Effects of OsAOX1a Deficiency on Mitochondrial Metabolism at Critical Node of Seed Viability in Rice

Mitochondrial alternative oxidase 1a (AOX1a) plays an extremely important role in the critical node of seed viability during storage. However, the regulatory mechanism is still poorly understood. The aim of this study was to identify the regulatory mechanisms by comparing OsAOX1a-RNAi and wild-type (WT) rice seed during artificial aging treatment. Weight gain and time for the seed germination percentage decreased to 50% (P50) in OsAOX1a-RNAi rice seed, indicating possible impairment in seed development and storability. Compared to WT seeds at 100%, 90%, 80%, and 70% germination, the NADH- and succinate-dependent O2 consumption, the activity of mitochondrial malate dehydrogenase, and ATP contents all decreased in the OsAOX1a-RNAi seeds, indicating that mitochondrial status in the OsAOX1a-RNAi seeds after imbibition was weaker than in the WT seeds. In addition, the reduction in the abundance of Complex I subunits showed that the capacity of the mitochondrial electron transfer chain was significantly inhibited in the OsAOX1a-RNAi seeds at the critical node of seed viability. The results indicate that ATP production was impaired in the OsAOX1a-RNAi seeds during aging. Therefore, we conclude that mitochondrial metabolism and alternative pathways were severely inhibited in the OsAOX1a-RNAi seeds at critical node of viability, which could accelerate the collapse of seed viability. The precise regulatory mechanism of the alternative pathway at the critical node of viability needs to be further analyzed. This finding might provide the basis for developing monitoring and warning indicators when seed viability declines to the critical node during storage.


Introduction
Mitochondrial redox homeostasis is a key factor affecting seed viability. During the imbibition process of seeds with high viability, the number of mitochondria, as well as the inner membrane and cristae of the mitochondria are significantly improved, and proteins and genes involved in energy and material metabolism, enzyme activity of TCA cycle, electron transport chain and oxidative phosphorylation are also significantly improved [1,2]. However, in the process of seed aging, there are a number of deleterious changes that inhibits the repair of mitochondrial structure and function; the total number of mitochondria (the outer membrane and inner membrane) are difficult to distinguish, and membrane integrity+ decrease the number of matrix, cristae is less than that of high viability seeds and the shape is irregular. The proportion of mitochondria increased and the motility decreased, and the physiological functions, such as electron transport chain, oxidative phosphorylation and marker enzymes, were also lower than normal seeds, and Furthermore, the expression of the core subunit of the membrane arm of the mETC complex I was significantly up-regulated, electron and proton transfer ability was improved, and ATP generation was maintained [5,27]. Previous work confirmed that the cytochrome pathway, the accumulation of ROS, and mitochondrial protein carbonylation are the processes that are inhibited during the critical node of rice seed viability, however, AOX1a can be induced to maintain mitochondrial redox homeostasis and enhance the function of ATP generation. Given that the molecular mechanisms underlying rice seed conservation are still unclear, this study will explore the regulatory mechanism of rice seed conservation longevity from the perspective of mitochondrial activity regulation using wild-type and OsAOX1a-RNAi rice seeds for artificial aging.

Comparison of Survival Curves between WT and OsAOX1a-RNAi Seeds after Aging Treatment
We found that the grain length is smaller in seeds with lower levels of OsAOX1a expression ( Figure 1a). Compared with WT seeds, the seed width, length, and 100-grain weight of OsAOX1a-RNAi seeds had decreased by 1.9%, 2.2% and 8.2%, respectively (Figure 1b-d). These results show that the accumulation of dry matter in OsAOX1a-RNAi seeds was less than that in the WT seeds.
It has been reported that the redox homeostasis of the critical node of rice seed bility was significantly altered, inhibiting the cytochrome pathway, and the expressio OsAOX1a was significantly up-regulated compared to gene expression in unaged se Furthermore, the expression of the core subunit of the membrane arm of the mETC c plex I was significantly up-regulated, electron and proton transfer ability was improv and ATP generation was maintained [5,27]. Previous work confirmed that the cytochro pathway, the accumulation of ROS, and mitochondrial protein carbonylation are the p cesses that are inhibited during the critical node of rice seed viability, however, AO can be induced to maintain mitochondrial redox homeostasis and enhance the functio ATP generation. Given that the molecular mechanisms underlying rice seed conserva are still unclear, this study will explore the regulatory mechanism of rice seed conser tion longevity from the perspective of mitochondrial activity regulation using wild-t and OsAOX1a-RNAi rice seeds for artificial aging.

