Comparative Transcriptome Combined with Morphophysiological Analyses Revealed Carotenoid Biosynthesis for Differential Chilling Tolerance in Two Contrasting Rice (Oryza sativa L.) Genotypes

doi:10.21203/rs.3.rs-3332662/v1

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Comparative Transcriptome Combined with Morphophysiological Analyses Revealed Carotenoid Biosynthesis for Differential Chilling Tolerance in Two Contrasting Rice (Oryza sativa L.) Genotypes

https://doi.org/10.21203/rs.3.rs-3332662/v1

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published 25 Nov, 2023

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Early spring cold spells can lead to leaf chlorosis during the rice seedling greening process. However, the physiological and molecular mechanisms underlying the rice greening process under low-temperature conditions remain unknown. In this study, comparative transcriptome and morphophysiological analyses were performed to investigate the mechanisms mediating the responses of the Koshihikari (Kos) and Kasalath (Kas) rice cultivars to chilling stress. According to their growth-related traits, electrolyte leakage, and chlorophyll fluorescence parameters, Kos was more tolerant to low-temperature stress than Kas. Moreover, chloroplast morphology was more normal (e.g., oval) in Kos than in Kas at 17°C. The comparative transcriptome analysis revealed 610 up-regulated differentially expressed genes that were common to all four comparisons. Furthermore, carotenoid biosynthesis was identified as a critical pathway for the Kos response to chilling stress. The genes in the carotenoid biosynthesis pathway were expressed at higher levels in Kos than in Kas at 17°C, which was in accordance with the higher leaf carotenoid content in Kos than in Kas. The lycopene β-cyclase and lycopene ε-cyclase activities increased more in Kos than in Kas. Additionally, the increases in the violaxanthin de-epoxidase and carotenoid hydroxylase activities in Kos seedlings resulted in the accumulation of zeaxanthin and lutein and mitigated the effects of chilling stress on chloroplasts. These findings have clarified the molecular mechanisms underlying the chilling tolerance of rice seedlings during the greening process.

Rice

carotenoid biosynthesis

chloroplast development

cold tolerance

greening process

In early spring, rice seedlings emerging from the soil are susceptible to cold stress, which increases the likelihood of frost damage and disease (Andaya and Tai 2007). The newly developed tray-overlaying seedling raising mode for transplanting rice addresses the problems of uneven emergence, rotten seedlings, and serious diseases caused by cold stress in early spring (Zhu et al 2018). However, during the transfer of rice seedlings from a warm (i.e., temperature controlled) dark room to cold conditions outdoors, some seedlings remain susceptible to low-temperature stress in early spring, resulting in leaf chlorosis (Fukuda et al 2021; Yoshida et al 1996)

Plant responses to low temperatures are mediated by a complex mechanism involving Ca²⁺ signaling, reactive oxygen species (ROS) homeostasis, phytohormones, and MAPK signaling (Das and Roychoudhury 2014; Wang et al 2021a). Plants produce a variety of antioxidants to counteract the oxidative stress induced by low temperatures, including carotenoids, tocopherols, ascorbic acid, glutathione, and antioxidant enzymes (Havaux 2014; Yun et al 2010). Additionally, when seedlings kept in darkness are exposed to light, their incompletely developed chloroplasts cannot process the sudden increase in light, which leads to photo-oxidative stress (Hideg et al 2006; op den Camp et al 2003). During this process, carotenoids, which function as antioxidants, also serve as pigments that protect chloroplasts from photo-oxidative stress exacerbated by low temperatures (Dhami and Cazzonelli 2020; Maslova et al 2021; Nisar et al 2015; Sun et al 2022). The significant correlations among low-temperature stress, carotenoid biosynthesis pathways, and phytohormone signaling have been investigated in many plants (Dhami and Cazzonelli 2020; Li et al 2021; Pan et al 2022; Yang et al 2021). A transcriptome-based acclimatization analysis of the photo/thermo-sensitive genic male sterile rice line Yu17S indicated that carotenoid biosynthesis and phytohormone signaling are closely related to cold tolerance (Pan et al. 2022).

