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Article

Chlorophyll Deficiency by an OsCHLI Mutation Reprograms Metabolism and Alters Growth Trade-Offs in Rice Seedlings

by
Byung Jun Jin
1,
Inkyu Park
2,
Sa-Eun Park
3,
Yujin Jeon
4,
Ah Hyeon Eum
4,
Jun-Ho Song
4,* and
Kyu-Chan Shim
3,*
1
Global Institute for Advanced Nanoscience & Technology, Changwon National University, Changwon 51140, Republic of Korea
2
Department of Biology and Chemistry, Changwon National University, Changwon 51140, Republic of Korea
3
Department of Crop Science, Chungnam National University, Daejeon 34134, Republic of Korea
4
Department of Biology, Chungbuk National University, Cheongju 28644, Republic of Korea
*
Authors to whom correspondence should be addressed.
Agriculture 2025, 15(17), 1807; https://doi.org/10.3390/agriculture15171807
Submission received: 29 July 2025 / Revised: 22 August 2025 / Accepted: 22 August 2025 / Published: 24 August 2025
(This article belongs to the Section Crop Genetics, Genomics and Breeding)
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Abstract

Chlorophyll biosynthesis is essential for photosynthesis and plant development. Disruptions in this pathway often manifest as pigment-deficient phenotypes. This study characterizes the morphological, anatomical, and physiological consequences of a chlorophyll-deficient rice mutant (yellow seedling, YS) caused by a loss-of-function mutation in the OsCHLI gene, which encodes the ATPase subunit of magnesium chelatase. Comparative analyses between YSs and wild-type green seedlings (GSs) revealed that YSs exhibited severe growth retardation, altered mesophyll structure, reduced xylem and bulliform cell areas, and higher stomatal and papillae density. These phenotypes were strongly light-dependent, indicating that OsCHLI plays a crucial role in light-mediated chloroplast development and growth. Transcriptome analysis further revealed global down-regulation of photosynthesis-, TCA cycle-, and cell wall-related genes, alongside selective up-regulation of redox-related pathways. These results suggest that chlorophyll deficiency induces systemic metabolic reprogramming, prioritizing stress responses over growth. This study highlights the multifaceted role of OsCHLI in plastid maturation, retrograde signaling, and developmental regulation, providing new insights for improving photosynthetic efficiency and stress resilience in rice.