Comparison of Survival Curves between WT and OsAOX1a-RNAi Seeds after Aging Treatment
We found that the grain length is smaller in seeds with lower levels of OsAOX1a pression (Figure 1a). Compared with WT seeds, the seed width, length, and 100-g weight of OsAOX1a-RNAi seeds had decreased by 1.9%, 2.2% and 8.2%, respectively ( ure 1b-d). These results show that the accumulation of dry matter in OsAOX1a-RN seeds was less than that in the WT seeds. Figure 1. Comparison of wild-type (WT) and OsAOX1a-RNAi rice seeds (a) with respect to w (b) length (c) and 100-grain weight (d). Data represent the mean ± standard deviation of three in pendent experiments. Asterisks indicate a significant difference between OsAOX1a-RNAi tr genic plants and WT controls via Student's t-test: * p < 0.05.
The initial germination rates of the WT and OsAOX1a-RNAi seeds were 98.5% 96.5%, respectively. Under artificial aging treatment at 40 °C and 75% relative humid for 30 d, the survival curves of each of the two groups showed the typical inverse S-sha curve with an initial plateau phase transition state (known as the critical node (CN)), lowed by a rapid decline in viability. The shape of the survival curve and most portantly, the time point at which seed survival (germination) declined to 50% (P50), fered between the two groups ( Figure 2). The plateau phase of the survival curve Comparison of wild-type (WT) and OsAOX1a-RNAi rice seeds (a) with respect to width (b) length (c) and 100-grain weight (d). Data represent the mean ± standard deviation of three independent experiments. Asterisks indicate a significant difference between OsAOX1a-RNAi transgenic plants and WT controls via Student's t-test: * p < 0.05.
The initial germination rates of the WT and OsAOX1a-RNAi seeds were 98.5% and 96.5%, respectively. Under artificial aging treatment at 40 • C and 75% relative humidity for 30 d, the survival curves of each of the two groups showed the typical inverse S-shaped curve with an initial plateau phase transition state (known as the critical node (CN)), followed by a rapid decline in viability. The shape of the survival curve and most importantly, the time point at which seed survival (germination) declined to 50% (P50), differed between the two groups ( Figure 2). The plateau phase of the survival curve was similar between the WT and OsAOX1a-RNAi groups. The germination rate of each of the two types of seed fell gradually to 90.0%, 80.0% and 70.0% after 6, 9, and 12 d of artificial aging treatment  Figure 2a), respectively. However, the vigor index of OsAOX1a-RNAi seeds during AA treatment was always significantly lower than that of the WT group (Figure 2b). Furthermore, once the critical node was reached (at 12 days of aging), the germination rate of the OsAOX1a-RNAi seeds began to decline rapidly compared to the WT seeds. The time required for the seed germination percentage to decrease to 50% (P50) was estimated to be 13.6 d for OsAOX1a-RNAi and of 14.8 d WT seeds. These results indicated that the storability of OsAOX1a-RNAi seeds significantly decreased compared to the WT seeds. similar between the WT and OsAOX1a-RNAi groups. The germination rate of each of the two types of seed fell gradually to 90.0%, 80.0% and 70.0% after 6, 9, and 12 d of artificial aging treatment (Figure 2a), respectively. However, the vigor index of OsAOX1a-RNAi seeds during AA treatment was always significantly lower than that of the WT group (Figure 2b). Furthermore, once the critical node was reached (at 12 days of aging), the germination rate of the OsAOX1a-RNAi seeds began to decline rapidly compared to the WT seeds. The time required for the seed germination percentage to decrease to 50% (P50) was estimated to be 13.6 d for OsAOX1a-RNAi and of 14.8 d WT seeds. These results indicated that the storability of OsAOX1a-RNAi seeds significantly decreased compared to the WT seeds. Figure 2. Seed viability curves showing the time for the seed germination percentage decrease to 50% (P50 (a)) and the vigor index (b) of wild-type and OsAOX1a-RNAi rice following artificial aging treatment. Data represent the mean ± standard deviation of three independent experiments. "……" represents the time when germination percentage decreases to 50%. Asterisks indicate a significant difference between OsAOX1a -RNAi transgenic plants and WT controls via Student's t-test: * p < 0.05; **, p < 0.01; ***, p < 0.001.

