Iron (Fe) is an essential plant micronutrient. The main valence forms are Fe (II) and Fe (III) (Ilbert and Bonnefoy, 2013). Although the total iron concentration in soils is high, it exists mostly in the oxidized Fe (III) form in aerated soils; this form is not directly plant-available (Guerinot and Yi, 1994; Briat et al., 2015). Plants have evolved two different molecular strategies to obtain sufficient Fe from the rhizosphere to adapt to the low-Fe environments. Dicots and non-graminaceous monocots have evolved different Fe uptake mechanisms (strategy I) (Romheld and Marschner, 1986; Hell and Stephan, 2003; Kobayashi and Nishizawa, 2012) compared with graminaceous plants (strategy II) (Romheld and Marschner, 1986; Connorton et al., 2017). Strategy I plants (such as tobacco) secrete phenolic compounds via phenolics efflux transporters and pump H⁺ out into the rhizosphere via the plasma membrane H⁺-ATPase, thus increasing iron solubility by chelation and contributing to Fe³⁺ reduction via acidification (Kim and Guerinot, 2007). Upon transfer of the dissolved Fe³⁺ from the rhizosphere to the root surface, ferric chelate reductase (FRO) reduces it to Fe²⁺ (Robinson et al., 1999), and then Fe²⁺ is absorbed into root cells by high-affinity iron-regulated transporters (IRTs) (Eide et al., 1996; Vert et al., 2002; Brumbarova et al., 2015).

Cellular iron combines with various organic substances as a structural/functional component, such as in cytochromes, catalase, peroxidases, heme, ferredoxin, and iron-sulfur clusters (Balk and Schaedler, 2014; Vigani and Murgia, 2018); these compounds are essential in chlorophyll synthesis, oxygen transport, redox reactions, nitrogen assimilation, photosynthesis, and electron transport in plants (Balk and Schaedler, 2014). For example, heme, cytochrome, iron-sulfur clusters, and ferredoxin are key components of the photosynthesis system and contribute to the photosynthetic and respiratory electron transport chains (Sousa et al., 2018; Kroh and Pilon, 2020). Therefore, iron deficiency not only leads to the inhibition of chlorophyll synthesis and diminished photosynthetic efficiency (Nelson and Ben-Shem, 2004; Msilini et al., 2011), but also results in the interruption of the respiratory electron transport in plant mitochondria and tricarboxylic acid cycle metabolism, hampering plant growth (Fernie et al., 2004).

Flue-cured leaves from common commercial tobacco [e.g., Yunyan87 (Y87)] usually display yellow appearance. However, a type of spontaneous variant that originated from flue-cured varieties has a reddish hue in flue-cured leaves and has historically been referred to as Cherry Red tobacco (Wada, 1956; Hall et al., 1965). It is reported that Cherry red leaves contain high levels of nornicotine as the principal alkaloid, whereas nicotine is the principal alkaloid in common tobacco with flue-cured yellow leaves (Wada, 1956; Hall et al., 1965). Compared with the common tobacco, high concentrations of nornicotine in the Cherry Red tobacco leaves are associated with a strong expression of nicotine N-demethylase gene CYP82E4, which plays a key role in nicotine to nornicotine conversion (Siminszky et al., 2005). Nornicotine was suggested to be a substrate reacting with o-quinones (produced by oxidation of chlorogenic acid and caffeic acid by polyphenol oxidase) to generate red color (570 nm wavelength) in Cherry Red tobacco (Weeks et al., 1993).

During several years of field observations, we identified a spontaneous Cherry Red variant (CR60) that originated from flue-cured cultivar Yunyan87 (Y87). In CR60, the CYP82E4 expression was activated by an unknown mechanism, resulting in significantly increased accumulation of nornicotine in leaves (Song et al., 2021), similarly to the previous report (Siminszky et al., 2005). However, apart from CYP82E4 expression and correspondingly high levels of nornicotine, the growth characteristics and physiological/molecular properties of CR60 are largely unknown.

In this study, we found that CR60 accumulates higher concentrations of iron in comparison with Y87 under field conditions, implying the difference in iron uptake and transport between the two genotypes. Therefore, we further analyzed the physiological and molecular changes in CR60 and Y87 exposed to different concentrations of iron under hydroponic conditions. The results showed that CR60 exhibited higher iron deficiency tolerance than Y87, which might be attributed to higher expression of several genes associated with iron uptake, chlorophyll biosynthesis and electron transport chain in the photosynthetic system of CR60.

As a micronutrient, iron plays an irreplaceable role in regulating plant growth and metabolism (Kobayashi and Nishizawa, 2012). Iron is involved in chlorophyll synthesis and serves as a cofactor in numerous enzymes (Balk and Schaedler, 2014; Kroh and Pilon, 2020). It has long been known that the photosynthetic capacity of higher plants is affected by iron deficiency. The primary response associated with the insufficient availability of iron is the loss of chlorophyll and interveinal chlorosis of young leaves (Miller et al., 2008). In this work, we found that the first fully expanded apical leaves of Y87 exhibited an obvious interveinal chlorosis after 21 days of iron deficiency treatments; by contrast, CR60 remained green and contained high concentrations of chlorophyll, indicating that CR60 is more tolerant to iron deficiency stress than Y87 (Figure 2). A comparative transcriptome analysis between CR60 and Y87 further showed that the differences in chlorophyll synthesis and photosynthesis might be responsible for their phenotypic differences (Figures 3C–F, 4B,C).

