1. Introduction
Excessive greenhouse gas emissions from human activities raise the global temperature, and high ambient temperatures lead to biochemical and physiological changes in plants, thereby affecting plant growth and development. High temperatures cause protein denaturation and aggregation, inhibiting protein function and compromising membrane integrity. Reactive oxygen species (ROS) are subsequently generated when high-energy state electrons are released from heat-disrupted membrane-associated processes such as photosynthesis [
1,
2]. ROS are highly reactive and toxic, and they can cause oxidative damage to cells [
3,
4]. To counter the threat of oxidative damage under various environmental stresses, plants have developed ROS-scavenging mechanisms to eliminate ROS [
5,
6]. By combining antioxidant enzymes with antioxidants, plant cells can detoxify hydrogen peroxide and superoxide [
7,
8]. Several pieces of evidence indicate that antioxidant enzymes and antioxidants are associated with the plant heat tolerance [
7,
9–
12].
Ferredoxins (FDXs) in chloroplasts are electron transfer proteins that deliver reducing equivalents from photosystem I (PSI) in photosynthetic organisms [
13]. Electrons from reduced FDXs are accepted by FDX-NADPH-oxidoreductase (FNR) to generate NADPH, which is required for carbon assimilation in the Calvin cycle [
14,
15]. FDXs can also donate electrons to nitrite reductase (NiR), sulfite reductase (SiR) and fatty acid desaturases (FADs) for nitrogen and sulfur assimilation as well as fatty acid desaturation [
16,
17]. In addition, FDXs are key regulators of FDX-thioredoxin reductase (FTR) in thioredoxin systems [
14]. Moreover, FDXs are components in the water-water cycle, a ROS-scavenging pathway, and generate ascorbate and peroxiredoxin to protect the photosynthetic apparatus [
18–
20].
FDX transcripts have been observed to decrease under drought, cold, or salt stress conditions in
Arabidopsis [
21]. The amount of FDX is also decreased in tobacco under various stresses [
22]. Decreasing FDX by antisense RNA in transgenic plants causes leaf yellowing under high light stress, and the ROS level is increased in FDXs-limiting plants [
23,
24]. These results suggest that the expression of FDXs is down regulated by abiotic stress, resulting in increased the ROS level and subsequent oxidative damage to cells. In addition, ectopic expression of a cyanobacterial flavodoxin, which is a functional analog of FDXs found in cyanobacteria and some algae, decreases the ROS level in transgenic tobacco and enhances plant tolerance to heat, high light, chilling, drought, UV radiation, and iron starvation [
22,
25,
26]. However, ectopic expression of a cyanobacterial FDX in tobacco chloroplasts does not improve the tolerance of transgenic plants to oxidative and chilling stresses [
27]. Although the level of foreign cyanobacterial FDX has been shown to decrease in the manner of an endogenous FDX in transgenic tobacco under stress [
27], whether FDX functions under adverse environment stresses remains uncertain.
The single-celled green alga
Chlamydomonas reinhardtii is an excellent photosynthetic model organism for examining physiological responses of cells under abiotic stresses [
28]. Recent studies on hydrogen production by FDXs and hydrogenase in
Chlamydomonas have proposed methods for potentially generating clean energy [
29–
31]. Previous studies have shown that
Chlamydomonas contains six FDXs, PETF, and FDX2—FDX6 [
32,
33]. Although the expression levels of
PETF, and
FDX2–
FDX6 vary under hypoxia, iron- and copper-deficient conditions [
34], PETF is a major photosynthetic ferredoxin in chloroplasts and performs a function in electron transfers between PSI and FNR [
34,
35]. In this study, we generated transgenic
Chlamydomonas overexpressing
PETF to clarify whether increasing FDX gene expression levels enhance the tolerance of algae to heat stress.
3. Discussion
Reduction of ROS level is a major biotechnology strategy used to protect plants from various abiotic stresses [
38–
40]. In the study reported herein, ROS produced by
C. reinhardtii cause oxidative damage, which results in cell death under heat treatment at 42 °C for 40 min. Three
Chlamydomonas transgenic lines overexpressing
PETF were generated, and ROS levels in these transgenic lines were significantly reduced even after heat treatment. These transgenic algae presented highly thermotolerant phenotypes that are correlated to the transgene
PETF expression levels. These findings indicate that overexpression of the
PETF gene decreases ROS levels and contributes to the tolerance of heat stress. However, plant responses to heat stress are highly complex, and have effects on protein denaturation, membrane destabilization, metabolic equilibration, and redox homeostasis [
9,
41]. Integration of different protective mechanisms contributes to plant tolerance under heat stress, and the complex protective networks can be facilitated by overexpression of
PETF.
The
Chlamydomonas genome contains six ferrdoxin (FDX) genes and the expression of each FDX gene is responsive to different environmental stress and nutrient conditions [
34]. For example, transcription of
FDX2 was upregulated by H
2O
2, although the FDX2 protein was rapidly damaged after H
2O
2 treatment. On the other hand, the expression of the
FDX5 transcript was responsive to O
2, copper, and nickel supplementation [
34]. The most abundant
FDX transcript found in
Chlamydomonas grown in TAP medium under normal growth condition is
PETF, and its expression remains constitutive in most tested conditions, including under H
2O
2 treatment [
34]. In this study, it was demonstrated that
PETF mRNA decreased slightly after heat treatment, and Terauchi
et al. (2009) showed that PETF protein is not significantly degraded under oxidative stresses [
34]. Therefore, it was proposed that high-level
PETF mRNA can be maintained and translated to functional
PETF, which contributes to transgenic algae resistant to heat stress. Moreover, it is known that monodehydroascorbate (MDA) is a major sink of photosynthetic electrons and can be reduced to ascorbate by FDX in cells [
28,
37]. Our results showed that the ratios of reduced ascorbate in the P1-5, P1-7, and P1-10 lines were higher than that of CC125 (
Figure 1D), suggesting that the PETF-transgenic lines contained more functional PETF and reduced more ascorbate than the non-transgenic line did.