1. Introduction

Rice (Oryza sativa L.) is a staple crop feeding over half the global population and depends on efficient light capture and conversion for high productivity [1,2]. Chlorophyll is a central pigment for light energy capture and photochemical conversion in plants, and its biosynthesis is tightly coordinated with chloroplast development and cellular differentiation [3,4]. In rice, chlorophyll deficiency leads to severe impairments in seedling growth, architecture, and productivity, underscoring the fundamental role of chlorophyll in sustaining photosynthesis and development [5,6,7]. Understanding the genetic and physiological regulation of this pathway can guide the breeding of stress-tolerant, high-yielding rice varieties under climate challenges [8].
The chlorophyll biosynthetic pathway in plastids involves over 15 conserved enzymatic steps [8]. A crucial regulatory step within this pathway is catalyzed by the magnesium chelatase complex, which mediates the insertion of a divalent magnesium ion (Mg2+) into protoporphyrin IX to produce Mg-protoporphyrin IX—a direct precursor in the chlorophyll biosynthesis branch [9,10]. This multimeric complex consists of three core subunits—CHLI, CHLD, and CHLH—that coordinate ATP hydrolysis and substrate binding [11]. Among these, CHLI provides ATPase activity and regulates complex assembly and substrate delivery [11]. Furthermore, recent studies on mutations of CHLI orthologs have also shown the mode of action when under environmental stress through thioredoxin (Trx) and gene expression regulation. A study on Arabidopsis revealed that the CHLI1 subunit is redox-regulated by thioredoxin. Accordingly, Mg-chelatase activity can be modulated through redox signaling pathways [12]. Related to this, two chloroplast thioredoxin systems, ferredoxin-dependent Trx reductase (FTR) and the NADPH-dependent Trx reductase C (NTRC), are differentiated to separately respond on light intensity [13]. The xan-ATPh.chli-1/xan-h.chli-1 mutant line, the missense barley mutant of CHLI, causes a cold-sensitive pale-green phenotype, highlighting the subunit’s role in chlorophyll synthesis and environmental responsiveness [14]. In rice, single-base mutant (G529C) exhibited the relation of thioredoxin, and the 2-bp deletion mutant of the dinucleotide repeat region (AG4) showed altered photosynthesis and carbohydrate metabolism pathway genes [6,15]. This multifaceted diversification suggests the necessity of broadening the study of the role of CHLI in linking Trx-mediated regulation of photosynthesis, stress responses, ROS degradation, and light-dependent phenotypes in plants.
More recently, accumulating evidence has suggested that micro-phenotypes serve as the key to reveal the hidden relationships between genotypes and complicated macro-phenotypes, serving as critical indicators of plants responding to environmental stimulations and internal physiological regulation [16]. In particular, the leaf cuticular features directly influence transpiration, gas exchange, and pathogen defense, making them highly relevant for breeding resilient rice varieties. It prompts scientists to confirm whether CHLI mutant plants, exhibiting alterations in chlorophyll metabolism and stress responses, also show noticeable changes in micro-phenotypes. The application of high-resolution imaging modalities, such as scanning electron microscopy (SEM) and confocal microscopy, along with AI-powered phenotyping pipelines, has enabled the precise quantification and comparative analysis of these microscopic traits across genotypes [16,17].
In this context, genetic and phenotypic studies on the rice ortholog of magnesium chelatase subunit CHLI, OsCHLI, provide a critical clue to establish the effect of molecular defects of CHLI on phenotypic outcomes. OsCHLI is essential for chlorophyll biosynthesis in rice [11,18] and loss-of-function mutations result in severe phenotypes such as yellow or albino seedlings, stunted growth, and lethality due to impaired photosynthetic capacity [6,15]. While previous studies have reported mutations disrupting OsCHLI structure and function [6,12,18,19], their broader impacts on plant physiology, anatomy development light response, and stress-related signaling remain unclear. In particular, the roles of OsCHLI in systemic growth regulation, plastid–nuclear (retrograde) signaling, and light-dependent development are largely unexplored [19], underscoring the need for integrative studies linking molecular defects to whole-plant outcomes, and those integrating environmental light regimes with integrative multi-scale anatomical and transcriptomic analyses are particularly essential.
In this study, we examined the physiological and anatomical effects of a loss-of-function mutant in the rice chlorophyll synthesis-regulating gene OsCHLI to find new clues of the role of OsCHLI, which have not been established in previous studies [6,15]. We compared developmental traits of the mutant and wild-type seedlings under different photo periods, from macro- to micro-phenotypes, including epidermal cells, stomata, mesophyll structure, chloroplast organization, and photosynthesis-related gene expression. The OsCHLI-deficient yellow seedling (YS) showed severe chlorophyll loss and stunted growth under light, but no significant morphological differences from green seedlings (GS) in darkness. YS had smaller stomata, higher stomatal and papillae densities, thicker minor vein epidermis, and reduced bulliform cell and xylem areas, while GS displayed compact mesophyll and regular chloroplast arrangement. Transcriptome analysis revealed the down-regulation of photosynthesis-, TCA cycle-, and mitochondrial electron transport-related genes, alongside the up-regulation in the OPP pathway, antioxidant defenses, and parts of amino acid metabolism. These results underscore the pivotal role of OsCHLI in linking light-dependent chlorophyll biosynthesis with leaf structure, metabolism, and overall seedling development in rice.

2. Materials and Methods

2.1. Plant Material and Phenotype Evaluation of Seedling Traits

The normal green seedling (GS) and chlorophyll-deficient mutant yellow seedling (YS) were derived from a cross between the introgression line CR5029 and Hwaseong (O. sativa). A 2 bp deletion in the OsCHLI gene was identified from previous study, causing of the chlorophyll-deficient phenotype in YS [6]. To investigate changes in micro-phenotypes and the effect of light on seedling growth, a segregating line (CR1034-2) showing segregation for GS and YS was used in this study.
A total of 30 seeds were sown in a sowing plate (5 × 5 cm) and grown under both light and dark conditions at 30/25 °C (day/night) with a 12/12 h photoperiod in a growth chamber (HB-301L-3, Hanbaek Scientific Co., Bucheon-si, Gyeonggi-do, Republic of Korea). For the dark condition, seedlings were grown in a box completely wrapped in aluminum foil. After two weeks, seedling phenotypes were evaluated because YS plants died two to three weeks after germination due to the absence of photosynthesis.

2.2. Macro-Phenotype Evaluation of Seedling Traits

Two weeks after sowing, shoot length (mm), root length (mm), shoot fresh weight (mg), and root fresh weight (mg) of GS and YS plants grown under light and dark conditions were measured. Roots and shoots were separated, and each trait was evaluated individually.

2.3. Leaf Micro-Phenotype Analysis

To investigate micro-phenotypes of GS and YS leaves, the samples were fixed in FAA solution (40% formalin: 40% glacial acetic acid: 70% ethyl alcohol) for 7 days. The leaf samples were dehydrated using a graded ethanol series (50%, 70%, 90%, 95%, and 100% ethanol) at room temperature for 1 h per ethanol concentration. The dehydrated material was immersed in liquid carbon dioxide (CO2) for critical point drying (CPD, SPI-13200JE-AB, SPI Supplies, West Chester, PA, USA). The dried leaves were mounted on the aluminum stubs using a double-sided adhesive conductive carbon disk (05073-BA, SPI Supplies, West Chester, PA, USA). All samples were gold-coated using an ion-sputtering device (208HR, Cressington Scientific Instruments Ltd., Watford, UK), and all samples were observed using a low-voltage field emission scanning electron microscope (JSM-7600F, JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV with a working distance of 4.5–6.5 mm.

