Cucumber Possesses a Single Terminal Alternative Oxidase Gene That is Upregulated by Cold Stress and in the Mosaic (MSC) Mitochondrial Mutants

Abstract

Alternative oxidase (AOX) is a mitochondrial terminal oxidase which is responsible for an alternative route of electron transport in the respiratory chain. This nuclear-encoded enzyme is involved in a major path of survival under adverse conditions by transfer of electrons from ubiquinol instead of the main cytochrome pathway. AOX protects against unexpected inhibition of the cytochrome c oxidase pathway and plays an important role in stress tolerance. Two AOX subfamilies (AOX1 and AOX2) exist in higher plants and are usually encoded by small gene families. In this study, genome-wide searches and cloning were completed to identify and characterize AOX genes in cucumber (Cucumis sativus L.). Our results revealed that cucumber possesses no AOX1 gene(s) and only a single AOX2 gene located on chromosome 4. Expression studies showed that AOX2 in wild-type cucumber is constitutively expressed at low levels and is upregulated by cold stress. AOX2 transcripts and protein were detected in leaves and flowers of wild-type plants, with higher levels in the three independently derived mosaic (MSC) mitochondrial mutants. Because cucumber possesses a single AOX gene and its expression increases under cold stress and in the MSC mutants, this plant is a unique and intriguing model to study AOX expression and regulation particularly in the context of mitochondria-to-nucleus retrograde signaling.

Keywords: Alternative oxidase; Cucumis sativus; Gene structure; Mitochondrial mutants.

Fig. 1

The structure of the cucumber AOX2 gene (NCBI Gen Bank Acc. No. AY258276). The cucumber AOX2 gene (2,035 bp) possesses four exons and three introns with 55 bp 5′ UTR and 200 bp 3′ UTR regions. P1 and P2 are degenerated primers used to amplify conserved 446 bp fragment of AOX gene (called ‘probe’, used in hybridization experiments)

Fig. 2

Southern blot hybridizations from hybridization of nick-translated 446 bp AOX clone (‘probe’, Fig. 1) as an evidence for the presence of a single AOX gene in cucumber. a Cucumber line B, melon ‘Iroqouis’, watermelon ‘Dixielee’, Cucurbita moschata ‘Butternut’, and Cucurbita pepo ‘Dark Green Zucchini’ DNA digested with EcoRI and b cucumber line B DNA digested with nine different restriction enzymes

Fig. 3

Phylogenetic tree of 42 AOX proteins from 12 different higher plant species of the Cucurbitales, Fabales, and Brassicales; fungi (N. crassa and C. albicans); green algae (C. reinhardtii); eubacteria (N. aromaticivorans); protists (T. brucei), and animalia (C. gigas). Classification of higher plants, C. albicans, and C. reinhardtii AOX proteins is according to Considine et al. (2002). Classification of AOX proteins from other organisms is consistent with the protein sequence description at the NCBI GeneBank. The tree was obtained by the Neighbor-Joining method with 1,000 bootstrap replicates using the MEGA6 program (Tamura et al. 2013). Branches are drawn in proportion to genetic distance according to the scale shown in the bottom of the figure. The tree was constructed according to sequence data indicated in the Supplementary file 1

Fig. 4

Amino acid sites unique to AOX2 in cucumber, melon, and watermelon. An alignment of the conservative fragment (101–354 aa) of AOX2a–c consensus sequence (Costa et al. 2014) and cucumber, melon, and watermelon corresponding AOX fragments appear with eight specific amino acids residues. Indicated amino acids sites were rated as exhibiting relevant differences using the ‘sequence harmony’ (SH) methodology (Feenstra et al. 2007). In addition, those presenting the strong reliability parameters in SH ≤ 0.13 are marked with an asterisk. Figure was prepared by using the model proposed by Costa et al. (2014). Four highly conserved AOX active regions (LET, NERMHL, LEEEA, and RADE__H) were marked by frames, glutamic acid (E) and histidine (H) amino acid residues involved in iron-binding were indicated by black circles (Berthold et al. 2000). Capital letters at the specific sites represent the most frequently seen amino acids. Consensus symbols used in the AOX2a–c consensus sequences: exclamation mark is I (Isoleucine), or V (valine); percent sign is F (phenylalanine), or Y (tyrosine); number sign is N (asparagine), D (aspartic acid), Q (glutamine), or E (glutamic acid). For more details, the reader is referred to the Supplementary file 2

Fig. 5

Expression of AOX2 in field grown plants of cucumber and in leaves and flowers of control line B (wild-type) and mutant MSC16. Revealing higher amounts of Aox2 transcripts by Northern-blot analysis a and AOX2 protein by Western blot b in MSC16. a Relative amounts of alternative oxidase transcript in total RNA extracts. Approximate sizes of Aox2 transcript in kilobases is shown on right. Gel picture at bottom shows ethidium bromide stained rRNA showing equal loading of RNA samples. b Relative amounts of alternative oxidase and porin in total protein extracts. Approximate sizes of polypeptides in kilodaltons are shown on right. Porin was used as internal control

Fig. 6

Expression AOX2 in cucumber line B after cold treatment. Both analyses RT-qPCR (a) and Western blot (b and c) revealed that cucumber AOX2 is cold-responsive. Rapid increase (172-fold, p < 0.001) of Aox2 transcript abundance after chilling was observed (a) and the significantly highest amount of AOX2 protein (6.3-fold increase, p < 0.001) was proved 48 h (2 days) after chilling (b and c). Differences between stages after chilling were referenced to the state before chilling marked as ‘0(ctrl)’. The following ranges of P value were defined: *p < 0.05, **p < 0.01, and ***p < 0.001. a RT-qPCR results obtained with CsAox2FL_F and CsAox2R101 primers designed to 5′ end of Aox2. Primers specific to UBI-ep and TIP41 were used as reference genes (Supplemental tables S1 and S2). The diagram shows the average relative normalized expression to control (pre-chilling). Bars represent standard error of the mean (SEM). b Western blot confirmed cold regulation of AOX2. Alpha-tubulin was used as an internal control. Approximate size of polypeptides in kilodaltons are shown on right. c Densitometry quantification of Western blot analysis (n = 4) performed using ChemiDoc XRS+ system (Bio-Rad Laboratories). Relative amount of AOX2 was normalized to alpha-tubulin

Fig. 7

Expression of AOX2 in cucumber control line B (wild-type) and MSC lines. AOX2 is upregulated in MSC lines 3, 12, and 16 both before and after chilling compared to control line B (B_WT). a Results of RT-qPCR analysis performed with primers designed to 5′ end of Aox2 (CsAox2FL_F and CsAox2R101). Primers specific to M2 and mdhG were used as reference genes (Supplemental table S1 and S2). The diagram shows Aox2, the average relative expression in MSC mutants before chilling, referenced to the control line B (B_WT, referred to as 1). Bars represent standard error of the mean (SEM). Statistical differences were confirmed using t Student test and P value < 0.05 was considered as statistically significant. The following P value ranges were defined: *p < 0.05, **p < 0.01, and ***p < 0.001. b AOX2 expression pattern in the MSC mutants under cold stress. Ponceau S staining was used as a loading control. Approximate size of AOX2 in kilodaltons is shown on right