Temperature-mediated shifts in chlorophyll biosynthesis in leaves of chlorophyll b-lacking rice (Oryza sativa L.)

Extreme temperatures have become a threat to crop yields. To maintain plant growth and yield, chlorophyll (Chl) biosynthesis plays a crucial role in adaptation to temperature stress. This study investigated the influence of temperature on the biosynthesis and characteristics of pigments (Chl a, Chl b, and carotenoids) in the leaves of Chl b-lacking mutant rice (Chlorina 1, ch1) and wild-type rice (Norin No.8, wt). The ch1 showed thinner stacked grana caused by a decrease in thylakoid membranes per granum at 15 °C, whereas the destacked grana were observed at 35 °C after 12 h incubation. However, the grana are stacked normally, along with the absence of Chl b, and a significantly decreased amount of Chl a in both wt and ch1 were observed after heat stress exposure, demonstrating that light-harvesting complex II proteins are involved in grana stacking. Ch1 was sensitive to 15 °C during the first 4 h of incubation but it subsequently adapted to the cold environment. In addition, there were no significant differences in the photosynthesis between wt and ch1 after 12 h incubation at 35 °C. Differentially expressed gene (DEGs) analysis revealed that GluRS expression decreased, which resulted in a decline in Chl biosynthesis in wt and ch1 at 35 °C. At 8 h and 12 h, there were no significant differences in the expression of DEGs involved in Chl biosynthesis and degradation between wt and ch1 at 15 °C. ALAD expression in wt and ch1 at 15 °C decreased until it was undetectable. These findings suggested that ch1 may adapt to temperatures ranging from 15 °C to 35 °C.


Introduction
Extreme temperatures caused by climate change, including cold stress and heat stress, affect the development, growth, and seasonal behaviors of plants, and can consequently impact crop yield (Raza et al., 2019;Ding et al., 2020). Under unsuitable growth temperature, thermomorphogenesis occurs and produces rapid changes in morphology. Vernalization and cold stratification processes can be observed as increases in flowering or seed germination under cold, moist temperatures, and may trigger cold acclimatization. In contrast, global warming frequently accelerates heat stress, which affects a part of the plant life cycle, particularly photosynthesis and carbon assimilation. High-temperatures have been found to decrease net photosynthetic rate, which is attributed to either stomatal or nonstomatal limitation (Hassan, 1999;Shangguan et al., 1999;Yordanov et al., 1999;Kadam et al., 2014). Rubisco activase, a heat-stable enzyme in higher plants involved in carbon assimilation and metabolism in the chloroplast stroma, was found to be inactivated at higher temperatures (Salvucci and Crafts-Brandner, 2004;Sharkey, 2005). Moreover, the key physiological indicator of heat stress was found to be loss and detachment of thylakoid membrane integrity (Gounaris et al., 1984;Semenova, 2004;Yamamoto et al., 2008;Ding et al., 2020). In addition, photosystem II (PS II) is the most heat-sensitive component within the chloroplast thylakoid membrane (Hu et al., 2020).
The function of the electron transport, cleavage of the D1 reaction center-binding protein in PSII, and oxygenevolving complex were found to be altered by heat stress (Havaux and Tardy, 1997;Klimov et al., 1998;Yamane et al., 1998;Wahid et al., 2007;Allakhverdiev et al., 2008;Lin et al., 2013). Thus, the chloroplast was suggested as a key sensor to detect elevated temperatures because it is highly sensitive to damage from heat stress (Yu et al., 2012). Chlorophylls (Chl) and carotenoid (Car) are the predominant plastid pigments with approximately 10 9 tons of Chl being biosynthesized and degraded annually (Rüdiger, 1997;Eckhardt et al., 2004;Liu et al., 2007). Chl biosynthesis pathways have been well-documented (Nguyen et al., 2020) and are summarized in Figure 1.
Chl is extremely important during photosynthesis because they play a central role in light absorption and energy transfer (Fromme et al., 2003). Chl a is the most abundant pigment and presents in all higher plants. The second-most abundant pigment is Chl b, which accounts for approximately one-quarter of the total chlorophyll in higher plants, and differs from Chl a only in the presence of a formyl group at the C-7 position instead of a methyl group. Chl a plays an important role in both light absorption and primary photochemical reactions, whereas Chl b is involved only in light absorption (Michel et al., 1983;Voitsekhovskaja and Tyutereva, 2015;Landi et al., 2020). Furthermore, Chl b also plays a major role in sustaining the accumulation of polypeptides associated with pigments and can serve as an intermediate to improve the efficiency of energy transfer between Chl a and carotenoid (Car) (Bellemare et al., 1982;Terao et al., 1985;Allen et al., 1988). In addition, the binding of Chl b to the antenna proteins is needed for stable anchoring of the antenna complexes in thylakoid membranes (Voitsekhovskaja and Tyutereva, 2015).