Assessment of Mitochondria Status during Artificial Aging Treatments
To gain deeper insight into the differences in mitochondria status between OsAOX1a-RNAi and WT seeds during artificial aging treatments, mitochondria were extracted from seeds that were allowed to imbibe for 48 h at 100%, 90%, 80%, and 70% germination, respectively. The mitochondrial electron transport capacity was evaluated by monitoring NADH and succinate-dependent O2 consumption ( Table 1). The respiratory rate of mitochondrial significantly decreased with the decrease in seed viability in both seed groups. Compared to the WT seeds at 100%, 90%, 80%, and 70% germination, the NADH-dependent O2 consumption of the OsAOX1a-RNAi seeds decreased by 62%, 61%, 70% and 53%, respectively; the succinate-dependent O2 consumption were decreased by 21%, 28%, 54% and 19%, respectively. In addition, we measured the abundance of AOX1 and Cyt c, which plays a crucial role in mitochondrial electron transport ( Figure 3). Compared to the WT seeds, the abundance of AOX1 and Cyt c in the OsAOX1a-RNAi seeds was always lower during the artificial aging treatment. These results suggested that the capacity for electron transport was inhibited in the mitochondria of the OsAOX1a-RNAi seeds after imbibition, indicating a decrease in ATP supply.

Assessment of Mitochondria Status during Artificial Aging Treatments
To gain deeper insight into the differences in mitochondria status between OsAOX1a-RNAi and WT seeds during artificial aging treatments, mitochondria were extracted from seeds that were allowed to imbibe for 48 h at 100%, 90%, 80%, and 70% germination, respectively. The mitochondrial electron transport capacity was evaluated by monitoring NADH and succinate-dependent O 2 consumption ( Table 1). The respiratory rate of mitochondrial significantly decreased with the decrease in seed viability in both seed groups. Compared to the WT seeds at 100%, 90%, 80%, and 70% germination, the NADH-dependent O 2 consumption of the OsAOX1a-RNAi seeds decreased by 62%, 61%, 70% and 53%, respectively; the succinate-dependent O 2 consumption were decreased by 21%, 28%, 54% and 19%, respectively. In addition, we measured the abundance of AOX1 and Cyt c, which plays a crucial role in mitochondrial electron transport ( Figure 3). Compared to the WT seeds, the abundance of AOX1 and Cyt c in the OsAOX1a-RNAi seeds was always lower during the artificial aging treatment. These results suggested that the capacity for electron transport was inhibited in the mitochondria of the OsAOX1a-RNAi seeds after imbibition, indicating a decrease in ATP supply.
In addition, the mitochondrial malate dehydrogenase (MDH) activity and ATP content were measured for both the OsAOX1a-RNAi and WT seeds at 100%, 90%, 80% and 70% germination. MDH activity and ATP content were found to decrease significantly with the decrease in seed viability in both the seed groups ( Figure 4). However, compared to the WT seeds, the MDH activity of the OsAOX1a-RNAi seeds decreased by 14.4%, 14.3%, 13.18% and 24.8%, at 100%, 90%, 80%, and 70% germination, respectively, while ATP content decreased by 47.2%, 60.5%, 53.8% and 51.8%, respectively. These results indicate that the mitochondrial metabolism in the OsAOX1a-RNAi seeds after imbibition was weaker than that of the WT group. Table 1. Rate of oxygen uptake (nmolO 2· min −1 mg protein −1 ) by mitochondria in wild-type and OsAOX1a-RNAi seeds at germination percentage of 100%, 90%, 80%, and 70% after 48 h imbibition. O 2 consumption rates are presented as means ± SD (n = 3).  In addition, the mitochondrial malate dehydrogenase (MDH) activity and ATP tent were measured for both the OsAOX1a-RNAi and WT seeds at 100%, 90%, 80% 70% germination. MDH activity and ATP content were found to decrease signific with the decrease in seed viability in both the seed groups ( Figure 4). However, comp to the WT seeds, the MDH activity of the OsAOX1a-RNAi seeds decreased by 1 14.3%, 13.18% and 24.8%, at 100%, 90%, 80%, and 70% germination, respectively, ATP content decreased by 47.2%, 60.5%, 53.8% and 51.8%, respectively. These resu dicate that the mitochondrial metabolism in the OsAOX1a-RNAi seeds after imbib was weaker than that of the WT group.  In addition, the mitochondrial malate dehydrogenase (MDH) activity and ATP content were measured for both the OsAOX1a-RNAi and WT seeds at 100%, 90%, 80% and 70% germination. MDH activity and ATP content were found to decrease significantly with the decrease in seed viability in both the seed groups ( Figure 4). However, compared to the WT seeds, the MDH activity of the OsAOX1a-RNAi seeds decreased by 14.4%, 14.3%, 13.18% and 24.8%, at 100%, 90%, 80%, and 70% germination, respectively, while ATP content decreased by 47.2%, 60.5%, 53.8% and 51.8%, respectively. These results indicate that the mitochondrial metabolism in the OsAOX1a-RNAi seeds after imbibition was weaker than that of the WT group.

The Abundance of Complex I Subunits during Artificial Aging Treatments
Mitochondrial electron transfer chain Complex I is upstream of the AOX pathway and is classified into five functional sections: N module, Q module, P module, CA domain and GLDH domain. The abundance of Complex I subunits was compared between OsAOX1a-RNAi and WT seeds at germination rates of 100%, 90%, 80% and 70% ( Figure 5).