2.4. Leaf Anatomical-Phenotype Analysis

To examine cross sections of GS and YS leaves, the samples were dehydrated using a tertiary butyl alcohol (TBA) series and embedded in paraffin with an automatic tissue processor (Leica EG1150H, Leica Microsystems, Wetzlar, Germany). The embedded paraffin blocks were sectioned into 10 μm slices using a manual rotary microtome (HistoCore MULTICUT, Leica Biosystems, Nussloch, Germany). The sections were then mounted on glass slides and double-stained with Fast Green FCF and Safranin O using an automatic slide stainer. The permanent slides were scanned using a digital slide scanner (3DHistech Pannoramic Desk II DW, 3DHistech Kft., Budapest, Hungary), and the images were observed and captured using the viewer program (CaseViewer software version 2.4.0, 3DHistech Kft., Budapest, Hungary). A total of nine anatomical phenotypes were investigated, including leaf thickness on major veins (LTma, μm), leaf thickness on minor veins (LTmi, μm), epidermis thickness on major veins (ETma, μm), epidermis thickness on minor veins (ETmi, μm), interveinal distance between major veins (IDVma, μm), interveinal distance between minor veins (IDVmi, μm), central bulliform cell area (B, μm2), total area of xylem per major vein (X, μm2), and phloem area per major vein (P, μm2).
All micro-phenotypical and ultrastructural quantitative characteristics of scanned and captured images were digitally measured using Digimizer version 6.3.0 (MedCalc Software, Ostend, Belgium). The terminology of leaf cuticular characteristics followed Ellis [20,21].

2.5. Transcriptome Analysis

In our previous study, RNA-seq analysis was performed to identify differentially expressed genes (DEGs) between GS and YS [6]. A total of 8327 DEGs were identified based on the criteria of log2 fold change (log2FC) ≥ ±2 and a false discovery rate (FDR) < 0.05 (Supplementary Table S1). To further investigate genes associated with seedling growth and micro-phenotypic traits, MapMan (Ver. 3.7.0) analysis was performed to visualize transcriptomic changes across major metabolic pathways [22].

2.6. Statistical Analysis

Student’s t-test was conducted using Microsoft Excel. For the macro-phenotype analysis, more than 15 seedlings of GS and YS were evaluated for each trait, and the experiments were replicated three times. For micro- and anatomical phenotypes, three independent biological replicates were analyzed. Box plots were generated using Python (Ver. 3.13).

3. Results

3.1. Light-Dependent Phenotypic Differences Between Green Seedlings (GSs) and Yellow Seedlings (YSs)

Previously, we reported that the loss of function mutant OsCHLI rice exhibited reduced chlorophyll content and stunted growth [6]. Although the YS mutant was grown under intermediate light conditions (12 h light/12 h dark), it was queried whether the YS mutant phenocopied those of plants grown under a light-limited environment. Accordingly, we hypothesized that the effect of loss of function OsCHLI could be expressed in a synergistic manner under absolute dark conditions. To confirm this, we conducted a comparative analysis of the growth characteristics of green seedling (GS) and yellow seedling (YS) mutants grown under light and dark conditions, respectively. Under light conditions, GSs exhibited vigorous shoot elongation and healthy green leaves, whereas YS displayed typical etiolated phenotypes, including pale-yellow leaves, indicative of chlorophyll deficiency and stunted growth, as previously described [6] (Figure S1A). Quantitative analysis confirmed that GSs had significantly greater shoot length, shoot weight, and root weight (p < 0.001), as well as root length (p < 0.01), compared to those of YSs under light conditions (Figure 1A–D). However, these phenotypic differences were not observed when both seedling groups were grown under dark conditions. The morphological and growth characters of GSs and YSs were comparable both qualitatively and quantitatively under dark conditions showing no significant differences in shoot and root parameters, respectively (Figure S1B and Figure 2E–H). These results demonstrate that the phenotypic differences between GS and YS are light-dependent, indicating that the regulatory role of OsCHLI in seedling development is closely associated with light-mediated chlorophyll biosynthesis and growth.

