Although the impact of agronomical and environmental growing conditions on the level of PhytoPs and PhytoFs have been already described in rice crops (Pinciroli et al. 2018, 2019), there is a gap of knowledge on the evolution of the PhytoP and PhytoF concentration during grain development. In this regard, obtaining accurate information on the evolution of the quantitative profile of PhytoPs and PhytoFs during this stage would be much informative, providing theoretical support for further improvement of agronomical management. The present work aims to study the changes on the concentration of these oxylipins in rice grains during the ontogenesis, as an approach that would contribute strongly to identify relationships between the concentration of PhytoPs and PhytoFs, and hormonal regulation of oxidative stress, and its impact of final product quality.
Total content of phytoprostanes and phytofurans in unripe and ripe rice grains
The assessment of rice grains of three distinct genotypes at different ripening stages (unripe and mature grains) on their total content of PhytoPs and PhytoFs noticed that these plant oxylipins' concentration varies significantly depending on the genotype considered and ontogenetic stage.
In respect to total PhytoPs, the relative abundance in unripe and mature rice grains of the genetic resources under study presented the following decreasing order: ‘R45’ (2006.4 ng.g− 1 dw) > ‘R52’ (1898.7 ng.g− 1 dw) > ‘Yerua’ (1616.1 ng.g− 1 dw) for unripe rice grains, and ‘Yerua’ (1713.2 ng.g− 1 dw) > ‘R52’ (1445.5 ng.g− 1 dw) > ‘R45’ (420.4 ng.g− 1 dw) for mature grains. As demonstrated previously regarding other crops, namely almonds (Carrasco-Del Amor et al. 2015), melon leaves (Yonny et al. 2016), rice (Pinciroli et al. 2017), cocoa (León-Pérez et al. 2019), olive oil (Carrasco-Del Amor et al. 2017; Collado-González et al., 2015b, 2015c; Domínguez-Perles et al. 2018; García-García et al. 2019) or, the diversity of the genetic background within a single species of higher plant can be responsible for specific profiles of fatty acids and resistance to biotic and abiotic stresses that, in turn, give rise to different quantitative profiles of plant oxylipins. The results retrieved in the current work reinforce this hypothesis, providing information that also extent this fact to the oxylipins level of rice grains during development.
In more detail, concerning the changes in the total PhytoPs concentration during ripening, the cv. ‘Yerua’ presented equivalent concentration of total PhytoPs in unripe and mature grains. In contrast, in the advanced lines ‘R52’ and ‘R45’, the abundance of these plant oxylipins in mature grains decreased significantly relative to those in the immature stage (by 24 and 79%, respectively) (Fig. 1A).
On the other hand, when comparing the concentration of total PhytoPs of rice grains between genotypes at both maturation stages, it was found that, regarding unripe samples harvested at 14 DAH, no significant differences were found between genotypes, which exhibited values raging between 1616.1 and 2005.4 ng.g− 1 dw. Nonetheless, regarding mature grains, the significantly highest concentration corresponded to the cv. ‘Yerua’ (1713.2 ng.g− 1 dw) that surpassed the concentrations found in the advanced lines ‘R52’ and ‘R45’ by 18.5 and 307.5%, respectively (Fig. 1A).
Besides PhytoPs, according to the concentration of total PhytoFs generated in the rice grains, the genotypes evaluated evidenced the following decreasing trend: ‘Yerua’ (37.0 ng.g− 1 dw) > ‘R52’ (27.6 ng.g− 1 dw) > ‘R45’ (32.2 ng.g− 1 dw) for unripe grains and ‘Yerua’ (38.1 ng.g− 1 dw) > ‘R52’ (22.0 ng.g− 1 dw) > ‘R45’ (17.4 ng.g− 1 dw) for mature grains. These plant oxylipins have also been studied in other plant seeds, namely pine nuts, walnuts, chia seeds, flax, rice, pea, almonds, pistachio and cocoa clones, where have been found in the concentration range 0.3–28.0 ng.g− 1 (Carrasco-Del Amor et al. 2016, 2017; Cuyamendous et al. 2016; Domínguez-Perles et al. 2018; León-Pérez et al. 2019; Pinciroli et al. 2019; 2020; Pino Ramos et al. 2019). The lower cellular content of PhytoFs observed could be due to the fact that the synthesis of this family of compounds is prioritized under a partial pressure of oxygen greater than the pressure present in plant cells during embryogenesis (Cuyamendous et al. 2015, Fessel et al. 2002).