The carotenoid biosynthesis pathway is initiated following the synthesis of geranylgeranyl pyrophosphate through the MEP pathway. Phytoene synthase (PSY), which is the primary rate-limiting enzyme of the carotenoid biosynthesis pathway, catalyzes the synthesis of 15-cis-phytoene (Gao et al 2010; Ruiz-Sola and Rodriguez-Concepcion 2012). Subsequently, all-trans-lycopene is synthesized from 15-cis-phytoene via reactions catalyzed by phytoene desaturase, ζ-carotene isomerase, ζ-carotene desaturase, and phytoene desaturase. Next, β- and α-carotene are synthesized by the cyclization of all-trans-lycopene mediated by lycopene ε-cyclase (LCYE) and lycopene β-cyclase (LCYB), respectively. Previous research indicated that LCYE and LCYB control a major branch of the carotenoid biosynthesis pathway (Kang et al 2018; Moreno et al 2016; Ruiz-Sola and Rodriguez-Concepcion 2012). Four carotenoid hydroxylases (CHs), which are classified as β-ring hydroxylases (BCHs) and cytochrome P450-type hydroxylases (CYP97A/CYP97C), hydroxylate α- and β-carotene to produce lutein and zeaxanthin. In addition, β-carotene and lutein, which are key components of photosynthetic membranes, form a pigment–protein complex essential for photoprotection (Ding et al 2021; Jahns and Holzwarth 2012; Leonelli et al 2017). The production of zeaxanthin is catalyzed by zeaxanthin epoxidase (ZEP) and reversed by violaxanthin de-epoxidase (VDE). This process, known as the xanthophyll cycle, is one of the primary mechanisms protecting plants from the detrimental effects of excessive light (Gao et al. 2010; Wang et al 2021b). Abscisic acid synthesis, which is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED), is considered to be crucial for carotenoid degradation. Rice (Oryza sativa L.) is a thermophilic crop (optimal growth temperature: 20–33°C) in the family Poaceae and is a major contributor to global food security.

There has recently been an increase in the frequency of abnormally low temperatures in early spring (Gao et al 2022; Zhang and Wang 2022). Leaf chlorosis is caused by the inhibition of chlorophyll biosynthesis and oxidative damage when rice seedlings are exposed to cold conditions (Fukuda et al. 2021; Zhao et al 2020). Moreover, the seedlings of some rice cultivars do not turn green after they are transplanted from a darkened chamber in which the temperature is controlled to a field with a lower temperature. In a recent study involving the indica rice cultivar Kasalath, chlorotic leaves were detected on plants kept at low temperatures, indicative of the sensitivity of this variety to cold stress (Fukuda et al. 2021). In contrast, Koshihikari is a cold-tolerant rice variety (Gazquez et al 2020; Rayee et al 2020). Therefore, these two rice varieties were selected as appropriate materials for studying the physiological and molecular mechanisms against chilling stress. Previous analyses of the chilling tolerance of these two rice varieties focused on physiological factors, such as photosynthetic capacity (Gazquez et al 2018), water use efficiency (Gazquez et al 2015), and secondary metabolism (Fukuda et al. 2021). Accordingly, the morphophysiological and molecular mechanisms that explain the difference in the cold tolerance of these two rice varieties should be examined in more detail. In this study, Kasalath and Koshihikari were compared in terms of their cold tolerance, chloroplast structure, and chlorophyll fluorescence. Furthermore, a comparative transcriptome analysis was carried out to investigate the molecular mechanisms responsible for cold tolerance. Our results provide further evidence of the importance of carotenoid biosynthesis in the leaf greening process of Poaceae plants exposed to low temperatures.

Kos exhibited higher tolerance to low-temperature stress

The phenotypes of Kas and Kos were compared at 17 and 26°C (Fig. 1A). The Kas and Kos seedlings grew normally and turned green at 26°C. However, at 17°C, the Kos seedlings turned green, but the Kas leaves appeared yellowish-white and yellowish-green during the greening process. These results suggested the greening process was slower for Kas than for Kos under chilling stress.

The shoot length, root length, dry weight and REL also differed between the two cultivars at 17 and 26°C (Fig. 1B). The trait values of both cultivars were lower at 17°C than at 26°C, especially the shoot length. Among the examined traits, the shoot length increased by 229.70% (Kas) and 536.72% (Kos) on day 4 (compared with the shoot length on day 0) at 26°C. However, the shoot length increased by only 58.98% (Kas) and 320.62% (Kos) on day 4 (compared with the shoot length on day 0) at 17°C. Accordingly, the shoot length decreased more for Kas than for Kos under low temperature. Furthermore, for both cultivars, REL was increased due to the low temperature. Moreover, REL was higher for Kas than for Kos at 17°C, indicating Kos was more tolerant to chilling stress than Kas.

The chlorophyll and carotenoid contents of the treated seedlings were measured (Fig. 2). After 4 days, the chlorophyll a and b content were 5.33- and 1.54-fold higher in Kas than in Kos at 17°C, whereas the contents of chlorophyll a and b were only 0.94- and 0.63-fold lower in Kas than in Kos at 26°C. The data indicated that the contents of chlorophyll a and b were increased in Kos (compared with those in Kas) on day 4 at 17°C. These results reflected a higher chlorophyll biosynthesis in Kas in response to low temperature.