Rice (Oryza sativa L.) is one of the most important food crops in the world. However, its yield is affected by the extreme temperatures caused by climate change and global warming (Horie, 2019). Under extreme temperatures, the photosynthetic machinery of rice undergoes modifications to adapt to the stress. Therefore, studies have focused on investigating the influence of temperature stress on the photosynthetic machinery of rice (Soda et al., 2018). Understanding the cold and heat tolerance in rice is one of the solutions for improving the rice yield (Cruz et al., 2013;Fahad et al., 2018). The breeding of mutated plants is achieved by effective treatments such as transfer-DNA insertion mutagenesis, physical mutagenesis (ionizing radiation), and chemical mutagenesis (ethyl methane sulphonate and EMS) (Liu et al., 2016). These mutations play an important role in evaluating plant life cycles, crop yield, plant characteristics, gene identification, and gene mapping genetics (Huang et al., 2014). Studies have reported that Chl-deficient mutants are light- (Hopkins et al., 1980;Markwell et al., 1986;Allen et al., 1988;Greene et al., 1988) and temperature-sensitive (Yang et al., 1990;Markwell and Osterman, 1992). Moreover, temperature sensitivity is a general characteristic of Chldeficient Melilotus alba (Yang et al., 1990) and Arabidopsis thaliana (Markwell and Osterman, 1992). Previous 3 studies have introduced and elucidated the transcriptomic profiles and photosynthetic properties of Chl blacking mutant rice (Chlorina 1, ch1) and wild-type rice (Norin No.8,wt) (Nguyen et al., 2020). However, the sensitivities under temperature stress on the Chl biosynthesis mechanism of Chl b-lacking rice remains unclear.
To further investigate these characteristics, the present study aimed to examine the influence of growth temperature on physiological characteristics and differentially expressed genes (DEGs) encoding 12 enzymes that play important role in Chl biosynthesis at 15 °C and 35 °C in both wt and ch1 rice. The interactions between wt and ch1 and temperature were also discussed in this work.

Materials and Methods
Plant materials and growth condition Seeds of wt and ch1 were graciously provided by Dr. Tomio Terao (Department of Applied Physiology, National Institute of Agrobiological Resources, Tsukuba Science City, Japan). The seeds were sown and observed in a growth chamber (Firstek, Taiwan), and the seedlings (3 plants) were grown for 42 d at 25 °C at a relative humidity (RH) of ≥ 80% and a 12/12 photoperiod with the light intensity of 2000 µmol s -1 m -2 . Then, sets of wt and ch1 plantlets were incubated at different temperatures (15 °C or 35 °C) for 12 h under the same RH, light intensity, and photoperiod. Leaves (3 leaves per plant) were then collected every 4 h, frozen in liquid nitrogen, and kept at -80 °C for next uses.
Transmission electron microscopy The ultrastructure of wt and ch1 leaf samples was observed using transmission electron microscopy.
Each set of leaves were trimmed into small fragments (0.5 × 0.5 × 0.5 mm) and sample fragments were fixed in 2.5% glutaraldehyde at 4 °C for 24 h followed by in 1% OsO4 for 2 h. All fragment sets were then fixed up to 70 nm using a Leica EM UC6 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany), stained using 1% (w/v) lead citrate and 1% (w/v) uranyl acetate (Spurr, 1969), and photographed using a Phillip Tecnai 12 transmission electron microscope (JEOL Ltd., Japan).
Quantitative RT-PCR (qPCR) Total RNA was extracted from wt and ch1 leaves using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. Total RNA (µg) extracted from wt and ch1 was subjected to cDNA synthesis using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostic Systems, Branchburg, NJ, USA) and oligo (dT) primer. Primer sets were designed using Primer Premiere software version 6.0 (Pre-miereBiosoft, Palo Alto, USA) (Table 1). qPCR was performed using the StepOne Plus Real-Time PCR system (Applied Biosystems, Life Technologies Inc, Italy) with iTaq Universal SYBR Green Supermix reagent (Bio-Rad, CA, USA) to analyze 12 genes involved in Chl biosynthesis and degradation (Nguyen et al., 2020). Relative gene expression values were calculated as 2 -∆Ct , where ∆Ct was calculated as the difference between the cycle threshold (Ct) of a target gene and the reference gene. The fold change in each gene in leaf tissues was calculated as 2 -∆Ct mutant/2 -∆Ct wild-type.