The Abundance of Complex I Subunits during Artificial Aging Treatments
Mitochondrial electron transfer chain Complex I is upstream of the AOX p and is classified into five functional sections: N module, Q module, P module, CA d and GLDH domain. The abundance of Complex I subunits was compared b OsAOX1a-RNAi and WT seeds at germination rates of 100%, 90%, 80% and 70% 5). In the OsAOX1a-RNAi seeds, the abundance of 75 kDa and 51 kDa subunits, t subunits in the N module, were always lower than the corresponding values in seeds during the artificial aging treatment. In addition, the abundance of the 75 kD unit in the WT seeds did not significantly change during the aging treatment, ho that in the OsAOX1a-RNAi seeds was gradually down-regulated. The abundance 51 kDa subunit was down-regulated during treatment, however, the correspondin in the OsAOX1a-RNAi seeds was too low to be resolved. Together, these results i that the capacity of electron transfer in OsAOX1a-RNAi was inhibited, especially time point of critical node of seed viability.
The abundance of the Nad3, Nad4, Nad4L and Nad6 subunits, the core subu the P module, showed different patterns between the OsAOX1a-RNAi and WT see ing the aging treatment. The abundance of Nad4 subunit was too low to be reso either of the two seed groups. The abundance of Nad3 and Nad6 subunits OsAOX1a-RNAi seeds was always higher than that in the WT seeds during agin ment. In addition, the abundance of Nad3 did not significantly change, and the abu of Nad6 was gradually down-regulated in both the seed groups during the agin ment. The abundance of the Nad4L subunit in the OsAOX1a-RNAi seeds was lower than that in the WT during the aging treatment. In addition, the abund Nad4L subunit in the WT seeds was gradually down-regulated during the agin ment, but was not significantly changed in the OsAOX1a-RNAi seeds.
The abundance of the 39 kDa subunit and the Nad7 subunit, the core subunit In the OsAOX1a-RNAi seeds, the abundance of 75 kDa and 51 kDa subunits, the core subunits in the N module, were always lower than the corresponding values in the WT seeds during the artificial aging treatment. In addition, the abundance of the 75 kDa subunit in the WT seeds did not significantly change during the aging treatment, however, that in the OsAOX1a-RNAi seeds was gradually down-regulated. The abundance of the 51 kDa subunit was down-regulated during treatment, however, the corresponding value in the OsAOX1a-RNAi seeds was too low to be resolved. Together, these results indicate that the capacity of electron transfer in OsAOX1a-RNAi was inhibited, especially at the time point of critical node of seed viability.