Besides the differences due to the genetic background, the ripening process also seems to have a critical influence on the final concentration of PhytoFs in mature grains. In this respect, in ‘Yerua’, the ripening process did not affect the concentration of total PhytoFs (37.6 ng.g− 1 dw, on average, in both maturation stages) significantly, while in ‘R52’ and ‘R45’, which presented a concentration of total PhytoFs at the unripe stage of 29.9 and 19.7 ng.g− 1 dw, respectively, during maturation experienced a decrease by 20.0 and 46.0%, respectively. However, this reduction was statistically significant only for ‘R45’ (Fig. 1B).
The overall results regarding total PhytoPs and PhytoFs suggest a higher content of PhytoPs in the advanced lines (‘R52’ and ‘R45’) in the initial stages when grain filling occurs (unripe grains). This is in agreement with the fact that, in the embryogenesis stage, the intense mitochondrial respiration rate generates high amounts of ROS, responsible for the oxidation of fatty acids making part of the cellular structures toward the synthesis of plant oxylipins (Bailly 2004). In this aspect, the totipotent protoplasts during the intense cell division occurring in embryogenesis have also been associated with the cellular machinery responsible for maintaining the redox balance (Bailly 2004). On the opposite, in later stages and especially when the grain is dry and inactive, the respiratory activity decreases even becoming practically null (León-Pérez et al. 2019), thus minimizing ROS production and the oxidation of fatty acids toward the synthesis of PhytoPs and PhytoFs. This has been further demonstrated by several authors, who have found an intense fall in the activity of protective enzymes responsible for the regulation of the redox balance (superoxide dismutase (SOD), catalase (CAT), peroxidase (POD)), by neutralizing the harmful effect of ROS after 14 or 21 DAH (Panda et al. 2013; Zhao et al. 2007). In agreement with this, this period could be required to reach the physiological maturity of seeds, as well as their final size, which would entail a decrease in metabolic activity.
In this regard, the intense generation of ROS would be defining the high level of oxylipins, in this case, by auto oxidation. This diversity of molecules could act as stress biomarkers. This mechanism would be equivalent to activating the enzymatic system that unfolds to neutralize ROS and restore the cellular redox balance.
However, this fact seems to be closely dependent on the genetic resource considered and its specific physiological characteristics (Collado-González et al. 2020; Medina et al. 2020). In this regard, after maturation, the grain of the cv. ‘Yerua’ did not show a reduction in the concentration of both families of stress biomarkers monitored in the present work (PhytoPs and PhytoFs). Besides, the slow senescence that characterizes this genotype is associated with preserving active green leaf tissues during the advanced grain-filling period. Possibly, this intense metabolic activity is also present regarding the metabolism of the seed, which constitutes a cornerstone for the presence of oxylipins related to limited damage to the cell membranes and the maintenance of the redox balance. On the other hand, in the advanced rice lines, the excess of energy may not dissipate efficiently, and ROS may be generated. The ROS produced speed-up the leaf senescence process, upon which the leaves turn yellow, showing severe damage to the photosynthetic apparatus (Panda et al. 2013). Senescence is an oxidative process that involves the overproduction of ROS (Zhao et al. 2007). In the frame of this cellular process, ROS cause cell damage via lipid peroxidation that is generally considered as a major contributor to the senescence syndrome (del Río et al. 1998). In this aspect, it has been reported that during the period featured by fast growth, as well as in plants with a significant biomass producing capacity, the antioxidant enzymes’ activity toward preventing the production of ROS augments (Panda et al. 2013).