Statistical analysis
The pigment contents and relative gene expressions (qPCR results) of wt and ch1 were statistically analyzed using least significant difference (LSD) t-tests at p≤ 0.05. All tests were performed by SAS version 8.0 (Research Triangle Park, NC, USA).

Results
Effects of temperature on pigment contents The 35~40 cm and 25~30 cm height wt and ch1 plantlets were obtained after 6 weeks incubation at 25 °C, respectively. Each set of wt and ch1 plantlets was transferred to a growth chamber with a controlled temperature of 15 °C (wt-15, ch1-15) or 35 °C (wt-35, ch1-35) for 12 h. The leaves were taken for analysis every 4 h. The results showed that leaf coloration differed between wt and ch1 at both 15 °C and 35 °C. From 0 -12 h, the wt leaves remained dark green to light green, whereas the ch1 leaves ranged from light green to yellow-green ( Figure 2) at both 15 °C and 35 °C. The tip of ch1 became dry compared to wt leaves after 12 h at 35 °C, whereas both ch1 and wt remained healthy at 15 °C. Overall, the wt leaves accumulated roughly twice the amount of total Chl as ch1 leaves at the start of incubation (0 h). Over 12 h at 15 °C, the total Chl content of wt-15 and ch1-15 decreased remarkably from 4.5 mg g -1 and 2.63 mg g -1 to 0.9 mg g -1 and 0.32 mg g -1 , respectively. Total Chl also significantly decreased in wt-35 and ch1-35 at 35 o C, from 4.31 mg g -1 to 0.34 mg g -1 and from 2.66 mg g -1 to 0.17 mg g -1 , respectively ( Figure 3A). Meanwhile, Chl a content also decreased in all samples ( Figure 3B). Chl b was not detected in ch1 in any treatment over 12 h, whereas Chl b significantly decreased in wt before it became absent at 8 h (for 35 o C treatment) or 12 h (for 15 o C treatment) ( Figure 3C). Hence, Chl b was absent from all ch1 leaves, leading to Chl a/b of ∞ over 12 h in both treatments, whereas the Chl a/b in wt increased to ∞ at 12 h (for 15 °C treatment) and 8 h (for 35 °C treatment) (Table 2). However, the Car content accumulated in wt leaves was roughly twice to four times greater than that in ch1 at 0 h. However, the Car content of ch1-15 slightly increased from 0.42 to 0.45 mg g -1 , while Car content in ch1-35 significantly dropped from 0.42 to 0.28 mg g -1 . Besides, the Car content of the wt-15 and wt-35 increased rapidly at 15 °C (0.71 mg g -1 ) and 35 °C (0.67 mg g -1 ), respectively ( Figure 3D).  To examine the influences of temperature on chloroplast development, the chloroplast ultrastructures of wt and ch1 grown at different temperatures were analyzed. At the start of both temperature treatments, chloroplasts from the mesophyll cells of wt leaves exhibited a normal structure, including distinct thylakoid membranes and stromal lamellae with small starch granules and one or two plastoglobuli ( Figure 4A).
