Exploring the Involvement of the Alternative Respiratory Pathway in Pisum sativum L. Seed Germination ^†

Abstract

Organic agriculture, recognized as a more sustainable agricultural system, strongly depends on the use of highly resilient genotypes. Seed resilience, with increased tolerance to germination that provide vigorous seedlings under environmental stresses, currently represents one of the most important agronomical traits. Seed germination involves the activation of several metabolic pathways, including cellular respiration. Alternative oxidase (AOX), a key enzyme in the alternative respiratory pathway, plays a crucial role in regulating cell reprogramming by controlling metabolic transitions related to the cellular redox state and the variable carbon balance. The involvement of the alternative respiratory pathway during germination was explored by analysis of PsAOX gene/protein expression. Seeds of four Pisum sativum L. cultivars (‘Respect-1′, ‘S134′, ‘G78′, and ‘S91′) were imbibed in sterile tap water for 16 h and metabolic parameters were measured by calorespirometry (heat and CO₂ emission rates) in a multi-cell differential scanning calorimeter in isothermal mode at 25 °C. The involvement of PsAOX was evaluated by transcript quantification (PsAOX1, PsAOX2a, and PsAOX2b) through RT-qPCR, and by analyzing PsAOX expression through Western blot. The results demonstrate that cv. ‘S91′, characterized by a low germination rate, exhibited the lowest metabolic heat and CO₂ emission rate. However, contrary to expectations, PsAOX transcript accumulation and PsAOX protein expression were significantly higher for ‘S91′ than for the other cultivars. These results indicate that higher levels of PsAOX (transcript and protein) could be linked to lower metabolic rates for embryo growth when seed germination is compromised.

1. Introduction

Legumes have been referred as the basis of a healthy diet, representing the most prominent source of food protein [1]. Among cultured legumes, peas (Pisum sativum L.) are one of the most widely spread crops in Europe, playing a very important role in human nutrition and animal feed [2]. Considering the importance of this grain legume together with the selection of organic agriculture as a more sustainable agricultural system, the development of more resilient cultivars has been recognized as a key strategy. Seed germination and seedling establishment are among the most critical stages and key factors for successful crop production [3]. In order to support the imposed needs, several breeding programs have been implemented, aiming to develop new cultivars characterized by seeds with greater plasticity toward environmental pressures, allowing them to more efficiently germinate. The European project LIVEESED [4], which integrates efforts by seed production companies and research/breeding institutes, is one example of the high interest in seed research.

Considering that germination involves the activation of several metabolic pathways, including cellular respiration, to provide the required energy, the involvement of the alternative respiratory pathway during germination was explored. Alternative oxidase (AOX) is a mitochondria inner membrane enzyme with a key role in the alternative respiratory pathway, in which this enzyme introduces a branch into the electron transport chain (ETC) at the ubiquinone pool. In this branch, AOX allows the transport of electrons to oxygen directly from the ubiquinone, preventing excessive reduction of the downstream complexes [5]. The involvement of this pathway in plant response to a diversity of environmental stresses and cell reprogramming events has been demonstrated [6,7], mainly associated with the ability to contribute to the control of reactive oxygen species (ROS) and thereby reducing oxidative damage [8]. In addition, a link between AOX gene expression and metabolic and respiratory parameters was established in different plant species in biological systems/stress conditions [9]. Considering that seed germination involves the activation of several metabolic pathways, metabolic and respiratory changes were monitored by using calorespirometry. Calorespirometry simultaneously measures metabolic heat rates (R_q) and CO₂ emission rates (R_CO2) of biological samples and has been used as a screening tool to assess metabolic and respiratory changes associated with cell reprogramming events [10].

The present research investigates the involvement of PsAOX on efficient seed germination, and explores the link between AOX and metabolic/respiratory changes.

2. Methods

Seeds of four P. sativum L. cultivars (‘Respect-1′, ‘S134′, ‘G78′, and ‘S91′) provided by LIVESEED partners were selected for calorespirometry measurements. Previously, seeds were imbibed in sterile tap water for 16 h. Calorespirometric parameters (heat and CO₂ emission rates) were measured at 25 °C using a multi-cell differential scanning calorimeter in isothermal mode. A total of 16 biological replicates (16 seeds) were considered per cultivar. The heat (R_q) and CO₂ emission rates (R_CO2) were determined according to Rodrigues et al. [11]. Germination rates were recorded 6 days after calorespirometric measurements.