3.2. Comparative Leaf Micro- and Anatomical Phenotypes Between GS and YS

We used normal chloroplast-containing GS leaves and chloroplast-deficient YS leaves, which were confirmed in previous studies, as samples (Figure S2). O. sativa’s leaves were amphistomatic with dumbbell-shaped stomata and platelet epicuticular wax types, both GSs and YSs (Figure 2, Table 1). Moreover, two seedling phenotypes, GSs and YSs, shared trichomes, including hooks (ho), micro-hairs (mi), cuticular papillae (pa), prickle (pr), and silica body (sb), on both surfaces. On the adaxial surface, the stomata length and width of YSs were significantly smaller than those of GSs (Figure 3A,B; p < 0.001 and p < 0.01). However, YSs exhibited significantly higher stomatal and papillae densities than those of GSs (Figure 3D,E; p < 0.001), while the stomatal area was significantly smaller in YSs (Figure 3C). This alteration of anatomical phenotypes was consistently observed on abaxial surface of both leaves of GSs and YSs. Not only was the stomatal size of YSs, including length and width, significantly smaller than that of GSs (Figure 3F,G; p < 0.001), but also the stomata and papillae density was also significantly higher than that of GSs (Figure 3I,J; p < 0.001). The area of stomata was also significantly larger in GSs, rather than in YSs, as observed on abaxial surface (Figure 3H; p < 0.001).
In addition, we identified nine anatomical phenotypes between GSs and YSs (Figure 4; Table S2). Among these phenotypes, epidermis thickness on minor veins, bulliform cell area, and total area of xylem per major veins were significantly different (all p < 0.001; Table S2). YSs had a significantly thicker epidermis on minor veins; however, the bulliform cell and xylem area were significantly smaller compared to GSs.

3.3. Loss of Function of OsCHLI-Induced Aberrant Mesophyll Development and Chloroplast Arrangement

To investigate the anatomical effects of OsCHLI loss of function, transverse sections of leaf blades from GSs and YSs were examined using light microscopy and slide scanning data. In GS leaves, both the mid-vein region and the lamina exhibited densely packed mesophyll tissues, with numerous stained parenchyma cells arranged compactly throughout the interveinal areas (Figure 4A,B). Chloroplasts were distributed regularly around the bundle sheath and mesophyll layers, indicating well-organized chloroplast positioning and normal tissue differentiation. In contrast, YS mutants displayed severely altered leaf anatomy. Mid-vein and major bundle sheath regions of YS leaves showed increased intercellular space, enlarged and irregularly shaped mesophyll cells, and loosely packed tissue structure (Figure 4C). Similar abnormalities were observed in lamina and minor bundle sheath areas, where chloroplast distribution appeared sparse and disorganized, suggesting impaired plastid development (Figure 4D). Collectively, the reduced cellular density and disorganized chloroplast positioning observed in YS tissues underscore the essential role of OsCHLI in promoting proper mesophyll differentiation and maintaining the anatomical integrity of rice leaves.

3.4. Yellow Seedling Mutants Showed Altered Expression of Metabolomic Pathway

In this study, the YSs exhibited a wide range of phenotypic alterations, including growth retardation, changes in leaf anatomy, and distinct micro-morphological traits such as altered stomatal and papillae patterns. To gain a more comprehensive understanding of the molecular mechanisms underlying these macro- and micro-phenotypic differences, we conducted transcriptome analysis. By examining global gene expression changes, we aimed to connect structural and physiological phenotypes with the metabolic pathways affected by chlorophyll deficiency. To characterize the metabolic consequences of the yellow seedling phenotype, we conducted transcriptome profiling followed by pathway visualization using a global metabolic overview map (Figure 5). Out of 8327 annotated genes, 8276 were successfully mapped to rice metabolic pathways, with 1109 differentially expressed genes (DEGs) visualized across various functional modules (Figure 5). A pronounced down-regulation was observed in genes associated with photosynthesis, particularly those involved in light reactions, photosynthetic electron transport, and photorespiration, consistent with the YS phenotype and absence of chlorophyll. The suppression of chloroplast-associated transcripts highlights the severe impairment in photochemical energy conversion in the YSs. Concomitant with the loss of photosynthetic activity, transcripts in the TCA cycle and mitochondrial electron transport chain were also broadly down-regulated. This suggests a reduction in mitochondrial respiration, probably reflecting diminished energy demand or carbon substrate availability in the absence of active photosynthesis. Interestingly, the oxidative pentose phosphate (OPP) pathway and ascorbate–glutathione cycle displayed selective up-regulation, indicating an increased need for NADPH and antioxidant defenses, potentially as a response to photo-oxidative stress or redox imbalance in the YS. Genes involved in amino acid metabolism, including those related to proline, arginine, glutamine, and branched-chain amino acids, exhibited variable expression patterns. Notably, components of proline biosynthesis, often linked to abiotic stress responses and osmotic regulation, were up-regulated, possibly as a compensatory mechanism. In lipid metabolism, several genes responsible for fatty acid synthesis and membrane lipid remodeling were differentially expressed, indicating changes in membrane structure or signaling processes. Moreover, the cell wall biosynthesis module was markedly down-regulated, consistent with reduced cellular expansion and growth retardation in YSs (Figure 4). Genes within secondary metabolism, including the phenylpropanoid and flavonoid pathways, were up-regulated, likely contributing to stress adaptation or signaling under impaired chloroplast development.
We then focused on cell wall precursor metabolism to further understand how carbon fluxes were altered in YSs. MapMan-based visualization of sugar nucleotide biosynthesis revealed significant transcriptional reprogramming in pathways that supply essential precursors for cell wall polysaccharides (Figure S3). Several genes involved in the conversion of D-glucose-6-phosphate and D-glucose-1-phosphate to UDP-sugars, such as UDP-D-glucose, UDP-D-galactose, and UDP-D-glucuronic acid, were significantly down-regulated in YS. These molecules serve as activated sugar donors for cellulose, hemicellulose, and pectin biosynthesis, suggesting a restriction in cell wall construction. This is consistent with the observed growth retardation and impaired cellular expansion in YS seedlings.
Notably, genes involved in the production of GDP-l-fucose and UDP-l-rhamnose, key sugars in cell wall side chains and glycoproteins, also showed reduced expression, further supporting a global limitation in structural carbohydrate biosynthesis. In contrast, specific up-regulation was observed in pathways related to myo-inositol and GDP-D-mannose metabolism, possibly indicating redirection of carbon flux toward alternative sugar derivatives under metabolic stress. Collectively, these transcriptomic changes reflect a comprehensive reallocation of metabolic resources in YS, prioritizing stress mitigation and redox balance over growth and cell wall biosynthesis.