Kos presented a better-maintained photosystem morphological structure under chilling stress

The chloroplast ultrastructure of the rice seedlings was observed to clarify the effect of chilling stress (Fig. 3). Chloroplast development was inhibited in both cultivars at 17°C, resulting in irregular chloroplast morphology and uneven stacking of cystoid stroma lamellae. The chloroplasts of Kos had more thylakoids stacked into grana than the chloroplasts of Kas. In terms of morphology, the chloroplasts in Kos were more normal (i.e., oval) than the chloroplasts in Kas at 17°C. Hence, the abnormal development of the chloroplasts and thylakoid membrane structures were the dominant factors explaining the leaf chlorosis of the Kas seedlings exposed to chilling stress.

A Pulsed amplitude modulation (PAM) chlorophyll fluorescence meter was used in this study to investigate whether leaf photosynthetic activities were affected by the low-temperature treatment. The maximum (F_v/F_m) and actual photosynthetic capacity of PSII (Φ_PSII) of both cultivars were significantly lower at 17°C than at 26°C. More specifically, the F_v/F_m value of Kas at 26°C was 1.43- folds higher than those at 17°C, whereas in Kos, it was only 1.04- folds higher than those at 17°C. Moreover, F_v/F_m was significantly lower for Kas than for Kos at 17°C, but there was no significant difference between Kas and Kos at 26°C. These results suggested the photosystem morphological structure and the PSII reaction center energy conversion efficiency maintained better in Kos than in Kas exposed to chilling stress.

Carotenoid biosynthesis was identified as a key pathway by the comparative transcriptome analysis of the leaves under chilling stress conditions

To further investigate the molecular mechanisms underlying the differences in the greening process of the two rice genotypes that underwent the low-temperature treatment, we conducted a comparative transcriptome analysis using the high-throughput sequencing system. During the temperature treatments (17 and 26°C), a total of 30 libraries were constructed for the two examined rice genotypes (Kas and Kos). This period is crucial for determining whether rice seedlings can withstand the effects of cold stress. After removing the low-quality reads, more than 40,000,000 clean reads were retained for each sample. The transcriptome sequencing profiles for each sample are provided in Table S2.

The PCA of the RNA-seq data revealed principal components (PCs) 1, 2, and 3 accounted for 25.2%, 17.1%, and 10.5% of the total variability, respectively (Fig. 5A). Genotypes were the key factor affecting gene expression profile. Kas and Kos were separated by the first principal component (PC1). Compared with PC1, the treatment factor (PC2) had a smaller effect, especially on the expression levels of the two genotypes at 26°C (Fig. 5A). According to PC3, for both genotypes, the day 2 and 4 samples were clustered together and separated from the day 0 samples. Thus, the expression of many genes was induced in the seedlings during the period between day 0 and day 2. Under low temperature, gene expression levels on days 2 and 4 were generally higher in Kos than in Kas, suggesting Kas was more sensitive to low-temperature stress than Kos.

After the seedlings were exposed to light for 2 and 4 days, the DEGs on days 2 and 4 (relative to the corresponding expression on day 0) were detected for each genotype [Kos17-(2d/0d), Kos17-(4d/0d), Kas17(2d/0d), and Kas17-(4d/0d)]. The DEGs between Kas and Kos at various time-points [(Kas17/Kos17)-0d, (Kas17/Kos17)-2d, and (Kas17/Kos17)-4d] were also detected (Fig. 5B). A total of 4,278 DEGs (2,879 up-regulated and 1,399 down-regulated) and 4,686 DEGs (2,313 up-regulated and 2,373 down-regulated) were detected in Kas17-(2d/0d) and Kas17-(4d/0d), respectively (Fig. 5C), which was more than the 3,607 DEGs (2,079 up-regulated and 1,528 down-regulated) and 4,508 DEGs (2,399 up-regulated and 2,109 down-regulated) detected in Kos17-(2d/0d) and Kos17-(4d/0d), respectively (Fig. 5B).

The analysis of the up- and down-regulated DEGs among different sets detected 951 DEGs in both Kas17-(2d/0d) and Kas17-(4d/0d), of which 604 genes were up-regulated and 347 genes were down-regulated (Fig. 6B). There were 918 common DEGs in Kos17-(2d/0d) and Kos17-(4d/0d), among which 554 genes were up-regulated and 354 genes were down-regulated. Additionally, 989 genes were detected as DEGs in Kas17-(2d/0d), Kas17-(4d/0d), Kos17-(2d/0d), and Kos17-(4d/0d), of which 610 genes were up-regulated and 379 genes were down-regulated.

The GO and KEGG enrichment analyses of the DEGs revealed differences in the molecular mechanisms affected by low-temperature stress between the two genotypes (Fig. 6A and C). The DEGs were classified in three main GO categories (i.e., BP, CC, and MF) (Fig. 6A). The enriched GO terms among the DEGs in both genotypes were related to oxidative stress, including oxidation-reduction process, response to oxidative stress, cellular oxidant detoxification, and hydrogen peroxide catabolic process. Photosynthesis-related GO terms were significantly enriched among the DEGs in Kas17-(2d/0d) and Kas17-(4d/0d), including photosynthesis-light harvesting, photosynthesis-light reaction, photosynthetic electron transport in PSI, and PSII assembly. However, they were not significantly enriched among the DEGs in Kos17-(2d/0d) and Kos17-(4d/0d). These results reflected the oxidative stress response involving the chloroplasts of both Kas and Kos as well as the positive changes to the photosystems in Kas.