The abundance of the Nad3, Nad4, Nad4L and Nad6 subunits, the core subunits in the P module, showed different patterns between the OsAOX1a-RNAi and WT seeds during the aging treatment. The abundance of Nad4 subunit was too low to be resolved in either of the two seed groups. The abundance of Nad3 and Nad6 subunits in the OsAOX1a-RNAi seeds was always higher than that in the WT seeds during aging treatment. In addition, the abundance of Nad3 did not significantly change, and the abundance of Nad6 was gradually down-regulated in both the seed groups during the aging treatment. The abundance of the Nad4L subunit in the OsAOX1a-RNAi seeds was always lower than that in the WT during the aging treatment. In addition, the abundance of Nad4L subunit in the WT seeds was gradually down-regulated during the aging treatment, but was not significantly changed in the OsAOX1a-RNAi seeds.
The abundance of the 39 kDa subunit and the Nad7 subunit, the core subunits in the Q module, was always higher in the OsAOX1a-RNAi seeds than in the WT seeds during the aging treatment. The subunits of GLDH and γ-carbonic anhydrase (γ-CA) are the non-conserved subunits of Complex I. The abundance of GLDH subunit was gradually up-regulated in both types of seeds during the treatment. The abundance of the γ-CA subunit in the OsAOX1a-RNAi seeds was always lower than that in the WT during the aging treatment.

Low AOX1a Impaired Seed Development and Storability
AOX1a plays an extremely important role in maintaining growth and promoting plant survival under adverse conditions. Many studies have shown that the expression of AOX1a is up-regulated during a number of critical processes in plant growth and development, including seed germination, stress response, flowering and pollination [16,28,29]. The expression level of AtAOX1a under suitable conditions had no significant effect on plant vegetative growth, but under low temperature condition, the growth of AtAOX1a mutant leaves was shown to be slower and weaker than that of the WT group [30]. Similarly, ripening, respiration, and ethylene production were severely impaired in transgenic LeAOX-RNAi tomato plants [31]. In this study, the seed width, length, and 100-grain weight of OsAOX1a-RNAi seeds were all less than the corresponding measures in the wild-type seeds ( Figure 1). The dry matter accumulation of seeds was found to be positively correlated with the expression level of OsAOX1a, and the down-regulation of OsAOX1a impaired the regulation of cell life activities and seed development. Although the germination percentage of OsAOX1a-RNAi rice seeds after being aged 3, 6 and 9 d did not show a significant difference compared to that of the WT group, the vigor index of OsAOX1a-RNAi seeds during the aging treatment was always significantly lower than that of the WT group ( Figure 2). In addition, the P50 of WT is significantly longer than that of OsAOX1a-RNAi seeds. These results indicated that the down-regulation of OsAOX1a ultimately reduced seed storability. Regulating the expression of AOX1a to adapt to various stresses in the growth environment can help promote plant growth and developmental stability [32][33][34]. However, OsAOX1a-RNAi plant is unable to regulate the expression of OsAOX1a to cope with various stresses encountered during natural growth conditions. Therefore, it can be concluded that AOX1a is closely involved in the regulation of seed development and storability of the seeds.