4. Discussion

In this study, we investigated the functional consequences of a loss-of-function mutation in OsCHLI, a key subunit of the magnesium–chelatase complex involved in chlorophyll biosynthesis. Previous studies have shown that the gun5 mutant in Arabidopsis and the Chlorina-1 (OsCHLD) and Chlorina-9 (OsCHLI) mutants in rice exhibit chlorophyll-deficient phenotypes and disrupted plastid development, implicating these genes in not only pigment synthesis but also plastid-to-nucleus signaling and developmental regulation [6,18]. However, the precise anatomical and physiological impact of OsCHLI dysfunction in rice has remained largely unexplored. By characterizing a yellow seedling (YS) mutant carrying a frameshift mutation in OsCHLI [6], we demonstrate that loss of function of OsCHLI severely disrupts not only light-dependent tissue development, but also mesophyll cell patterning, chloroplast positioning, light-dependent tissue development, and regulation of metabolomic pathway genes. Furthermore, this study extends prior genetic research by incorporating light dependency, anatomical traits, and a metabolic systems perspective. Although the gene itself is not newly identified, this study offers novel insights into physiological responses, stress signaling, and light–development interactions. Overall, our results not only provide advanced insights to the current understanding of OsCHLI function in chlorophyll biosynthesis, but also its broader roles in light-mediated tissue patterning and metabolic regulation. This integrative approach advances previous studies by linking OsCHLI dysfunction to specific anatomical disruptions and global physiological consequences in rice.

4.1. Multifaceted Role of OsCHLI in Light-Triggered Plastid Maturation, Plastid-to-Nucleus Signaling, and Growth Regulation

Chlorophyll biosynthesis and chloroplast development are exquisitely light-dependent processes that sustain plant growth and energy conversion [4,20]. Compared to GSs, the YS plants exhibit pale-yellow, biomass-poor, and globally stunted growth under light conditions, whereas, under continuous darkness, both GSs and YSs are similarly etiolated (Figure S1). Previous studies have established that chlorophyll biosynthesis and chloroplast differentiation are strongly light-dependent processes [4,23]. However, unlike general chlorophyll-deficient mutants that exhibit defects irrespective of illumination, our findings reveal that OsCHLI loss-of-function phenotypes are manifested specifically under light, while GS and YS are phenotypically indistinguishable in darkness. This distinction suggests the possible role of OsCHLI as a crucial regulator of the etioplast-to-chloroplast transition [24,25], a process that requires rapid re-activation of tetrapyrrole biosynthetic genes and is tightly coupled with retrograde signaling [26,27,28]. Consistent with the Arabidopsis GUN5 mutant, where disruption of the Mg-chelatase H subunit perturbed nuclear photosynthetic gene expression through impaired plastid-to-nucleus signaling [5], our results in rice indicate that OsCHLI dysfunction similarly compromises retrograde communication, thereby amplifying growth and developmental defects under light. Collectively, our results provide interpretation that OsCHLI contributes not only to Mg-insertion but also to retrograde communication, possibly via plastid–nucleus membrane contact sites [10,20].
The contrasting light-dependent phenotype between GSs and YSs could be explained the dual role of OsCHLI in both chlorophyll biosynthesis and plastid-to-nucleus signaling (Figure 1). Under moderate light (12h), failure to insert Mg into protoporphyrin IX not only halts pigment accumulation but also attenuates the retrograde signals necessary to reprogram nuclear gene expression, thereby amplifying developmental defects [19,29]. Additionally, OsGATA16 has been shown to directly activate CHLI transcription in rice [27], while CHLI1 phosphorylation fine-tunes enzyme activity in Arabidopsis [18,30]. In other studies in Arabidopsis, CHLI homologs further diversify enzyme kinetics [31,32], and translational checkpoints as well as post-translational modifications are shown to safeguard against photo-oxidative damage [33,34,35]. These multilayered controls suggest that OsCHLI operates at the junction of transcriptional, translational, and post-translational regulation. Nevertheless, our study did not directly test how OsCHLI responds across light intensities, photoperiods, or spectral qualities, which represents a limitation. Future studies employing time-series transcriptomic and proteomic analyses, together with profiling of chlorophyll intermediates and retrograde signaling molecules [36], will be essential to pinpoint the exact regulatory tier at which OsCHLI exerts control.

