Global regulation of reactive oxygen species scavenging genes in alfalfa root and shoot under gradual drought stress and recovery

Abstract

Reactive oxygen species (ROS) production and scavenging in plants under drought stress have been studied intensively in recent years. Here we report a global analysis of gene expression for the major ROS generating and scavenging proteins in alfalfa root and shoot under gradual drought stress followed by one-day recovery. Data from two alfalfa varieties, one drought tolerant and one drought sensitive, were compared and no qualitative differences in ROS gene regulation between the two were found. Conserved, tissue-specific patterns of gene expression in response to drought were observed for several ROS-scavenging gene families, including ascorbate peroxidase, monodehydroascorbate reductase, and peroxiredoxin. In addition, differential gene expression within families was observed. Genes for the ROS-generating enzyme, NADPH oxidase were generally induced under drought, while those for glycolate oxidase were repressed. Among the ROS-scavenging protein genes, Ferritin, Cu/Zn superoxide dismutase (SOD), and the majority of the glutathione peroxidase family members were induced under drought in both roots and shoots of both alfalfa varieties. In contrast, Fe-SOD, CC-type glutaredoxins, and thoiredoxins were downregulated.

Keywords: :

• reactive oxygen species
• drought
• alfalfa

Reactive oxygen species (ROS) are produced unavoidably in plant cells as a consequence of oxygen metabolism. Major forms of ROS include the superoxide anion (O₂^-), hydrogen peroxide (H₂O₂), hydroxyl radical (•OH), and singlet oxygen (¹O₂), which differ in the extent of reduction or activation of oxygen.

Under optimal growth conditions, ROS generation and scavenging in plant cells are maintained in a dynamic balance.^1,2 However, when plants are stressed, ROS generation tends to exceed scavenging and accumulated ROS may oxidize lipids, proteins and nucleic acids^1,3,4 and further cause programmed cell death.^5,6 Aside from negative effects, the role of ROS as signaling molecules has gained increasing attention in recent years.^4,7–9

Overproduction of ROS in plants under drought stress is well documented^10–12 and in most cases a general increase in plant antioxidant enzyme activities was measured under drought.^10,13,14 However, the majority of these studies monitored one or few ROS scavenging enzymes in the above ground tissues and the data were collected at limited time points.

Here, we present a genome-wide analysis of the expression of the major ROS generating and scavenging genes in alfalfa root and shoot under gradual 11-d soil drought stress followed by one-day recovery (Table S1). Indirect ROS scavengers including alternative oxidase and ferritin, and three protein redox regulating proteins were also included in the analysis. Genes involved in the metabolism of ROS-scavenging chemicals were not included.

Results of two alfalfa varieties, Wisfal (M. sativa ssp. falcata var Wisfal), a drought tolerant variety, and Chilean (M. sativa ssp. sativa var Chilean), a drought sensitive variety, were compared. The data were extracted from Kang et al.¹⁵ and gene expression heat maps were constructed with Multi Array Viewer (www.tm4.org/mev/). Medicago truncatula ROS generating and scavenging genes were identified by homology to Arabidopsis^2,16,17 and updated according to annotations in UniProt (www.uniprot.org) and the M. truncatula Gene Expression Atlas (mtgea.noble.org/v2/). The corresponding M. truncatula Affymetrix probeset IDs were identified through Legoo (www.legoo.com).

Plasma membrane NADPH oxidase, also called respiratory-burst oxidase (RBO), catalyzes the production of O₂^-. NADPH oxidase is involved in various plant biotic and abiotic stress responses.^9,18–20 The expression of most NADPH oxidases increased in both roots and shoots of alfalfa under drought, especially under severe drought stress (D11; Fig. 1). NADPH oxidase expression appeared to be more sensitive to drought in the drought-sensitive variety, Chilean, than in drought-tolerant Wisfal. On the other hand, the expression of the important ROS generator in the peroxisome, glycolate oxidase (GLO), declined rapidly upon drought stress in the root. In the shoot, reduction in GLO expression only became apparent at severe drought stress (Fig. 1). Previous studies on GLO regulation under drought stress found no consensus, with some finding reduction in GLO activity^11,21 consistent with our results, and others finding an increase in GLO gene expression²² and protein activity^23–25 under drought.