Low AOX1a Impaired Mitochondrial Activity during Seed Imbibition
AOX1a is a key component in the mitochondrial alternative chain, whose inactivation could lead to a different response [35,36]. In the present study, we found that in the OsAOX1a-RNAi seeds, compared to the WT seeds, the NADH-and succinate-dependent O 2 consumption were both reduced during the artificial aging treatment, and the expressions of the abundances of AOX1 and Cyt c were inhibited (Table 1 and Figure 3). These results are consistent with the fact that the respiratory capacity of AOX1a mitochondria was significantly restricted by the impairment of the alternative-terminal oxidase, the cytochrome oxidase, or both [21,37]. Compared to our previous study, the capacity of O 2 consumption of the pure mitochondria was higher than the crude mitochondria in the present study, which were isolated from the imbibed rice seed after artificial aging treatment. In addition, the MDH activity and ATP content were reduced in the OsAOX1a-RNAi seeds compared to the WT seeds during treatment (Figure 4). These results are consistent with the reported lower activity of the malate-oxaloacetate shuttle in the AOX1a mutant compared to the WT seeds [38]. MDH catalyzes the interconversion of malate and oxaloacetate in mitochondria [39,40]. MDH activity was shown to be reduced in rice seeds during artificial aging treatment [5]. These results indicate that ATP production was severely inhibited in the OsAOX1a-RNAi seeds at the critical node of viability. ATP supply was the core factor for seed germination. The insufficient supply of ATP in OsAOX1a-RNAi seeds at the critical node of viability might lead to a rapid loss of seed viability. AOX1a was important to maintain mitochondrial ETC and energy performance [41]. When considering the pub-lished findings as well as the results of the current study, it appears that mitochondrial metabolism may be inhibited in the OsAOX1a-RNAi seeds after imbibition.

Low AOX1a Altered Mitochondrial Electron Transfer Chain Complex I during Seed Imbibition
In our previous study, the alternative pathway of ETC was significantly induced, whereas the cytochrome pathway was inhibited at the critical node of seed viability in rice [5]. Complex I is the entry site for electrons into the ETC through the oxidation of NADH, which is composed of multiple subunits, including three core modules: N (NADH oxidation site) and Q (ubiquinone reduction site) modules, and P (proton transfer site) module [42][43][44]. When Complex I activity is inhibited, the subunits of NADH dehydrogenase II belonging to the P module, and AOX are upregulated under stress conditions, thus forming an alternative pathway for maintaining cellular respiration and energy supply [45,46]. Our previous study had shown that the P module was significantly induced, whereas the N module was inhibited at the critical node of seed viability in rice [27]. In the present study, we also found that the core subunits of the N module were down-regulated, whereas the subunits of the P module were up-regulated in both OsAOX1a-RNAi and WT at critical node of seed viability ( Figure 5). AOX and the subunits of the N module display dramatic and coordinated responses to stress treatments [47,48]. However, the abundance of Complex I subunits of the WT seeds was higher than that of the OsAOX1a-RNAi seeds, which suggests that the capacity of ETC of the WT seed was significantly higher than that of OsAOX1a-RNAi seed at the critical node of seed viability; therefore, the capacity of ATP production in the WT seeds was higher than that in the OsAOX1a-RNAi seed seeds. This might be the reason for the more rapid loss of viability of the OsAOX1a-RNAi seeds compared to the WT after critical node.