However, the chloroplasts of ch1 leaves presented indistinct or absent stromal lamellae, indistinct thylakoid membranes, and abundant vesicles and plastoglobuli ( Figure 4A). The stacked thylakoid grana of the ch1 leaves were also thinner than those of the wt leaves ( Figure 4A). After 12 h incubation, destacked thylakoid grana were observed in ch1-35 ( Figure 4C), while thinner and stacked thylakoid grana remained in ch1-15 ( Figure   4B). The expression of 11 of 12 genes related to Chl biosynthesis and degradation were analyzed by qPCR ( Figure 5). Among the genes, UROD, CPOX, and NOL were significantly up-regulated in ch1 at 0 h. Overall, the relative gene expression levels in the rice (both wt and ch1) grown at 35 °C were higher than that of rice grown at 15 °C. ALAD, CPOX, DVR, and NOL were not detected after 4 h incubation at 15 °C in ch1-15. In contrast, the expression levels of ALAD, CPOX, DVR, and NOL did not differ significantly over 12 h at 35 °C in ch1-35. 8 Figure 5. Expression of levels of 12 differentially expressed genes in wild-type and chlorophyll b-lacking rice Gene expression was measured using quantitative real-time PCR. Asterisks (*) indicate significant differences in expression levels between the wt and ch1 (p ≤ 0.05)

Discussion
Temperature stress results in changes in plant growth and physiology, and consequently, limits agricultural productivity (Kadam et al., 2014;Xin et al., 2015;Petrov et al., 2016). Both heat and cold stresses cause multi-step damages to the photosynthetic apparatus of plants. Cold stress has been shown to inhibit photosynthesis in the albino rice mutant (Allen and Ort, 2001). Nevertheless, the molecular mechanisms involving in the influences of temperature stress on plant development are not completely understood in Chl 9 b-lacking mutant. In this study, wt and ch1 were grown at low and high temperatures to examine the responses of Chl b-lacking mutants to temperature stress. Compare to wt, ch1 plantlets were remarkably shorter and less healthy, with light green to yellow-green leaf coloration under both temperature treatments (15 °C and 35 °C).
Moreover, after 12 h incubation, the tips of ch1-35 leaves were dry compared to ch1-15 and wt-35, suggesting that high temperatures damaged plant development. This result is similar to that observed for heat-stress sensitive albino rice (Qiu et al., 2018).
When plants are exposed to extreme temperature, a high amount of reactive oxygen species (ROS) is produced, damaging the cellular structures and consequently causing plant senescence and death (Cheng et al., 2016). The peroxidation of membrane lipids caused by O2 and H2O2 resulted in destruction of chloroplast and degradation of plastid pigments (Zhao et al., 2011). In addition, chloroplast microstructure, photosynthetic metabolism, and energy production are damaged by cold and chilling conditions (Bilska and Sowiński, 2010), which inhibit photosynthesis and cause a serious threat to crop development. Under chilling stress, the efficiency of photosynthetic electron transfer in plants is drastically reduced, resulting in a burst of ROS that directly induces cellular oxidative damages and increases membrane rigidity (Nakashima et al., 2007;Chaves et al., 2009;Lawlor and Tezara, 2009;Xie et al., 2009). To respond to those damages, Car plays a vital role in the pathways that protect the photosynthetic apparatus from various extreme environmental factors. Under extreme heat conditions, Car mitigates the effects of intense heat by scavenging ROS produced during photooxidative stress (Strzałka et al., 2003). In addition, chloroplast plays a crucial role in photosynthetic machinery, where numerous metabolic reactions occur. However, it is effortlessly affected by environmental stressors, such as heat (Zhu, 2016). Heat stress causes partial changes in membrane fluidity, which is attributed to electron transport activity, and leads to photosynthesis inhibition (Raison et al., 1982;Havaux and Tardy, 1996;Murakami et al., 2000). In this study, total Chl decreased dramatically over 12 h in both temperature treatments, suggesting that temperature stress affects both wt and ch1. In higher plants, photosynthesis temperature is usually based on natural atmosphere encountering. Consequently, the optimum temperature range typically matches the average daytime temperature (Berry and Bjorkman, 1980;Poorter, 2004). Additionally, the photosystems, the photosynthetic machinery, principally PSII with its oxygen-evolving complex, the ATP producing process, and the carbon metabolism process, are the three major stress-sensitive site (Aro et al., 1993;Bukhov and Carpentier, 2000;Nishiyama et al., 2005;Mohanty et al., 2007;Allakhverdiev et al., 2008). The analyses of the chloroplast ultrastructure revealed differences in thylakoid membrane structure between wt and ch1 grown at both 15 °C and 35 °C for 12 h. The wt chloroplasts possessed typical thylakoid membranes that were efficient for harvesting and converting