Carotenoid-related GO terms, such as carotene catabolic process, carotene metabolic process, and carotenoid dioxygenase activity, were significantly enriched among the DEGs in Kos17-(2d/0d) and Kos17-(4d/0d), but not among the DEGs in Kas17-(2d/0d) and Kas17-(4d/0d). The following GO terms were also assigned to the DEGs in Kos17-(2d/0d) and Kos17-(4d/0d): cellular response to a toxic substance and carbon-oxygen lyase activity. A KEGG analysis was performed for the common up-regulated DEGs in Kos17-(2d/0d), Kos17-(4d/0d), Kas17-(2d/0d), and Kas17-(4d/0d) (Fig. 6C). Notable enriched KEGG pathways included carotenoid biosynthesis, phenylpropanoid biosynthesis, photosynthesis, and glyoxylate and dicarboxylate metabolism. Among these pathways, carotenoid biosynthesis was significantly enriched among the DEGs in Kos17-(2d/0d) and Kos17-(4d/0d), but not among the DEGs in Kas17-(2d/0d) and Kas17-(4d/0d). Thus, carotenoid biosynthesis may be critical for the cold tolerance of rice seedlings.

We also compared the expression levels of carotenoid biosynthesis-related genes between Kas and Kos (i.e., OsPSY, OsLCYE, OsLCYB, CYP97C2, OsVDE, and OsNCED5) (Fig. 7B). Among these genes, most showed a more pronounced up-regulation in the expression level of Kos at 17°C, whereas not in Kas. Specifically, after the 4-day treatment at 17°C, the OsLCYE, OsLCYB, and OsNCED5 expression levels in Kos increased by 193.24%, 105.75%, and 573.49%, respectively. These increases were greater than the corresponding increases in Kas (i.e., 133.69%, 42.56%, and 311.18% for OsLCYE, OsLCYB, and OsNCED5, respectively). Accordingly, the carotenoid synthesis-related gene expression levels were up-regulated substantially more in Kos than in Kas under low-temperature stress.

Low temperature significantly induced the expression levels of genes related to carotenoid synthesis in Kos (Fig. 7B). After the incubation at 17°C for 4 days, the OsLCYE, OsLCYB, CYP97C2, OsVDE, and OsNCED5 expression levels in Kos increased by 193.24%, 105.75%, 100.07%, 169.09%, and 573.49%, respectively. For the Kos seedlings incubated at 26°C for 4 days, the OsLCYE, OsLCYB, CYP97C2, OsVDE, and OsNCED5 expression levels increased by 153.90%, 76.91%, 44.35%, 49.21%, and 136.57%, respectively. These results indicated that the low-temperature treatment significantly up-regulated the expression of carotenoid synthesis-related genes in Kos, providing additional evidence that carotenoid biosynthesis is a key pathway for enhancing the cold tolerance of rice seedlings.

Carotenoid biosynthesis is critical for conferring cold tolerance

The activities of the key enzymes mediating carotenoid biosynthesis were examined. After a 2-day treatment at 17°C, there were significant increases in the PSY, LCYB, VDE, CH, and NCED activities in Kos. In contrast, there were no significant changes in the PSY, LCYB, VDE, CH, and NCED activities in Kas. More specifically, the LCYB, PSY, VDE, CH, and NCED activities in Kos at 17°C were 2.99-fold, 1.62-fold, 2.02-fold, 2.34-fold, and 1.59-fold higher on day 2 than on day 0, respectively. However, in Kas at 17°C, the LCYB, PSY, VDE, and NCED activities were only 0.73-fold, 0.81-fold, 0.50-fold, and 1.59-fold higher on day 2 than on day 0, respectively. It is evident that the rate of increase in the activities of key enzymes under chilling stress in Kos were significantly higher than those of Kas.

The contents of β-carotene, lutein, zeaxanthin and violaxanthin were significantly higher in Kos than in Kas on day 4 of the incubation at 17°C, which were 3.36-fold, 2.61-fold, 2.66-fold, and 1.43-fold higher in Kos than in Kas at 17°C, respectively (Fig. 8B). However, at 26°C, they were only 1.32-fold, 1.01-fold, 1.00-fold, and 0.74-fold higher in Kos than in Kas, respectively. The greater accumulation of carotenoid biosynthesis related substances in Kos than in Kas at 17°C suggests carotenoids may contribute to enhance the response of seedlings to low-temperature stress.