4.2. Structural Adaptations and Cellular Responses Associated with Chlorophyll Deficiency

We found that YSs are characterized by a smaller size and a higher density of stomata in this study. In our observations, YSs exhibited notably impaired growth under light conditions. This growth inhibition is presumed to result from chloroplast deficiency, which leads to reduced energy production and metabolic activity within the chloroplast, ultimately constraining overall cell growth and expansion [37]. Consequently, a reduction in the size of mesophyll and epidermal cells may have contributed to an apparent increase in stomatal density. However, the direct mechanistic link between chloroplast deficiency and increased stomatal number remains unclear and warrants further investigation.
The leaves of O. sativa have rough surfaces containing silica-accumulating nipple-like structures called cuticular papillae [38,39]. The shape of papillae, which protrude from epidermal cells, largely determines the brightness and color intensity like conical cells of petal organs [40,41]. Bright green leaf (blg) mutants, whose locus encodes OsRopGEF10 (Os05g0454200), were found to have an absence of silica-accumulating cuticular papillae, resulting in a luminous green color [40]. In addition, the expression level of OsRopGEF10 was up-regulated in YS, with a logFC of 6.4, possibly as a compensatory response to impaired silica accumulation (Table S1). Thus, the papillae on the leaves of rice might promote the diffuse reflection of light; however, smooth epidermal surfaces reflect visible light more directly with less diffusion, making the leaves bright green [24,39]. In this study, chlorophyll deficiency in YSs resulted in a higher density of papillae on both leaf surfaces compared to the wild type. In the absence of chlorophyll, excessive light is directly exposed to the cells, increasing the risk of cellular damage. Therefore, we presumed that an increased density of papillae serves as an adaptive mechanism to regulate light intensity, protect tissues, block ultraviolet radiation, accumulate silica, and enhance resistance to pests and pathogens. Further research is needed to clarify the relationship between the mutant allele of OsCHLI and the formation and frequency of cuticular papillae.
Previous studies have suggested that a larger xylem size benefits leaf hydraulic conductance, stomatal conductance, and, ultimately, photosynthesis [42]. Consistent with this, our findings indicate that the small xylem area observed in YS is closely associated with reduced photosynthetic efficiency and altered chloroplast structure. Further studies utilizing various mutants will be necessary to elucidate the direct relationship between photosynthetic efficiency, chloroplast structure, and total xylem area. Bulliform cells, also known as hygroscopic or motor cells, are large, thin-walled, and highly vacuolated cells located in the leaf adaxial epidermis [43]. In monocotyledonous plants including rice, they play a crucial role in leaf rolling in response to drought and heat stress [44,45]. Leaf rolling reduces sunlight exposure and heat absorption; thereby, decreasing transpiration and helping conserve water [45]. In crop species, moderate leaf rolling is considered beneficial for improving yield, as it can enhance photosynthetic efficiency [46,47]. In this study, we also suggest that the small-sized bulliform cells observed in YSs may be associated with responses to drought and high temperature, as well as with photosynthetic efficiency. Further investigation is needed to determine whether OsCHLI, a magnesium chelatase involved in chlorophyll biosynthesis, also plays an active role in the development of xylem vessels or bulliform cells beyond its function in chlorophyll production.

4.3. OsCHLI-Dependent Alterations in Mesophyll Patterning and Chloroplast Positioning

Beyond pigment metabolism, we identify a novel role of OsCHLI in shaping mesophyll tissue organization and chloroplast positioning, thereby extending the prior knowledge of Mg-chelatase function. Our comparative microscopy revealed that YS leaves exhibit reduced mesophyll density, irregular parenchyma cells, and excessive intercellular space compared to GSs, along with sparse and disorganized chloroplast distribution around bundle sheaths and mesophyll layers. These results indicate that OsCHLI disruption impairs the coordination between plastid biogenesis and cell differentiation. Previous studies on rice Mg-chelatase mutants, including CHLI and CHLD, similarly reported defective chloroplast development accompanied by anatomical abnormalities [6,18,48]. However, our study goes further by linking these phenotypes explicitly to light-dependent regulation: GS and YS differences largely disappear under darkness, underscoring the conditional nature of OsCHLI function (Figure 1).
The mechanistic basis for these structural alterations may involve impaired plastid-to-nucleus signaling, as suggested by Chan et al. [49], which can misregulate nuclear-encoded genes involved in cell division, expansion, and wall modification. Additionally, light-mediated cues are known to influence mesophyll patterning and plastid positioning [37]. Hence, the YS abnormalities may also reflect compromised photoreceptor signaling. Physiologically, disorganized mesophyll layers restrict internal light scattering and CO2 diffusion, limiting photosynthetic efficiency, a phenomenon well established by Brodersen and Vogelmann [50]. The altered architecture could also impede water and nutrient transport, compounding growth defects. Together, these findings establish OsCHLI as a key regulatory node connecting chlorophyll metabolism, plastid signaling, and leaf anatomical patterning.
Despite these advances, our study has limitations. We did not directly quantify mesophyll conductance, chloroplast motility, or hormone-mediated effects, which could refine the causal link between OsCHLI function and tissue-level outcomes. Further integrative approaches combining transcriptomic profiling, hormone quantification, and plastid ultrastructure imaging under varying light regimes will be crucial for dissecting the signaling networks through which OsCHLI coordinates leaf development.