Figure 1. Expression profiles of selected ROS-generating genes in roots and shoots of Chilean and Wisfal subjected to drought stress and re-watering. Rboh, NADPH oxidase (respiratory burst oxidase homolog); GLO, glycolate oxidase. C, Chilean; W, Wisfal; R, root; S, shoot; CTL, control; D5, five-day drought; D8, eight-day drought; D11, 11-d drought; RW, one day after re-watering.

The expression of the major ROS scavenging genes was finely tuned at sub-family levels under drought stress (Fig. 2). SODs catalyze the dismutation of O₂^- to H₂O₂ and are located in various subcellular compartments in plants.^1,26 The expression of FeSOD (FSD, chloroplast) was generally repressed under drought stress in alfalfa while Cu/ZnSOD (CSD, chloroplast and cytosol) was induced (Fig. 2). MnSOD (mitochondria) transcript level was too low to be detected (Table S1). Earlier studies reported increase in SOD activity^11,27,28 and abundance²⁹ after drought stress, while MnSOD but not Cu/ZnSOD transcript was shown to be induced under drought in wheat.³⁰ However, both wheat³¹ and common bean³² SOD activity remained constant under moderate to severe drought.

Figure 2. Expression profiles of ROS-scavenging genes in roots and shoots of Chilean and Wisfal subjected to gradual drought stress and re-watering. AOX, Alternative oxidase; APX, ascorbate peroxidase; DHAR, dehydroascorbate reductase; MDHAR, monodehydroascorbate reductase; GPX, glutathione peroxidase; Grx, glutaredoxin; Prx, peroxiredoxin; Trx, thioredoxin; SOD, superoxide dismutase. C, Chilean; W, Wisfal; R, root; S, shoot; CTL, control; D5, 5-d drought; D8, 8-d drought; D11, 11-d drought; RW, one day after re-watering.

Ascorbate peroxidase (APX) is an important H₂O₂ scavenger at the expense of oxidizing ascorbate and glutathione in the ascorbate-glutathione cycle (Gill and Tuteja 2010).^33–35 Ascorbate and glutathione regeneration requires the activity of dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR). Transcript levels of APX were very high in the shoot under well-watered conditions (Table S1) but decreased gradually under drought, along with ascorbate levels,¹⁵ which implies that oxidized ascorbate was not fully recycled. In contrast, APX regulation in the root was weak and not consistent (Fig. 2). Interestingly, neither MDHAR nor DHAR had similar expression patterns to APX; MDHAR was induced in the shoot but repressed in the root while DHAR was induced in both root and shoot during drought. The transcript level of GRs was below the detection limit (Table S1). In earlier studies with similar experimental design as this study, the activity of APX, DHAR and MDHAR in pea were found to decrease significantly under severe drought stress,¹¹ while in wheat, their activity changes were correlated with both the drought sensitivity of the cultivar and severity of the stress.³⁶

Glutathione peroxidase (GPX) and Peroxiredoxin (Prx) are H₂O₂ scavengers which use glutathione as reductant. In drought-stressed alfalfa, Prxs were repressed in the shoot but induced in the root while most GPXs were induced in both root and shoot except GPX2 and GPX3 (Fig. 2). Interestingly, AtGPX2 and AtGPX3 are neighbors in the phylogenetic tree.³⁷ In agreement with our results, increase in GPX transcript level or activity under drought have been reported in several plant species.^31,32,38

Alternative oxidase (AOX) reduces ROS generation by the mitochondrial electron transport chain under stress.³⁹ AOX1 is induced in response to various biotic and abiotic stresses in plants.^2,39,40 However, under drought stress in alfalfa, AOX1A transcript level was below the detection limit (Table S1), while AOX2 and AOX4 were strongly regulated but in different ways (Fig. 2). Two of the AOX2 homologs were induced in the root of Wisfal but repressed in Chilean, which may contribute to greater drought tolerance in Wisfal.