Iron uptake and translocation are tightly regulated by its transport- and homeostasis-related genes in plants. In strategy I plants (e.g., tobacco), ferric-chelate reductase (FCR) catalyzes the reduction of Fe³⁺ to Fe²⁺, which is then assimilated into root cells via the high-affinity metal transporter IRT1 (iron-regulated transporter 1) (Eide et al., 1996; Robinson et al., 1999; Vert et al., 2002; Santi and Schmidt, 2009). NRAMP3 is a vacuolar metal transporter involved in homeostasis and transport of divalent metals (e.g., Fe²⁺, Mn²⁺ and Cd²⁺) (Thomine et al., 2003). Additionally, ferritin localized in plastids of plant leaves is an iron storage protein, and the expression of ferritin 1 (Fer1) is induced by iron application in arabidopsis (Ravet et al., 2009). In this study, we found that the expression of FRO2 and FRO8, two genes encoding ferric-chelate reductase, were induced in roots of both genotypes by Fe deficiency (Figure 5B). Additionally, IRT1 and NRAMP3 were also upregulated by Fe starvation in both leaves and roots of the two genotypes, whereas the expression of IRT1 was higher in roots of CR60 than Y87 (Figure 5B). Iron deficit reduced the expression of Fer1 in leaves of both CR60 and Y87, but no significant difference was found between the two genotypes (Figure 5A). These results indicate CR60 and Y87 share similar responses regarding Fe³⁺ reduction and iron storage under iron deficiency. The higher leaf accumulation of Fe in CR60 than Y87 (Figure 1B) might be attributed to higher expression of IRT1 in roots of CR60 than Y87 during Fe starvation (Figure 5B), which contributed to greater tolerance to iron deficiency stress in CR60 compared with Y87. In tomato plants, iron affects the expression of SlIRT1 via modulation of the DNA methylation of its promoter (Chen et al., 2022). Therefore, investigating whether epigenetic modulation is also involved in iron-mediated changes in the expression of these genes in tobacco is warranted.

Iron is required not only for chlorophyll biosynthesis and the maintenance of chloroplast structure and function, but also serves as an important component of cytochromes in the electron transport chain. Approximately 80% of iron is located in photosynthesizing cells and is directly required for the structural and functional integrity of thylakoid membrane and the biosynthesis of chlorophyll (Li et al., 2021). Visible chlorosis (a decrease in chlorophyll concentration) was found in Y87 leaves stressed by Fe deficiency for 21 days (Figures 2A,D), indicating that Fe starvation affected the biosynthesis of chlorophyll. In contrast, CR60 developed larger leaf area, remained greener and contained higher chlorophyll concentrations than Y87 after 21 days of Fe deficiency (Figures 2B–D). Congruent with these observations, our transcriptome and RT-PCR results showed that a large number of genes associated with photosynthesis, PS-I/II and chlorophyll metabolism were regulated by Fe deficiency (Figures 3, 4, 6 and Supplementary Tables 2–5). More specifically, the expression of NYC1, PRO, and GSA, key genes involved in the biosynthesis of chlorophyll and heme, was significantly higher in CR60 than Y87 under iron deficiency stress (Figure 6A), which was in accordance with higher chlorophyll concentrations measured in CR60 than Y87 (Figure 2D). Although iron starvation decreased the expression of PsbO, PC, and FD1 (all associated with the electron transfer chain in the photosynthetic system), the relative expression of these genes was significantly higher in CR60 than Y87 (Figure 6B). Additionally, the ATPase subunit II expression was induced by iron deficiency and was higher in CR60 than Y87. These results suggested that CR60 maintained greater chlorophyll biosynthesis and had higher photosynthetic rate under Fe starvation than Y87.

The CR60 showed higher tolerance to iron deficiency than Y87, as shown by significant differences in leaf growth and chlorophyll concentrations after 21 days of iron deficiency. The expression of IRT1 and several genes associated with chlorophyll biosynthesis (e.g., NYC1, PRO, and GSA) and the electron transport chain in photosynthesis (e.g., PsbO, PC, FD1, ATPase subunit II, and ATP1A1) was higher in CR60 than Y87, which could at least partially explain greater tolerance to Fe starvation in CR60 than Y87. Furthermore, our preliminary data showed that the concentrations of iron in CR60 leaves was correlated positively with the development of granular red dapples, indicating iron might be also related to the formation of Cherry Red leaves. Therefore, whether and how iron regulates the formation of Cherry Red tobacco are questions worth investigating in the future studies.

The data presented in the study are deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA), accession number PRJNA807089.

ZS conceived this study, analyzed the data, and wrote and revised the manuscript. FL and YZ performed the experiments, analyzed the data, and wrote the manuscript. XP, NC, and XS analyzed the data and revised the manuscript. ZR and QC analyzed the data, interpreted the results, and revised the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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