The involvement of PsAOX in seed germination was evaluated by gene expression analysis of the three P. sativum AOX genes (PsAOX1, PsAOX2a, and PsAOX2b) and by the analysis of PsAOX protein levels through Western blot. Samples of the four pea cultivars were collected at 16 h post imbibition and further homogenized using liquid nitrogen. A total of 12 samples, each one consisting of a pull of 4 seeds, were used per cultivar.

For PsAOX gene expression studies, cv. ‘Respect-1′ and cv. ‘S91′ were selected based on the results obtained by calorespirometry. Total RNA was extracted from the harvested material using the Maxwell^® 16 LEV simplyRNA Cells Kit (Promega) and integrity was evaluated through 1.2% agarose gel electrophoresis. Then, 1 µg of total RNA was used for cDNA synthesis using the SensiFAST^TM cDNA Synthesis Kit (Bioline). Transcript level accumulation of the three PsAOX genes was assessed in an ABI 7500 system using SensiFAST SYBR Lo-ROX kit (Bioline). PsPOB and PsSAR1 were used as reference genes for data normalization. Primer specificity was evaluated by visualization of a single peak at the dissociation curve and the efficiency (E) was calculated by the equation: E (%) = 10^{−(1/slope) − 1)} × 100 [12]. To obtain the slope value, a standard curve of a 4-fold dilution series was generated for each primer pair.

For analysis of PsAOX protein levels, all four cvs. were considered: ‘Respect-1′, ‘S134′, ‘G78′, and ‘S91′. Total protein content was extracted by phenol precipitation from 50 mg of plant material (adapted from [13]) and total protein quantification of 12 replicates of each cultivar was performed using the Pierce 660 nm Protein Assay Reagent (Thermo Scientific™). Western blot was performed to obtain a comparison of the levels of PsAOX protein between the cultivars. Briefly, after protein separation by SDS-PAGE (25 μg total protein from each sample) in 14% polyacrylamide gels using a Laemmli buffer system [14], proteins were transferred to a PDVF membrane by electroblotting using a Semi-Dry Trans-Blot Turbo Transfer (Bio-Rad) system. After transferring, blocking was performed with 5% non-fat milk powder in TBS with Tween 20 and the membrane was incubated with primary antibody anti-AOX1/2 (Agrisera AS04054; dilution: 1:1000) overnight at 4 °C. AOX bands were detected with an alkaline phosphatase-linked secondary antibody (anti-rabbit, Agrisera AS09607, 1:10,000 dilution), using a chemifluorescent substrate (ECF Plus Western Blotting Detection Reagents, GE, Healthcare). Membranes were placed in a transilluminator (Bio-Rad Gel-doc system) and a semi-quantitative analysis of band intensity was carried out using the software Bio-Rad Image Lab 5.2.1. Six replicates of each cultivar were used.

Statistical analyses were performed using SPSS version 22.0. Normality and homoscedasticity were checked in all data. One-way ANOVA followed by a Tukey HSD test was used for the comparison of calorespirometric parameters, germination rate, and PsAOX band volume between the four cultivars. Comparison of transcript levels of PsAOX1, PsAOX2a, and PsAOX2d between the two cultivars was performed using Student’s t-test. When data did not meet the assumptions to perform parametric tests, the Kruskal–Wallis or Mann–Whitney nonparametric tests were conducted. Statistical significance was considered at p < 0.05.

3. Results and Discussion

At 25 °C, the calorespirometric parameters Rq and R_CO2 (Figure 1) were significantly lower in cvs. ‘S91′ and ‘G78′ when compared to cvs. ‘Respect-1′ and ‘S134′. Later, at 6 days post imbibition, seeds of cvs. ‘G78′, ‘S134′, and ‘Respect-1′ presented significantly higher germination rates compared to the cv. ‘S91′. These results show that higher Rq and R_CO2 could be related with higher germination rates, as previously reported by Edelstein et al. [15], who observed that higher values of metabolic activity were associated with higher germination rates of melon seeds. With seed lots from onion and several Brassica species, a positive correlation has been shown between single seed respiration and seed quality [16].