Among all the gene families analyzed, ferritins stand out as the most strongly and uniformly upregulated family under drought stress (Table S1). Upon recovery from drought stress, ferritin expression decreased rapidly, revealing high sensitivity to drought. Ferritin is an effective Fe sequestrator and plays a crucial role in plant oxidative stress primarily by removal of free Fe, which catalyzes the Fenton reaction that produces highly reactive hydroxyl radicals.^41,42 Ferritin also serves as an active ROS scavenger because the oxidation of Fe(II) to Fe(III) consumes oxygen and H₂O₂ before it is stored in ferritin.⁴³ In plants, ferritin level is very low in mature leaves under normal growth conditions but it is induced by excess Fe application, ABA treatment and other oxidative stresses.^42,44,45 Ferritin mRNA accumulated in soybean nodules under drought stress⁴⁶ and in maize shoots under dehydration stress.⁴⁷ Ferritin protein abundance increased more than 2-fold in the root apical region of soybean under water stress.⁴⁸ Our data support the notion that removing free iron may be crucial for plant cells to survive drought induced oxidative stress.

Under drought stress, plant cells generate excessive ROS that oxidizes cellular proteins.^11,49,50 Oxidized proteins can be reduced by several families of “redoxins,” including thioredoxin (Trx), glutaredoxin (Grx), and Prx. However, almost all redoxin transcripts were repressed under drought stress in Arabidopsis.² In alfalfa, leaf 2-cys Prx protein abundance increased upon drought stress.⁵¹ In our experiments, most of the redoxins were downregulated under drought except the classical-type Grx and Prx in the root (Fig. 2). Note that Prx is a H₂O₂ scavenger in addition to being a protein redox regulator as mentioned above. The Trx gene family is large in alfalfa but almost all of these genes did not change activity or were downregulated in response to drought (Fig. 2). One exception, corresponding to Mtr.40458.1.S1, was induced by drought in alfalfa (Fig. 2), as was a homolog, CDSP32/AT1G76080.1, in potato.^52,53

In summary, ROS scavenging genes in alfalfa were highly responsive to drought and regulated in a conserved manner in a drought tolerant and a drought sensitive variety. It was intriguing to find many ROS-scavenging genes repressed under drought. However, the importance of a ROS-scavenging gene cannot be judged solely upon whether it is induced under stress. Some ROS-scavenging genes such as APXs are expressed at very high levels under optimal growth conditions (Table S1) and may still be the main ROS scavenger when slightly downregulated under drought stress. A systematic analysis of the changes in ROS-scavenging protein stability and activity under drought stress would be a useful complement to the results presented here. The promoter regions of alfalfa ROS-scavenging genes should also be analyzed to identify potential regulatory domains and transcription factor families that might control their tissue-specific and stress-responsive expression patterns. It is clear from our work that one cannot assume that it is always beneficial to increase the expression of ROS scavenging genes during stress, as many earlier studies have.¹ In future, it will be interesting to determine which drought-induced ROS genes contribute significantly to drought tolerance in alfalfa.

Abbreviations:
AOX	=	Alternative oxidase
APX	=	ascorbate peroxidase
DHAR	=	dehydroascorbate reductase
MDHAR	=	monodehydroascorbate reductase
GLO	=	glycolate oxidase
GPX	=	glutathione peroxidase
GR	=	glutathione reductase
Grx	=	glutaredoxin
Prx	=	peroxiredoxin
Trx	=	thioredoxin
Rboh	=	NADPH oxidase (respiratory burst oxidase homolog)
ROS	=	reactive oxygen species
SOD	=	superoxide dismutase

Acknowledgments

This work was supported by the Oklahoma Bioenergy Center (OBC) and The Samuel Roberts Noble Foundation. The authors would like to thank Dr. Mingyi Wang for assistance with data analysis and Eric Worley for valuable suggestions.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.