley 21).
Figure 4.
Figure 4.
Contents of Glutathione (A) and Total S (B) in the Analyzed Tobacco Lines. Plants were grown in the optimal AB medium (nS) or for 2 d in S-deficient conditions in the AB-S medium (–S). The statistically significant differences between the transformants and the parental line (wt) grown in the same conditions are marked with either a single (for p < 0.05) or double (for p < 0.01) asterisks. (A) The material was combined from five plants for each analyzed group; the mean data from five independent extractions with standard deviations (SD) are shown. (B) The material was combined from five plants for each analyzed group; the mean of three assays with SD values are shown.
Figure 5.
Figure 5.
Comparison of Root and Shoot Lengths in Seedlings Grown for 3 Weeks on AB Plates Maintained in a Vertical Position in Either the Optimal (nS) or S-Deficient (–S) AB-S Medium. The average data (with SD indicated) were calculated from 30–50 measured seedlings, depending on the line. The statistically significant (p < 0.05) differences between the transformants and the control (CNTR) are marked with asterisks. Similar results were obtained for two control lines, parental line (not shown), and a tobacco line transformed with GFP expression cassette (CNTR).
Figure 6.
Figure 6.
The Expression Levels of the Selected Genes in Mature Leaves of the Analyzed Tobacco Lines. In all real time RT–PCR (qRT–PCR) reactions, both Tac9 and UBQ2 served as references, except for the qRT–PCR shown in (A), in which only Tac9 was used as a reference gene. nS, optimal conditions; –S, 2 d of S-deficit. Each experiment was performed at least two times in triplicate using two independently isolated RNA templates, with similar results. The presented data are from one representative experiment performed in triplicate with SD indicated.
Figure 7.
Figure 7.
Principal Component Analysis of Metabolite Profiling Data for Parental Line (wt) and the Transgenic Lines Grown Either in the Optimal Medium (nS) or in the S-Deficient Medium (–S).

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