Effect of exogenous salicylic acid on the quantitative profile of phytoprostanes and phytofurans in unripe and mature rice grain
Moreover, total PhytoPs and PhytoFs, in this study, the abundance of the individual compounds belonging to both families of plant oxylipins was determined in the three rice genotypes, regardless of the harvest time and the supplementation with salicylic acid. These analyses allowed establishing the following decreasing order of average concentration: co-eluting ent-16-F1t-PhytoP and ent-16-epi-16-F1t-PhytoP (625.9 ng.g− 1 dw) > 9-epi-9-F1t-PhytoP (406.6 ng.g− 1 dw) > 9-F1t-PhytoP (377.4 ng.g− 1 dw) > 9-D1t-PhytoP (73.2 ng.g− 1 dw) > 16-B1-PhytoP (13.9 ng.g− 1 dw) > 9-epi-9-D1t-PhytoP (9.9 ng.g− 1 dw) > 9-L1-PhytoP (9.8 ng.g− 1 dw) (data not shown). When comparing these results with the information available in the literature on rice and other plant-based foods and foodstuffs (dry melon leaves, aged wine, grape nuts, olive oil, almonds, and macroalgae), it was noticed that, in agreement with the outcomes described in this work, compounds belonging to the F1-PhytoPs were the most abundant (Barbosa et al. 2015; Carrasco-Del Amor et al. 2015; Collado-Gonzalez et al. 2017; Domínguez-Perles et al. 2018; Marhuenda et al. 2015; Martínez-Sánchez et al. 2020; Yonny et al. 2016).
On the other hand, the concentration of individual PhytoFs in the three genotypes analyzed was found in the following decreasing order of average concentration: ent-16(RS)-9-epi-ST-Δ14-10-PhytoF (15.7 ng.g− 1 dw) > ent-16(RS)-13-epi-ST-Δ14-9-PhytoF (10.1 ng.g− 1 dw) > ent-9(RS)-12-epi-ST-Δ10-13-PhytoF (3.3 ng.g− 1 dw) (data not shown). This ranking matches with that observed in five indica-type rice genotypes (Pinciroli et al. 2019), as well as with the most abundant PhytoF described in melon leaves by Yonny et al. (2016) (ent-16(RS)-9-epi-ST-Δ14-10-PhytoF) .
Besides the basal concentration of individual PhytoPs and PhytoFs, the influence of 1 and 15 mM salicylic acid on the content of these oxylipins, in unripe and mature rice grains, was also evaluated in the ‘Yerua’ cv. and two advanced lines (‘R52’ and ‘R45’). As a result, significantly different effects were observed in the concentration of individual PhytoPs and PhytoFs (Figs. 2 and 3). This is of particular relevance because, nowadays, little is known about the oxidative state in the early stages of grain filling, even though the possible involvement of ROS in seed-filling processes has been well documented (Bailly 2004).
In this regard, developing embryos would potentially generate significant amounts of ROS, requiring strict control through antioxidant mechanisms (Bailly 2004). In unripe grains, a single application of 1 or 15 mM SA demonstrated that the basal concentration of individual PhytoPs and PhytoFs decreased reversely with the amount of SA applied. However, this response was only significant in respect to 9-epi-9-D1t-PhytoP (23.3, 10.1, and 9.7 ng.g− 1 dw for 0, 1, and 15 mM SA, respectively) (Fig. 2 Table A.1). The rest of the plant oxylipins did not show a response with a simple application of SA in that short period (6 days after application) and doses (1–15 mM SA) (Fig. 2 Table A.1).
On the contrary, when analyzing the effect of the application of 1 and 15 mM SA on the quantitative profile of PhytoPs and PhytoFs of mature grain, both 1 and 15 mM SA decreased the concentration of 7 out of the 11 oxylipins monitored (ent-16-F1t-PhytoP, ent-16-epi-16-F1t-PhytoP, 9-D1t-PhytoP, 9-L1-PhytoP, ent-16(RS)-9-epi-ST-Δ14-10-PhytoF, ent-9(RS)-12-epi-ST-Δ10-13-PhytoF, and ent-16(RS)-13-epi-ST-Δ14-9-PhytoF). At the same time, 9-F1t-PhytoP, 9-epi-9-F1t-PhytoP, 9-epi-9-D1t-PhytoP, and 16-B1-PhytoP did not respond significantly to the hormonal application (Fig. 2 Table A.1).
Before the harvest of unripe rice grains, a single SA application was made, six days before. Nonetheless, when harvesting the mature grain, rice plants had received two SA (1 or 15 mM) applications that raised the hormonal concentration of plants, causing a marked effect on the redox balance during the filling and drying of the grain.