Rice is a thermophilic crop sensitive to low temperatures (Fukuda et al. 2021; Fukuda and Terao 2015; Mackill and Lei 1997). Rice seedling leaves become chlorotic and appear white under cold conditions, especially during the greening process (Fukuda et al. 2021; Yoshida et al. 1996). After the exposure to light at 17°C, Kas leaves were yellow as they failed to turn green in the study. However, the Kos seedlings incubated at 17°C underwent a normal greening process, resulting in green leaves (Fig. 1). Furthermore, in response to the low-temperature treatment, the Kos leaves had relatively normal oval chloroplasts and more thylakoids than the Kas leaves (Fig. 3). The F_v/F_m and Φ_PSII values were significantly higher for Kos than for Kas after the 4-day exposure to light at 17°C, implying the photosystem morphological structure was maintained better in Kos than in Kas (Fig. 4).

The RNA-seq analysis and comparative transcriptome analysis were performed to investigate the physiological and molecular mechanisms underlying the differences in the leaf greening process between Kas and Kos. The carotenoid biosynthesis pathway was significantly enriched among the DEGs in Kos17-(2d/0d) and Kos17-(4d/0d), but not among the DEGs in Kas17-(2d/0d) and Kas17-(4d/0d) (Fig. 6C). The GO terms related to carotenoid synthesis and metabolism, such as carotenoid metabolic process, carotenoid catabolic process, and terpenoid biosynthetic process, were also significantly enriched among the DEGs in Kos17-(2d/0d) and Kos17-(4d/0d) (Fig. 6A). These results suggest the biosynthesis of carotenoid is more active in Kos than in Kas in response to chilling stress. Similar results have been identified in transcriptome analyses of tea (Li et al 2016; Yang et al. 2021), pepper (Li et al. 2021), and Chinese cabbage (Li et al 2019).

As photosynthetic pigments, carotenoids can expand the range of absorbable light during photosynthesis (Havaux 2014; Maslova et al. 2021), while also protecting light-capturing complexes, membranes, and proteins from excessive incident light via heat dissipation and free radical detoxification (Havaux 2014; Havaux et al 2007; Nisar et al. 2015). The inhibition of chlorophyll synthesis leads to the photosensitized production of singlet oxygen, resulting in ROS production (Hideg et al. 2006; Li and Kim 2022), especially when plants are transferred from darkness to light (op den Camp et al. 2003). Under low temperature, chlorophyll synthesis of rice seedling may be adversely affected during the greening process, thereby resulting in leaf chlorosis (Zhao et al. 2020). Carotenoids can protect plants from cold stress (Dall'Osto et al 2007; Kayess et al 2020). In current study, compared with Kas seedlings, the Kos seedlings with a greener phenotype showed higher carotenoid content at low temperature (Fig. 8B). The xanthophyll cycle and xanthophyll biosynthesis in the process of carotenoid biosynthesis have been revealed as the key physiological mechanism of uneven leaf color in rice, which were similar to this study (Hao et al 2022; Pan et al. 2022). According to RNA-seq analysis, expression of carotenoid-related genes increased significantly more in Kos than in Kas at 17°C; this result was confirmed by the qRT-PCR data (Fig. 7). OsPSY was the rate-limiting gene for carotenoid synthesis, and OsLCYE and OsLCYB acted as synthetic precursors for numerous carotenoids, controlling the production of α- and β-carotene (Cazzonelli et al 2009; Kang et al. 2018; Wang et al 2021c). The higher OsPSY, OsLCYB, and OsLCYE expression levels in Kos than in Kas at 17°C reflected the greater synthesis of carotenoids in Kos than in Kas in response to chilling stress. Furthermore, the LCYB and LCYE activities increased significantly in Kos on days 2 and 4 of the low-temperature treatment, which were also higher in Kos than in Kas (Fig. 8A). After the 4-day incubation at 17°C, the content of β-carotene in Kos was significantly higher than that in Kas (Fig. 8B), suggesting carotenoid biosynthesis prevented leaf chlorosis in the Kos seedlings incubated at 17°C.

In rice leaves, chloroplast synthesis is adversely affected by low temperatures, which also significantly decrease the tolerance to light, leading to increased susceptibility to photo-oxidative stress (Hideg et al. 2006). The xanthophyll cycle was employed against this stress, where zeaxanthin, a potent low-molecular-weight antioxidant, eliminated oxidative substances (Hideg et al. 2006; Jahns and Holzwarth 2012; Leonelli et al. 2017). When plants were exposed to light, VDE activity was increased and zeaxanthin production was promoted as part of the xanthophyll cycle (Wang et al. 2021b). In this study, the expression level of OsVDE was significantly up-regulated in Kos on days 2 and 4; this finding was supported by the qRT-PCR data (Fig. 7). Additionally, the content of zeaxanthin was significantly higher in Kos than in Kas after the 4-day incubation at 17°C In addition to zeaxanthin, β-carotene is a carotenoid that mitigates the harmful effects of photo-oxidative stress and serves as a precursor of zeaxanthin (Havaux 2014; Sun et al. 2022; Zhao et al 2022). In current study, the LCYB and VDE activities, which regulated the synthesis of β-carotene and zeaxanthin, increased significantly in Kos at 17°C (Fig. 8A). Moreover, there was a significant increase in the content of β-carotene in Kos incubated at 17°C (relative to the content in the Kos seedlings maintained at 26°C). In contrast, the β-carotene content was relatively low in Kas, implying Kos can synthesize more β-carotene at low temperatures than Kas (Fig. 8B). The accumulation of β-carotene and zeaxanthin may help alleviate the detrimental effects of chilling stress on chloroplasts, thereby promoting the leaf greening process.

