Glutaredoxin AtGRXS8 represses transcriptional and developmental responses to nitrate in Arabidopsis thaliana roots

Abstract

Glutaredoxins (GRXs) are small oxidoreductase enzymes that can reduce disulfide bonds in target proteins. The class III GRX gene family is unique to land plants, and Arabidopsis thaliana has 21 class III GRXs, which remain largely uncharacterized. About 80% of A. thaliana class III GRXs are transcriptionally regulated by nitrate, and several recent studies have suggested roles for these GRXs in nitrogen signaling. Our objective was to functionally characterize two nitrate-induced GRX genes, AtGRXS5 and AtGRXS8, defining their roles in signaling and development in the A. thaliana root. We demonstrated that AtGRXS5 and AtGRXS8 are primarily expressed in root and shoot vasculature (phloem), and that the corresponding GRX proteins display nucleo-cytosolic subcellular localization. Ectopic expression of AtGRXS8 in transgenic plants caused major alterations in root system architecture: Normal primary root development, but a near absence of lateral roots. RNA sequencing demonstrated that the roots of AtGRXS8-overexpressing plants show strongly reduced transcript abundance for many primary nitrate response genes, including the major high-affinity nitrate transporters. Correspondingly, high-affinity nitrate uptake and the transport of nitrate from roots to shoots are compromised in AtGRXS8-overexpressing plants. Finally, we demonstrated that the AtGRXS8 protein can physically interact with the TGA1 and TGA4 transcription factors, which are central regulators of early transcriptional responses to nitrate in A. thaliana roots. Overall, these results suggest that AtGRXS8 acts to quench both transcriptional and developmental aspects of primary nitrate response, potentially by interfering with the activity of the TGA1 and TGA4 transcription factors.

Keywords: Arabidopsis thaliana; glutaredoxin; nitrate; root development; signaling.

FIGURE 1

Expression domains of AtGRXS5 and AtGRXS8. (a) Histochemical GUS staining of 14‐day‐old seedlings expressing AtGRXS8promoter::GUS (left) and AtGRXS5promoter::GUS (right). Scale bars = 1 mm. (b) GUS‐stained cross‐sections of stem (bolt) vascular bundles from soil‐grown plants expressing AtGRXS8promoter::GUS (left) and AtGRXS5promoter::GUS (right). Scale bars = 40 µm. X = xylem, P = phloem. (c) GUS‐stained cross‐sections of primary roots from plants expressing AtGRXS8promoter::GUS (left) and AtGRXS5promoter::GUS (right). Scale bars = 100 µm. X = xylem, P = phloem. (d) Fluorometric GUS assays performed on total protein extracts from 5‐day‐old seedlings expressing AtGRXS8promoter::GUS and AtGRXS5promoter::GUS. Seedlings were grown on either nitrate‐replete complete media or nitrate‐free media. Values were normalized to set GUS activity on nitrate‐free media equal to 1.0. Averages ± SEM are shown, N ≥ 3. An asterisk indicates a significant difference (p ≤ .05) in GUS activity, as determined by Student's t test

FIGURE 2

Subcellular localization of the AtGRXS5 and AtGRXS8 proteins. (a) Confocal micrograph of epidermal cells from Nicotiana benthamiana leaves Agroinfiltrated with a 35Spro::YFP‐AtGRXS5 construct. (b) Confocal micrograph of epidermal cells from N. benthamiana leaves Agroinfiltrated with a 35Spro::YFP‐AtGRXS8 construct. Scale bars = 25 µm

FIGURE 3

Overexpression of AtGRXS8. Transcript levels of AtGRXS8 were quantified via real‐time RT‐PCR in 9 day old seedlings grown in liquid culture. Values were normalized to set AtGRXS8 transcript abundance in wild type (Columbia‐0) equal to 1.0. Averages ± SEM are shown, N ≥ 3. An asterisk indicates a significant difference (p ≤ .05) in AtGRXS8 transcript abundance compared to wild‐type, as determined by Student's t test

FIGURE 4

Root system phenotypes in transgenic plant lines overexpressing AtGRXS8 (lines H6, J2, G8). (a) Primary root lengths of 12‐day‐old seedlings grown on vertically oriented media. Averages ± SEM are shown, N ≥ 20. (b) Number of lateral roots in 12‐day‐old seedlings grown on vertically oriented media. Averages ± SEM are shown, N ≥ 20. An asterisk indicates a significant difference (p ≤ .05) compared with wild‐type, as determined by Student's t test. (c) Representative images of 12‐day‐old wild‐type (WT) and transgenic line H6 seedlings. Corresponding line drawings represent binned average root system architectures from 20 to 25 seedlings (Shahzad et al., 2018). Scale bars = 1 cm. (d) Number of lateral root primordia (stages IV–VIII; Péret et al., 2009) in 12‐day‐old seedlings grown on vertically oriented media. Averages ± SEM are shown, N ≥ 9

FIGURE 5

AtGRXS8 represses nitrate transporter gene expression. Transcript abundance of the noted nitrate transporter genes was measured by real‐time RT‐PCR in wild‐type and transgenic line H6 (which ectopically expresses AtGRXS8) seedlings. Total RNA was isolated from the roots of the 6‐day‐old seedlings, prior to lateral root emergence. All values were individually normalized to set transcript abundance in line H6 equal to 1.0. Averages ± SEM are shown, N ≥ 3. An asterisk indicates a significant difference in transcript abundance compared with wild type (p ≤ .05), as determined by Student's t test

FIGURE 6

Plant nitrate uptake and distribution is altered by ectopic expression of AtGRXS8. (a) High‐affinity nitrate uptake, as measured by quantifying root tissue nitrate content of 15‐day‐old plants grown on ammonium succinate media and then supplied with 0.1 mM KNO₃ for 45 min (Canales et al., 2017). Averages ± SEM are shown, N ≥ 20. (b) Tissue nitrate content of 11‐day‐old seedlings grown on vertically oriented plates of growth media containing 9 mM nitrate. Averages ± SEM are shown, N ≥ 9. Asterisks indicate significant differences (p ≤ .05) in tissue nitrate concentration compared with wild‐type (WT) tissue, as determined by Student's t test

FIGURE 7

Characterizing protein–protein interactions between TGA transcription factors and AtGRXS8 via yeast two‐hybrid assay. DO‐2 indicates media without tryptophan and leucine, which selects for yeast containing both bait and prey vectors. DO‐3 indicates media without tryptophan, leucine, and histidine, which selects for yeast containing both bait and prey vectors, and a protein–protein interaction between the bait and prey proteins. (a) Positive control (bait‐SMAD, prey‐SMURF‐ Colland et al., 2004). (b) Negative control (bait‐empty vector; prey‐TGA1). (c) Negative control (bait‐empty vector; prey‐TGA4). (d) Negative control (bait‐AtGRXS8; prey‐empty vector). (e) AtGRXS8‐TGA1 protein–protein interaction (bait‐AtGRXS8; prey‐TGA1). (f) AtGRXS8‐TGA4 protein–protein interaction (bait‐AtGRXS8; prey‐TGA4)