The AOX gene family is nucleus encoded, composed of one to six members distributed into two subfamilies (AOX1 and AOX2) [17]. In peas, the AOX gene family is composed of one AOX1 subfamily member (PsAOX1) and two AOX2 subfamily members (PsAOX2a and PsAOX2b) [18]. Based on the calorespirometric results, the cv. ‘Respect-1′ and cv. ‘S91′ were selected for PsAOX gene expression studies. Regarding the analysis of PsAOX gene expression performed 16 h post water imbibition, a higher transcript accumulation of PsAOX2b was observed in comparison to PsAOX1 and PsAOX2a. Additionally, transcript accumulation was significantly higher in cv. ‘S91′ when compared to cv. ‘Respect-1’ (data not shown).

Previous studies with tobacco and Arabidopsis have demonstrated the importance of AOX1 in photosynthesis-related pathways and respiration under light conditions [19]. Members of the AOX1-subfamily have also been associated with plant responses to abiotic stress factors [6,7]. At 16 h post imbibition, the photosynthetic apparatus is still not activated, which could explain the lower levels of PsAOX1 transcript. In addition to AOX’s involvement in ROS control, ROS levels were already reduced at the considered timepoint through the enzymatic ROS scavenging system and the action of antioxidant metabolites.

On the other hand, members of the AOX2 subfamily have been mainly associated with developmental processes [6]. The higher level of transcript accumulation achieved for PsAOX2a lead us to suggest a higher involvement of this gene in the germination process, particularly in the case of cv. ‘S91′.

The analysis of total protein concentration in the four P. sativum cultivars showed significantly higher protein concentration in cv. ‘Respect-1’ compared to cvs. ‘S134’, ‘G78’, and ‘S91’. No statistically significant differences in total protein concentration were observed between cvs. ‘S134’, ‘G78’, and ‘S91’ (data not shown). The PsAOX protein levels were evaluated 16 h post water imbibition by Western blot. The band corresponding to the PsAOX protein was detected in all cultivars and higher levels of protein were observed in cv. ‘S91′, as compared to cvs. ‘Respect-1′ (RS), ‘S134′, and ‘G78′. Although we were not able to analyze the different isoforms of the PsAOX protein through Western blot analysis, the results at the protein level confirm the results observed by transcript analysis, i.e., a higher PsAOX protein expression in the cultivar that presents the highest level of PsAOX gene expression.

The absence of a direct relationship between the total protein concentration and the level of the AOX protein could be expected, since the AOX is only a part of the total protein concentration [20]. In fact, the values of total protein concentration essentially reflect the concentration of storage proteins, which is variable among cultivars [21]. Moreover, studies focused on the protein composition of pea seeds showed that a large percentage of the identified proteins correspond to storage proteins (mainly albumin, legumin, and vicilin), with the rest being involved in the response to biotic and abiotic stress, energy production, metabolism, and storage of essential non-protein compounds [21].

Overall, seeds from the cultivar characterized by lower R_q and R_CO2 (cv. ‘S91′) coincided with lower vigor, exhibited significantly higher expression of the PsAOX genes (PsAOX1, PsAOX2a, and PsAOX2d), and PsAOX protein. From these results, we hypothesize that higher levels of AOX (transcript and protein) in germinating seeds are linked to lower metabolic rates and germination. Further studies will be required to validate this hypothesis.

4. Conclusions

To the best of our knowledge, the study presented here represents the first approach aiming to establish a link between respiratory parameters monitored by calorespirometry and PsAOX transcript accumulation/PsAOX protein expression during the germination of pea seeds. The present work suggests the involvement of AOX and the alternative respiratory pathway during seed germination and the applicability of calorespirometry to assess seed vigor, highlighting this method as a useful non-destructive phenotyping tool for selecting genotypes with superior germination capacity.