4.4. Metabolic Reprogramming Revealed by Transcriptome Analysis

Our transcriptome analysis uncovered a broad and coordinated shift in the expression of genes involved in energy metabolism, carbon flux, and structural biosynthesis in YS. The pronounced down-regulation of genes related to photosynthesis, the TCA cycle, and cell wall biosynthesis, alongside the selective up-regulation of redox-balancing pathways such as the oxidative pentose phosphate pathway and the ascorbate–glutathione cycle, collectively indicates a stress-adapted metabolic state. Similar results have been reported in the transcriptome study of leaf color mutants in various plant species. Notably, a transcriptome analysis of the green-revertible albino mutant W01S in rice revealed comparable patterns of transcriptional reprogramming, including broad down-regulation of genes associated with primary metabolism, energy production, and cell wall biosynthesis, along with enrichment of stress-related pathways such as oxidative stress responses and phytohormone metabolism [51]. In particular, the repression of genes related to the TCA cycle and UDP-sugar biosynthesis in W01S closely mirrors the metabolic signatures observed in YS, suggesting that impaired chloroplast function may universally trigger a conserved metabolic shift to mitigate oxidative stress while deprioritizing growth-related biosynthetic pathways. These parallels support the notion that chlorophyll-deficient phenotypes, regardless of their genetic origin, invoke systemic responses affecting not only photosynthetic capacity but also carbon allocation, cell wall formation, and energy balance at the whole-plant level [51]. Furthermore, a yellow-green leaf mutant in Brassica napus exhibited significant down-regulation of genes involved in photosynthesis, carbon metabolism, and energy production, while stress-related genes, including those associated with redox homeostasis, flavonoid biosynthesis, and heat shock protein-mediated repair, were up-regulated [52]. These findings indicate that chloroplast dysfunction consistently induces a systemic shift in gene expression across species, redirecting metabolic priorities from growth toward cellular protection. Such conserved responses highlight the central role of plastid integrity in maintaining cellular homeostasis and developmental coordination in higher plants. Moreover, this global transcriptional reprogramming reflects the central role of OsCHLI not only in chlorophyll biosynthesis but also in coordinating cellular energy balance and resource allocation in response to chloroplast dysfunction.
Genes related to light reactions, chlorophyll-binding proteins, and ATP-generating complexes were extensively repressed in YS, providing molecular evidence that supports the observed anatomical defects in mesophyll structure and reduced chloroplast density (Figure 5). Additionally, the transcriptional repression of sugar nucleotide biosynthesis genes correlates with the impaired development of vascular and epidermal tissues, including reduced xylem area and bulliform cell size (Figure 4), which are essential for water transport and drought responsiveness. These integrative findings demonstrate that loss of OsCHLI function affects not only pigment metabolism but also initiates systemic developmental reprogramming across multiple biological levels.

5. Conclusions

This study shows that loss of function of OsCHLI causes severe light-dependent growth defects in rice, including altered leaf anatomy, disrupted mesophyll structure, and impaired chloroplast positioning. Transcriptome profiling further revealed broad metabolic reprogramming, with suppression of photosynthesis and cell wall pathways and activation of redox-related processes. These results highlight OsCHLI as a key regulator linking chlorophyll biosynthesis, plastid signaling, and systemic metabolic adaptation. Future studies should explore how these mechanisms contribute to stress resilience and photosynthetic efficiency in rice.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15171807/s1: Figure S1: comparison of seedling morphology of green and yellow seedling grown under (A,C) light and (B,D) dark conditions; Figure S2: comparison of ultrastructure of mesophyll cells of green seedling (GS) (A) and yellow seedling (YS) (B); Figure S3: gene expression changes in cell wall precursor metabolism pathways between green and yellow rice seedlings. Table S1: list of differentially expressed genes detected between green seedlings and yellow seedlings; Table S2: comparison of leaf anatomical–phenotypes of green seedling (GS) and yellow seedling (YS).

Author Contributions

K.-C.S., I.P. and J.-H.S. contributed to the study’s conception and design; material preparation, data collection, and analysis were performed by B.J.J., S.-E.P., Y.J. and A.H.E.; the first draft of the manuscript was written by B.J.J. and K.-C.S.; I.P. and J.-H.S. edited the manuscript and provided advice on the experiments. All authors commented on previous versions of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Global-Learning & Academic Research Institution for Master’s Ph.D. students, and Postdocs (LAMP) Program of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (No. RS-2024-00445180 and No. RS-2024-00444460), the Rural Development Administration (RDA) of Republic of Korea (Grant No. RS-2025-02214096), and Korea Basic Science Institute (National research Facilities and Equipment Center) grant funded by the Ministry of Education (Grant No. 2023R1A6C101B022).