Materials and Treatments
The seeds of Japonica rice (Oryza sativa L. ssp. Japonica cv. "Nipponbare") were used in this study. In order to generate the interfering OsAOX1a construct, a 379-bp fragment was amplified from OsAOX1a full-length cDNA (AB004864), and then the fragment and its inverted repeat fragment were inserted downstream of a maize ubiquitin promoter at the BamHI (NEB), SacI (Takara Bio), KpnI (NEB), and SpeI (Promega) restriction sites of the modified pTCK303 vector [49]. Transgenic plants were generated by the constructs were transferred to Agrobacterium tumefaciens (strain EHA105), and were transformed into rice callus according to the method of Hiei et al. [50], which resulted to be positive in real-time PCR testing via Hyg R gene. The primers are shown in Table 2. Wild-type and transgenic lines were grown in the research fields located in the Hainan province under natural conditions.
The seeds were sealed in an aluminum foil bag at 40 • C for 7 days to break dormancy. The seed germination rate (%)and the vigor index were determined at 28 • C after 7 d in the dark. Artificial aging treatment was performed at 40 • C and 75% relative humidity for 21 days [5]. The aging curve was characterized by seeds withdrawn from the treatment every 3 days for each of the two genotypes (WT and OsAOX1a-RNAi). The time taken for the seed germination rate to decrease to 50% (P50) was calculated using the Avrami equation with OriginPro software 2021 [51].

Crude Mitochondria Purification
For mitochondrial purification, rice seeds were allowed to imbibe at 28 • C for 48 h in the dark, after which 1200 embryos were homogenized in grinding buffer (0.3 M mannitol, 2 mM EGTA, 0.5% (w/v) polyvinypyrrolidone-40, 0.5% (w/v) bovine serum albumin (BSA), 20 mM cysteine, pH 7.5) and the mixture was centrifuged at 2000× g for 5 min. The supernatant was removed, and the pellet was resuspended in a wash buffer (0.3 M sucrose, 10 mM TES, pH 7.5) and centrifuged again at 2000× g for 5 min. The resulting supernatant was collected and centrifuged at 12,000× g for 15 min. The supernatant was then discarded, and the pellet was resuspended in the wash buffer. The mixture was centrifuged at 2000× g for 5 min, the supernatant was removed, and the mixture was centrifuged again at 12,000× g for 15 min to obtain the mitochondria, from which 4 mg mitochondrial protein could be extracted. Each step of the experiment was performed at 4 • C, and the mitochondria were stored at −80 • C.

ATP Content Determination
ATP content was determined using ATP Content Assay Kit (Solarbio, Beijing, China) according to the instructions of the manufacturer. Mitochondria pellets were resuspended in 1 mL extraction buffer and centrifuged at 10,000× g for 10 min, after which 500 µL chloroform was added to the supernatant, and the mixture was centrifuged at 10,000× g for 3 min. The supernatant was collected for ATP detection.

Mitochondrial Malate Dehydrogenase Activity
Mitochondrial malate dehydrogenase activity was based on the reversible reaction of NAD + oxidizing L-malic acid to oxaloacetate, which was determined by monitoring the increase in absorbance at 340 nm and 25 • C, and 0.5 mg of mitochondrial protein was added to the reaction solution (0.1 M potassium phosphate, pH 7.5, 0.2 mM NADH, and 5 mM oxaloacetic acid).

Statistical Analyses
All data were analyzed as a one-variable general linear model procedure (analysis of variance) via SPSS (SPSS Inc., Chicago, IL, USA). Differences between OsAOX1a-RNAi transgenic plants and WT controls detected using the Student's t-test at the level of p < 0.05, 0.01, and 0.001 were considered as significant, and were labeled as *, **, and ***, respectively.

Conclusions
In conclusion, we explored the biological mechanisms of rice seed viability at the critical node, with the goal of providing a scientific basis for improved seed conservation.
The key finding of this study is that seed development and storability was shown to be impaired in OsAOX1a-RNAi seeds. In addition, mitochondrial metabolism and alternative pathways were severely inhibited in OsAOX1a-RNAi seeds at the critical node of viability. An insufficient level of ATP was available after imbibition, which led to the accelerated decrease in seed viability, compared to the wild type.