Lutein is a critical component of the chloroplast photosynthetic membrane, while also helping to protect the photosynthetic pigment system (Ding et al. 2021; Jahns and Holzwarth 2012). It has been reported that the decrease in the lutein content led to increased photosensitivity in Arabidopsis thaliana (Park et al 2002). In this study, the low-temperature treatment significantly up-regulated the expression of CYP97C2 in Kos (compared with the corresponding expression in Kas) on days 2 and 4 (Fig. 7B). The CYP97C2, OsLCYB, and OsLCYE genes encode the key regulators of lutein synthesis (Nisar et al. 2015; Sun et al. 2022). The activities of CH and LCYB were increased significantly in Kos under low-temperature stress (Fig. 8A). After the 4-day treatment at 17°C, lutein was significantly more abundant in Kos than in Kas (Fig. 8B). These results revealed the importance of lutein for photoprotection and the prevention of leaf chlorosis in Kos under low-temperature conditions.

In conclusion, comparative transcriptome and morphophysiological analyses were conducted for Kos and Kas, which had distinct greening phenotypes under low-temperature condition. The Kos seedlings underwent a normal greening process at 17°C and contained more chlorophyll and better developed chloroplasts than Kas, indicative of the greater chilling stress tolerance of Kos. The significant up-regulation of the expression of carotenoid biosynthesis-related genes in Kos is suggestive of the positive role of carotenoids in the greening process at low temperatures. The expression levels of the key genes involved in carotenoid biosynthesis, such as OsLCYB, OsLCYE, and OsPSY, were up-regulated in Kos. The increased PSY, LCYB, and LCYE activities as well as the accumulation of β-carotene, lutein, and zeaxanthin in Kos indicate carotenoid biosynthesis is crucial for preventing leaf chlorosis in Kos at low temperatures. This study provides valuable insights into the physiological and molecular mechanisms mediating the adaptation of rice to low temperatures, which may be useful for developing strategies for improving rice seedling quality.

Plant materials and experimental design

The cold-tolerant rice cultivar Koshihikari (Kos; Oryza sativa L., japonica) and the cold-susceptible cultivar Kasalath (Kas; Oryza sativa L., indica) were obtained from the China National Rice Research Institute. Healthy and intact seeds were surface-sterilized in 30% (v/v) H₂O₂ for 20 min. They were subsequently immersed in distilled water for 24 h and then incubated in darkness for 8 h on damp filter paper at 32°C. The seeds were manually transferred to a carbon-based composite seedling substrate specifically designed for rice in standard rice transplanter seedling trays, with each tray containing 90 g germinated seeds. The trays were moved into incubators with different temperatures when 80% of the seeds had germinated and the buds were approximately 10 mm long. The control group was incubated at 26°C (suitable growth temperature) with a light intensity of 120 µmol m^− 2 s^− 1, whereas the low-temperature group was incubated at 17°C with a light intensity of 120 µmol m^− 2 s^− 1. Seedling samples were collected on days 0, 2, and 4 for the analysis of various indices. Specifically, the shoot length, root length, and shoot weight of the Kas and Kos seedlings were measured, with three replicates per treatment, each comprising at least 30 randomly selected seedlings. Another sample of seedlings underwent a tap water wash and several distilled water rinses. These samples were drained, then kept at − 80°C for the estimation of additional parameters.

Determination of the carotenoid and chlorophyll contents and relative electrolyte leakage

Fresh leaves were collected from rice seedlings after 0, 2, and 4 days of the temperature treatments. The leaves were placed in a darkened container and soaked in 95% ethanol until they turned white. Subsequently, the absorbance of the solution at 470, 649, and 665 nm was determined using the SPECORD 200 spectrophotometer (Analytik, Jena, Germany). The chlorophyll a and b contents were calculated as previously described (Song et al 2023).