Concerning individual PhytoPs, the highest dose of SA assayed (15 mM) was the only with powerful enough to increase their concentration relative to untreated control significantly. In the case of PhytoFs, and especially regarding ent-9(RS)-12-epi-ST-Δ10-13-PhytoF and ent-16(RS)-13-epi-ST-Δ14-9-PhytoF of mature grains, even the lower dose of SA (1 mM) reduced the concentration of these oxylipins particularly, thus demonstrating their capacity to prevent oxidative stress during embryogenesis (Fig. 3 Table A.2).
In the light of these antecedents regarding other higher plants like tomato, the treatments with 1 mM SA seem to enhance the ROS production and consequently the lipid peroxidation reactions in vitro and photosynthetic tomato leaves (Poór 2020). Indeed, the induction of the ROS production by SA has been observed in treatment with different concentrations, in an array of plant species and models, such as in suspension culture of tobacco cells treated with 1 mM SA, in mitochondria isolated from tobacco leaves with 1 mM SA, in isolated tobacco cell mitochondria treated with 0.5 mM SA, in the appendix Sauromatum guttatum with SA 0.01 mM, in isolated soy cell mitochondria with 1 mM SA, in Orobanche seeds with 0.02 mM SA, in tobacco calluses with 0.02 mM SA and in purified mitochondria of the lupine yellow cotyledon treated with 1 mM SA (Poór 2020).
Recently, in rice plants, Wang et al. (2016) observed that the application of quinclorac damages the thylakoid membranes and that the pre-treatment of SA 0.07 mM (10mg L− 1) under quinclorac stress prevented the rupture of the thylakoid membrane by accelerating the production of chlorophyll and eliminating ROS, which indicates a key role in tolerance to SA-induced oxidative stress in rice plants.
Oxidative response of unripe and mature grains after the application of exogenous salicylic acid depending on rice cultivars
When analyzing the effect of the application of SA on the concentrations of the individual oxylipins (PhytoPs and PhytoFs) in unripe and mature grains, significant interactions for all individual PhytoPs at harvest time (ANOVA, p < 0.05), considering the ‘genotype’ as the source of variation were observed (Figs. 2 and 3).
Regarding individual PhytoPs in unripe grains and their evolution due to the exposition to different concentrations of SA, in general, the genotype ‘Yerua’ exhibited a decrease in their concentration. In this aspect, the differences found were only statistically significant for 9-epi-9-F1t-PhytoP (Fig. 2, p < 0.05). On the other hand, the mature grain of the ‘Yerua’ cv. the changes in individual PhytoPs observed affected 9-F1t-PhytoP, 16-B1-PhytoP, and 9-L1-PhytoP. Hence, while the concentration of 9-F1t-PhytoP was reduced significantly with the application of 1 mM SA, the concentration of 16-B1-PhytoP and 9-L1-PhytoP was only lowered significantly (by 53.1%, on average) when applying the highest dose (15 mM) (Fig. 2). In respect to the advanced line ‘R52’, in unripe grains, only the concentration of 9-epi-9-F1t-PhytoP was modified significantly by the application of SA, decreasing from 49.1 to 13.1 ng.g− 1 dw. However, this activity was observed when applying 1 mM SA. However, in mature grain, the concentrations of 9-F1t-PhytoP, ent-16-F1t-PhytoP + ent-16-epi-16-F1t-PhytoP, 9-epi-9-F1t-PhytoP, ent-9-D1t-PhytoP and 9-L1-PhytoP (299.6, 958.9, 7.3, 70.0, and 8.8 ng.g− 1 dw, respectively, under control conditions) decreased significantly after the application of 15 mM SA by up to 103.0% (Fig. 2). Finally, in respect to the advanced line ‘R45’, the application of 1 or 15 mM SA did not modify the concentration of individual PhytoPs in unripe grains significantly, while in mature grains of plants exposed to 1 mM SA, decreased concentrations of 16-B1-PhytoP and 9-L1-PhytoP from 5.96 to 3.82 ng.g− 1 dw and from 4.45 to 3.20 ng.g− 1 dw, respectively, were observed (Fig. 2).