light energy, whereas the ch1 chloroplasts possessed abnormal thylakoid membranes. Similarly, chloroplasts of green bamboo leaves contain abundant thylakoid membranes, whereas the thylakoid membranes of Chl b-lacking bamboo leaves are converted into numerous abnormal vesicles (Yang et al., 2015a). In Anthurium andraeanum, the mesophyll cells of wild-type plants possess normal chloroplasts containing small starch granules and, thus, large gaps among stroma thylakoids (Yang et al., 2015b). The results obtained in the present study also demonstrated differences in the chloroplast structures of wt and ch1 rice. The ultrastructure of ch1 (i.e. abundant plastoglobuli, indistinct thylakoid membranes, and reduced starch granules) indicated abnormal development and possibly a reduced accumulation of pigments, which would reduce light energy conversion efficiency and account for the coloration differences observed between wt and ch1. The number of thylakoid membranes in ch1 significantly reduced, leading to thinner stacked grana. Therefore, the enhancement in Chl a/b of ch1 might reflect abnormal chloroplast development and function. In addition, heat stress typically leads to a loss of thylakoid membrane integrity, particularly the unstacking of thylakoid membranes (Gounaris et al., 1983;Semenova, 2004;Allakhverdiev et al., 2008). In this work, the unstacked grana were observed in ch1-35 compared to ch1-15, suggesting that chloroplast development in chlorophyll b-lacking mutants is sensitive to high temperatures. The current study also found that grana stacking, along with an absence of Chl b, is normal in both wt and ch1 at 15 °C after 12 h, whereas unstacked grana were observed in ch1 at 35 °C. This result suggested that high temperatures affect chloroplast development in ch1. Studies have reported that grana stacking is affected both LHC II proteins and other factors (Nakatani and Baliga, 1985;Ouijja et al., 1988;Wood et al., 2019). The present study indicated that high temperatures might be one of the factors, along with LCHII, that affected grana stacking in rice. This study examined the expression level of 12 important genes that code enzymes involved in Chl biosynthesis in rice (Nguyen et al., 2020). Those genes include GluRS, ALAD, PBGD, UROD, CPOX, POR, DVR, CAO, HCAR, CHLG, NOL, and MgCH. Over 12 h, this study found no significant differences in the expression level of DEGs relative to Chl biosynthesis and degradation between wt-35 and ch1-35. This result suggests that ch1 is not sensitive to environmental temperatures ranging from 15 o C to 35 o C. GluRS expression decreased in wt-35 and ch1-35, and consequently, lead to a decrease in Chl biosynthesis in both wt-35 and ch1-35. This is because at the early stage of Chl biosynthesis, GluRS encodes glutamyl-tRNA reductase, which catalyzes the conversion of glutamyl-tRNA into glutamate-semialdehyde (GSA), an important intermediate in the Chl biosynthesis cycle (Verkamp et al., 1992;Zhao et al., 2014). More specifically, ch1 was observed to be sensitive to 15 °C at 4 h incubation but adapted to the cold environment at 8 h and 12 h. There was no significant difference in the expression of DEGs related to Chl biosynthesis and degradation between wt-15 and ch-15 at 8 h and 12 h. Moreover, even though GluRS expression rapidly increased, ALAD expression decreased until it could not be detected in wt-15 and ch1-15, suggesting that its production was blocked in the early stages of the experiment. Thus, total Chl of wt-15 and ch1-15 rapidly decreased over 12 h at 15 °C. This could be explained that ALAD encodes an aminolevulinic acid dehydratase, which catalyzes the reaction to condense two ALA to form porphobilinogen (Sassa, 1982). The intermediate porphobilinogen subsequently undergoes multiple enzymatic reactions to form Chl a and Chl b (Willows and Hansson, 2003;Masuda and Fujita, 2008). This study suggests that low-and high-temperatures may affect DEGs associated with chloroplast development rather than Chl biosynthesis. Although many studies reported that Chl-deficient mutants of plants such as Morus alba and Arabidopsis thalianaare sensitive to temperatures (Yang et al., 1990;Markwell and Osterman, 1992), this study showed that Chl b-lacking and wild-type rice adapted to environmental temperatures in ranging from 15 °C-35 °C.

Conclusions
This study reported the biosynthesis and characteristics of pigments in the leaves of wt and ch1 under temperature stress. Ultrastructure analysis in ch1 revealed the thinner stacked grana at 15 °C, whereas destacked grana were found at 30 °C after 12 h incubation. Under temperature stress, Chl b is undetectable, whereas a significant deficiency in Chl a was found in both wt and ch1. Photosynthesis capacity was insignificantly different in ch1 as compared to wt after 12 h incubation at 35 °C. However, ch1 was sensitive to 15 °C during the first 4 h of incubation. This study suggested that ch1 may adapt to temperatures ranging from 15 °C to 35 °C.