Relative electrolyte leakage (REL) was analyzed using a published method (Bajji et al 2002). Briefly, fresh leaves were cleaned with deionized water, cut into pieces, and then weighed before being added to tubes containing deionized water. The leaves were vacuum desiccated for 1 h before being placed at room temperature for 4 h. Throughout this period, the leaf samples were intermittently shaken. A conductivity meter (Mettler Toledo, Zurich, Switzerland) was used to determine the conductivity of the extraction solution.

The contents of key substances in the carotenoid biosynthesis, including β-carotene, lutein, zeaxanthin, and violaxanthin, were quantified as previously mentioned (Ma et al 2018; Yang et al. 2021). To extract compounds, acetone was added to fresh rice leaves (200 mg) prior to a 5-min sonication. The extraction using acetone was repeated multiple times. The resulting extracts were mixed with 1 mL acetone, filtered using a syringe filter, and assessed with an ultra-high-performance liquid chromatography (UHPLC) system. The UltiMate 3000 UHPLC system (Thermo Scientific Dionex, Sunnyvale, CA, USA) was employed to determine the β-carotene, lutein, zeaxanthin, and violaxanthin contents. The UHPLC separation and characterization were completed using the Compass C18 reversed-phase column (250 mm × 4.6 mm, 5 µm). The column temperature was 30°C and the walk-away time was 20 min. A Finnigan Surveyor PDA Plus Detector (Thermo, Hennigsdorf, Germany) was used to measure the UV-vis absorbance between 210 and 600 nm.

Chlorophyll fluorescence assay and examination of the chloroplast ultrastructure

The chlorophyll fluorescence of the rice seedlings was analyzed after a 4-day treatment at 17 or 26°C. Before the assay, the seedlings were placed in a darkened container for at least 20 min (dark adaptation), after which the IMAGING-PAM Multi-function Modulated Fluorescence Imaging System (Walz, Bavaria, Germany) was used to determine the fluorescence kinetic profile of the seedlings. The ImagingWinGige software was used to record and extract the following photosystem II (PSII) parameters: initial chlorophyll fluorescence (F_o) in dark-adapted leaves, maximum fluorescence (F_m) in dark-adapted leaves, maximum fluorescence (F_m′) in the light-adapted state, steady-state chlorophyll fluorescence (F_s) in the light-adapted state, effective quantum yield of PSII [i.e., Φ_PSII = (F_m′ − F_s)/F_m′], and potential maximum photosynthetic capacity of PSII [i.e., F_v/F_m = (F_m − F_o)/F_m]. To reduce the effect of external light, the measuring head was clothed with a black cover for all processes.

Chloroplast ultrastructural development in rice leaves was examined using a transmission electron microscope. Fresh leaf tissues (volume less than 1 mm³) were rapidly fixed in a 2.5% glutaraldehyde solution under vacuum conditions and then rested at 20°C for 2 h. They were then incubated at 4°C. The samples were rinsed three times with 0.1 M phosphate-buffered saline (PBS) (pH 7.4) for 15 min and then fixed in a solution consisting of 1% osmium acid and 0.1 M PBS (pH 7.4) for 5 h at 20°C. The samples were rinsed three times with 0.1 M PBS (pH 7.4) for 15 min before and after fixation, and then were dehydrated with different gradients of ethanol (30%, 50%, 70%, 80%, 90%, and 100%). Pure 812 embedding agent (SPI-Pon 812; Structure Probe, Inc., USA) was poured into embedding plates, after which the samples were added to the plates and dried overnight in an oven at 37°C before being polymerized in the oven at 60°C for 48 h. The samples were sliced (60–80 nm sections) using an ultrathin slicer (Leica Microsystems, Ltd., Wetzlar, Germany) and then stained for 15 min using a 2% uranyl acetate saturated alcohol solution and lead citrate. After drying overnight at room temperature, the chloroplast ultrastructure was examined and images were captured using the HT7700 transmission electron microscope (Hitachi, Tokyo, Japan).

RNA extraction and transcriptome sequencing

Total RNA was extracted from the shoots of the seedlings incubated at 26 or 17°C for 0, 2, and 4 days using the RNeasy^® Mini kit (Qiagen, Hilden, Germany). Because each treatment was performed in triplicate, 30 RNA samples were prepared. The RNA quality was evaluated using 1.0% agarose gel electrophoresis and confirmed (i.e., RNA integrity number > 8.0) using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The cDNA libraries were built using the NEB Next^® Ultra™ RNA Library Prep Kit for Illumina (NEB #E7530, USA). They were subsequently sequenced on the Illumina NovaSeq 6000 platform (Illumina Inc., CA, USA), which yielded an average of 6 billion clean reads per sample. To get rid of reads with adapters and poor-quality sequences, the raw reads were filtered. The high-quality reads were retained for the downstream analyses. To align the data, the reference genome and gene model annotation files were obtained from the Rice Genome Annotation Project (Kawahara et al 2013; Sakai et al 2013).