The different responses observed in three rice genotypes to exogenous SA agree with previous descriptions. They studied different rice genotypes and described a significant difference between photosynthetic parameters and the antioxidant activity of specific enzymes (Cao et al. 2009; Panda et al. 2013). This is in agreement with other works that described different efficiency to neutralize ROS by distinct genotypes (Enyedi et al. 1992; Pal et al. 2013). Indeed, in this regard, recently, a differential modification of the quantitative profile of oxylipins, when applying foliar fertilization in 5 rice genotypes has been specifically described (Pinciroli et al. 2019), reporting significant changes of ent-16-F1t-PhytoP, ent-16-epi-16-F1t-PhytoP, and 9-D1t-PhytoP.
With respect to the different concentrations of PhytoPs between immature and mature rice grain and the response to the exogenous SA applied, the genotype ‘Yerua’ did not present significant differences. On the other hand, in the advanced line ‘R52’, the concentration was different in mature grain regarding 9-F1t-PhytoP and 9-epi-9-D1t-PhytoP for all SA doses and in 9-epi-9-F1t-PhytoP, 9-D1t-PhytoP, 16-B1-PhytoP, and 9-L1-PhytoP when applying the highest amount of SA (15 mM). In contrast, in the advanced line ‘R45’, the concentration of all individual PhytoPs in mature grain was significantly lower than in unripe grain (Fig. 2).
Besides, the analysis of the concentration of individual PhytoFs in unripe grains of the ‘Yerua’ cv. evidenced that the concentrations of ent-16(RS)-9-epi-ST-Δ14-10-PhytoF, ent-16(RS)-13-epi-ST-Δ14-9-PhytoF, and 9-F1t-PhytoP were significantly reduced by the highest concentration of SA (15 mM) (Fig. 3). In mature grain, the lower concentration of SA (1 mM) was powerful enough to lower the concentration of all 3 PhytoFs monitored. Nonetheless, in unripe grains of both advanced lines under study, the application of SA did not produce any effect, while concerning mature grains, in ‘R52’, a decrease of the concentration of the three PhytoFs was observed as a result of the application of 15 mM SA. In the advanced line ‘R45’, the only PhytoF that evidenced a significant reduction of its concentration in plants exposed to 15 mM of SA was ent-16(RS)-13-epi-ST-Δ14-9-PhytoF that after SA treatment achieved 4.82 ng.g− 1.
When analyzing the quantitative profile of the individual PhytoFs at harvest time, a decrease of the mature grains produced compared to immature grains in the three genotypes was observed. However, in the genotype ‘Yerua’, this reduction was only significant concerning ent-16(RS)-13-epi-ST-Δ14-9-PhytoF after the application of 1 mM SA, while in ‘R52’ and ‘R45’, the significant differences observed were restricted to ent-16(RS)-9-epi-ST-Δ14-10-PhytoF and ent-16(RS)-13-epi-ST-Δ14-9-PhytoF, although in ‘R52’ as a result of the exposition to 15 mM SA and ‘R45’ concerning both 1 and 15 mM SA (Fig. 3).
The close relationship between the exogenous application of SA and the concentration of oxylipins observed in the results corroborates their link in the control of redox homeostasis. On this occasion, a parallel can also be made of the reduction of oxylipins when applying SA with the decline of enzymatic antioxidant activity. Wang et al. (2020) observed that SA pre-treatment further enhanced all enzymes' activity compared to the herbicide quinclorac treatment alone. Superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD) activities showed the same increasing tendency in the leaves of rice.
Effect of exogenous salicylic acid on yield and quality of rice grain
To evaluate the effect of applying the phytohormone SA on the yield and quality of rice in the genotypes under evaluation, it was assessed the number of grains per panicle and the percentage of chalkiness (often defined as the opaque parts in the endosperm (Zhang et al. 2009), since both parameters have a great economic repercussion, as well as the influence of exogenous SA on these parameters. The number of grains per panicle, together with the weight of the grain and the number of spikelets per panicle per growing area, define the productivity of the crop in kilograms per hectare (Kocher et al. 1990). Chalkiness is a defect that decreases the visual quality and generates a fragile structure in the grain, which increases the breakage during the mill, reducing, as a consequence, the percentage of whole grain, base of the commercialization of this cereal (Kocher et al. 1990).