In this study, three transgenic lines of
Chlamydomonas expressing the
PETF gene were obtained. The growth curve and chlorophyll content of transgenic lines did not have significant differences compared to that of non-transgenic lines under normal growth conditions. However, after heat treatment, the survival rates of
PETF-overexpressing lines increased significantly compared to that of non-transgenic line. In addition, chloroplasts in transgenic cells remained intact and exhibited little chlorophyll content decrease after heat treatment. Interestingly, chlorophyll b (Chl b) was more protected than chlorophyll a (Chl a) in transgenic lines. This report indicated that maintenance of chlorophylls by PETF would protect
Chlamydomonas against heat stress. However, the mechanism of PETF-mediated chlorophylls protection needs further investigation. Plant membrane systems including thylakoid membrane are known as direct targets of ROS under heat stress [
1]. Biosynthesis and degradation of chlorophyll determine the amount of chlorophyll present, and both processes are known to require FDX-dependent enzymes in plants [
42–
46]. Chlorophylls and their binding proteins form complexes when they are inserted into thylakoid membranes. When chlorophyll–protein complexes are dissociated, chlorophyll molecules enter the degradation pathway [
47]. Therefore, there is a strong correlation between thylakoid membrane stability and chlorophyll degradation under heat conditions. Moreover, several reports showed that degree of lipid saturation in membranes increases in plants under high temperature and thus reduces membrane stability [
48,
49]. Saturated fatty acid containing membrane glycerolipids are converted to unsaturated fatty acid by desaturases in plastids and endoplasmic reticulum (ER) [
50,
51], and the plastid desaturases required FDX to provide electrons for fatty acid desaturation [
42]. It is possible that overexpression of
PETF facilitates electron donation to desaturase for desaturation of fatty acids, and hence maintains membrane stability and chlorophyll content under heat stresses.
Chl b is synthesized from and can be reconverted to Chl a. The levels of Chl b are determined by the activity of three enzyme reactions; conversion of Chl a to Chl b by chlorophyllide a oxygenase (CAO), conversion of Chl b to 7-hydroxymethyl Chl a (HMChl a) by Chl b reductase (CBR), and conversion of HMChl a to Chl a by 7-hydroxymethyl-chlorophyll reductase (HCAR) [
52]. The CAO has been suggested to accept electrons from FDX to convert Chl a to Chl b, and both CBR and HCAR are FDX-dependent enzymes [
42]. Overexpression of
PETF in transgenic lines showed no alteration in levels of Chl a and Chl b, compared to the wild type, and it suggested that PETF contributes equally to both sides of conversion under normal growth condition. Chl b degradation is primarily performed via conversion of Chl a by CBR and HCAR and followed by two FDX-dependent enzymes, Pheide a oxygenase (PAO) and RCC reductase (RCCR) [
42]. It is proposed that PETF favors electron donation to PAO and RCCR, and accelerates Chl a degradation; therefore Chl b was protected more than Chl a in transgenic lines under heat stresses.
Mitochondria and chloroplasts have been clearly recognized as main sources of ROS in plant cells [
53]. In chloroplasts, increased levels of ROS are produced under adverse environmental conditions, such as drought, salt, high temperature and high-light, causing stress through the photosynthetic electron-transport chain (PETC) due to unsmooth electron flow [
53–
55]. In this study, the major ferredoxin PETF was overexpressed in expectations of reducing overproduction of high-energy electrons from PETC in
Chlamydomonas cells under heat stress and then decreasing ROS production to prevent cell damage. Consequently, transgenic algae overexpressing
PETF showed thermotolerance. Indeed, overexpression of chloroplast FNR, which is involved in the last step of PETC, increased tolerance to oxidative stress in tobacco [
56]. In addition, ectopic expression of a prokaryotic flavodoxin, an electron carrier flavoprotein not found in plants, targeted to chloroplast in transgenic tobacco plants is shown to increase tolerance against various abiotic stress [
22]. Similar hypotheses were tested in mitochondria of the mammalian cell line Cos-7 cells and results showed that ectopic expressed heterologous FNR and flavodoxin can protect Cos-7 cells against oxidative stress [
57]. On the other hand, ferredoxin can transfer electrons to generate ascorbate, which is employed by ascorbate peroxidase (APX) to scavenge H
2O
2[
18,
20,
58,
59]. Although electrons from ferredoxin can provide an alternative sink to generate O
2− from O
2 in the Mehler reaction, the reducing power of ferredoxin also acts for ascorbate reduction [
60]. Results aforementioned indicated that transgenic
Chlamydomonas lines overexpressing
PETF generated more reduced ascorbate than non-transgenic algae did, and it can donate electrons in ascorbate-mediated ROS scavenging to detoxify ROS generated under heat stress in chloroplast.