Analysis of differential gene expression

For each sample, gene expression levels were determined in terms of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) values using the RSEM software (version 1.2.15) (Li and Dewey 2011). Differentially expressed genes (DEGs) were detected using the DESeq2 software, with a false discovery rate < 0.01 and |log₂(fold-change)| >1 set as the thresholds for significance (Anders and Huber 2010). The PRCOMP function of the R package (version 3.5.0) was used to perform the principal component analysis (PCA). The PCA plot was visualized using SCATTERPLOT3D. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on the unigenes with up-regulated expression levels on days 2 and 4 of the low-temperature (17°C) treatment period (compared to the corresponding levels on day 0). The significantly enriched functional categories (Fisher’s test; p < 0.05) were visualized using ggplot2. After the Z-score normalization of the original RNA sequencing (RNA-seq) data, heatmaps were generated using the ‘pheatmap’ function of the R package.

Quantitative real-time PCR

The quantitative real-time PCR (qRT-PCR) analysis was performed using the 7500 Real-Time PCR System and the SYBR Premix Ex Taq™ kit to determine the relative expression levels of six genes encoding key enzymes for carotenoid synthesis (i.e., OsPSY, OsLCYE, OsLCYB, CYP97C2, and OsVDE, and OsNCED5). An endogenous actin-encoding gene (NM 197297) was used as the reference control for normalizing expression levels. The qRT-PCR data were analyzed using the 2^−ΔΔCt method (Pfaffl, 2001). For each gene, the expression level (i.e., mRNA level) for Kas at 17°C (designated as Kas-17) was set to 1. The qRT-PCR primers are detailed in Table S1.

Assays of the activities of carotenoid biosynthesis pathway enzymes

To quantitatively analyze enzyme activities, shoots were homogenized and centrifuged. The supernatants were collected and analyzed for the activities of PSY, LCYE, LCYB, CH, VDE, and NCED using commercial enzyme-linked immunosorbent assay kits (Meimian, China) as previously described (Wu et al 2020). Protein contents were analyzed according to the Bradford method (Bradford 1976; Jones et al 1989).

Statistical analyses

Data are herein presented as the mean and standard error. At least three biological replicates were included in all experiments. The data for the seedlings incubated at 26 or 17°C were statistically analyzed using the t-test (p < 0.05). The figures were made using Origin 9.1 (version 9.1) (Origin Lab, Northampton, MA, USA).

BCHs

β-ring hydroxylases

CHs

Carotenoid hydroxylases

DEGs

Differentially expressed genes

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

LCYB

Lycopene β-cyclase

LCYE

Lycopene ε-cyclase

NCED

9-cis-epoxycarotenoid dioxygenase

PAM

Pulsed amplitude modulation

PCA

Principal component analysis

PSY

Phytoene synthase

REL

Relative electrolyte leakage

ROS

Reactive oxygen species

VDE

Violaxanthin de-epoxidase

ZEP

Zeaxanthin epoxidase.

Author Contributions

Peng Zhang: Formal analysis, Software, Visualization, Writing—original draft. Xiang Wu: Investigation, Visualization. Yulin Chen: Data curation, Investigation. Guangmei Ji: Data curation, Formal analysis. Xinling Ma: Software, Validation. Yuping Zhang: Project administration, Resources. Jing Xiang: Methodology, Software. Yaliang Wang: Investigation, Resources. Zhigang Wang: Resources, Software. Liangtao Li: Conceptualization, Supervision. Huizhe Chen: Conceptualization, Funding acquisition. Yikai Zhang: Conceptualization, Funding acquisition, Project administration, Writing—review and editing.

Funding

This work was supported by the National Key Research and Development Plan of China (2022YFD1500404), “Leading Geese, Pioneer” Research and Development Project of Zhejiang Province (2022C02008; 2023C02004; 2022C02034), and Special Funds for the Construction of Modern Agricultural Technology System (CARS-01-22).

Availability of Data and Materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

Ethics Approval and Consent to Participate Not applicable.

Consent for Publication All authors are consent for publication.

Competing Interests The authors declare that they have no competing interests.

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Comparative Transcriptome Combined with Morphophysiological Analyses Revealed Carotenoid Biosynthesis for Differential Chilling Tolerance in Two Contrasting Rice (Oryza sativa L.) Genotypes

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Abstract

Figures

Background

Results

Kos exhibited higher tolerance to low-temperature stress

Kos presented a better-maintained photosystem morphological structure under chilling stress

Carotenoid biosynthesis is critical for conferring cold tolerance

Discussion

Conclusions

Materials and Methods

Plant materials and experimental design

Determination of the carotenoid and chlorophyll contents and relative electrolyte leakage

Chlorophyll fluorescence assay and examination of the chloroplast ultrastructure

RNA extraction and transcriptome sequencing

Analysis of differential gene expression

Quantitative real-time PCR

Assays of the activities of carotenoid biosynthesis pathway enzymes

Statistical analyses

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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