Table 1
Mean values of number of spikelets per panicle and chalkiness in mature grain of rice genotypes at different salicylic acid supplementation levels
Parameter
|
Genotype Z
|
Treatment Y
|
Interaction SA by G
|
Control
|
SA 1
|
SA 15
|
Spikelets per panicle
|
‘Yerua’
|
119.8 ± 1.7 a X
|
115.2 ± 6.3 a
|
120.4 ± 4.5 a
|
N.s.
|
|
‘R52’
|
112.1 ± 5.0 b
|
110.3 ± 1.3 b
|
132.2 ± 9.9 a
|
|
‘R45’
|
106.2 ± 5.5 a
|
107.8 ± 1.1 a
|
119.5 ± 5.4 a
|
Chalkiness (%)
|
‘Yerua’
|
18.6 ± 1.9 a
|
17.8 ± 1.9 a
|
15.6 ± 0.7 a
|
N.s.
|
|
‘R52’
|
22.6 ± 2.1 a
|
15.3 ± 2.0 ab
|
13.8 ± 0.7 b
|
|
‘R45’
|
15.4 ± 1.5 a
|
14.0 ± 1.7 a
|
11.7 ± 1.2 a
|
Z Genotypes, ‘Yerua’, Yerua PA; ‘R52’, R/03-5x desc/04-52-1-1; ‘R45’, R/03-5x desc/04-45-1-1. Y Control, without exogenous application of salicylic acid; SA1, salicylic acid 1 mM; SA15, salicylic acid 15 mM. Data presented as mean ± SEM (n = 3). X Values in the same row followed by different letters are significantly different at p < 0.05 according to one-way analysis of variance (ANOVA) and multiple range test of Tukey.
|
The analysis of the number of spikelets per panicle evidenced an increase of between 2.0 and 19.0% when applying exogenous SA at the highest dose (15 mM) in the three genotypes (Table 1). This difference was only statistically significant (p < 0.05) in the advanced line ‘R52’ when applying the highest dose of SA, which allowed recording the values of the number of spikelets per panicle 112.1, 110.3, and 132.2, on average, for the doses of SA of 0, 1, and 15 mM, respectively (Table 1). The number of spikelets per panicle did not present differences between genotypes, and its average value was 115.9 spikelets per panicle.
The application of SA also influenced the percentage of chalkiness. Although the physiological mechanisms involved in chalkiness are not fully understood, Zhang et al. described certain hormonal regulation (ethylene and 1-aminocylopropane-1-carboxylic acid) in the grains, which can affect the arrangement of the starch granules in the cells, leading to an increase in the dull parts in the endosperm (Zhang et al. 2009). In the case of damaged (central chalky) grains caused by the high-temperature stress, abnormal and round shape starch granules were loosely packed in the part of grain, and this part is whitely seen by irregular reflection of the light. The mechanism of grain chalkiness under high-temperature stress is considerably complicated. The temperature at the grain filling stage has shown to influence the starch composition in rice grains (Mitsui et al. 2013).
In the present work, chalkiness was equivalent in all genotypes (Table 1). However, the application of SA induced a decrease in the average values of the three genotypes. So, chalkiness was 18.9, 15.7, and 13.7% for 0, 1, and 15 mM SA, respectively, being the application 15 mM SA the only responsible for the differences retrieved relative to the control. When analyzing the effect of SA on each genotype, it was observed that, although all three genotypes improved the grain transparency, chalkiness value was only statistically lower in ‘R52’ (Table 1). The observed differences between the lines may be due to the endogenous content of SA and the different capacity to maintain the redox homeostasis (Pal et al. 2013). Although no trials have been reported of the influence of SA on the chalkiness, it is known that ascorbic acid is a major plant antioxidant that is likely responsible for changing redox homeostasis in critical developmental stages associated with grain filling and alters grain chalkiness in rice (Yu et al. 2015).
Furthermore, the chalking mechanism of a rice grain under the HT stress is discussed in terms of grain starch glycome, transcriptome, and proteome (Mitsui et al. 2013).