Data Availability Statement

The original data presented in this study are openly available in NCBI SRA at PRJNA935477.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agriculture 15 01807 g001
Figure 1. Comparison of seedling characteristics of green seedlings (GSs) and yellow seedlings (YSs) grown under (A–D) light and (E–H) dark conditions. Student’s t-test was conducted, and ** and *** indicate significant difference at p < 0.01 and p < 0.001. The ‘ns’ indicates not significant.
Figure 1. Comparison of seedling characteristics of green seedlings (GSs) and yellow seedlings (YSs) grown under (A–D) light and (E–H) dark conditions. Student’s t-test was conducted, and ** and *** indicate significant difference at p < 0.01 and p < 0.001. The ‘ns’ indicates not significant.
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Figure 2. Scanning electron microscope micrographs of leaf epidermis of green seedlings (GSs) and yellow seedlings (YSs). (A–F) GS; (G–L) YS. (A) Adaxial side of leaf blade. (B) Dumbbell-shaped stomata with papillae on the adaxial side. (C) Papilla with platelet epicuticular waxes on the adaxial side. (D) Abaxial side of leaf blade. (E) Dumbbell-shaped stomata with papillae on the abaxial side. (F) Micro-hair on the abaxial side. (G) Adaxial side of leaf blade. (H) Dumbbell-shaped stomata with papillae on the adaxial side. (I) Papillae with platelets on the adaxial side. (J) Abaxial side of leaf blade. (K) Dumbbell-shaped stomata complex with papillae on the abaxial side. (L) Hook on the abaxial side. ho, hook; mi, micro-hair; pa, cuticular papilla; pr, prickle; sb, silica body; st, stomata.
Figure 2. Scanning electron microscope micrographs of leaf epidermis of green seedlings (GSs) and yellow seedlings (YSs). (A–F) GS; (G–L) YS. (A) Adaxial side of leaf blade. (B) Dumbbell-shaped stomata with papillae on the adaxial side. (C) Papilla with platelet epicuticular waxes on the adaxial side. (D) Abaxial side of leaf blade. (E) Dumbbell-shaped stomata with papillae on the abaxial side. (F) Micro-hair on the abaxial side. (G) Adaxial side of leaf blade. (H) Dumbbell-shaped stomata with papillae on the adaxial side. (I) Papillae with platelets on the adaxial side. (J) Abaxial side of leaf blade. (K) Dumbbell-shaped stomata complex with papillae on the abaxial side. (L) Hook on the abaxial side. ho, hook; mi, micro-hair; pa, cuticular papilla; pr, prickle; sb, silica body; st, stomata.
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Figure 3. Comparison of number and structural differences of stomatal complex and papillae characters in (A–E) abaxial and (F–J) adaxial surfaces of green seedlings (GSs) and yellow seedlings (YSs). Student’s t-test was conducted, and ** and *** indicate significant difference at p < 0.01 and p < 0.001, respectively.
Figure 3. Comparison of number and structural differences of stomatal complex and papillae characters in (A–E) abaxial and (F–J) adaxial surfaces of green seedlings (GSs) and yellow seedlings (YSs). Student’s t-test was conducted, and ** and *** indicate significant difference at p < 0.01 and p < 0.001, respectively.
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Figure 4. Digital slide scanning micrographs of the leaf of green seedlings (GSs) and yellow seedlings (YSs). The red box is a magnified region. (A,B) GS. (C,D) YS. ab, abaxial surface; ad, adaxial surface; bu, bulliform cells; ph, phloem; ic, intercellular space; m, mesophyll; st, stomata; xy, xylem. IDVma, interveinal distance between major veins; IDVmi, interveinal distance between minor veins.
Figure 4. Digital slide scanning micrographs of the leaf of green seedlings (GSs) and yellow seedlings (YSs). The red box is a magnified region. (A,B) GS. (C,D) YS. ab, abaxial surface; ad, adaxial surface; bu, bulliform cells; ph, phloem; ic, intercellular space; m, mesophyll; st, stomata; xy, xylem. IDVma, interveinal distance between major veins; IDVmi, interveinal distance between minor veins.
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Figure 5. Transcriptome-based metabolic pathway overview map of rice seedlings showing differentially expressed genes (DEGs) between yellow seedling (YS) and green seedling (GS) phenotypes. Transcriptome data were visualized using MapMan. Each small square corresponds to one DEG assigned to the respective functional bin (e.g., cell wall, lipids, amino acids, etc.), and multiple boxes within a category represent different genes involved in that pathway. The color of each square indicates the log2 fold change between GS and YS: red denotes up-regulation and blue denotes down-regulation in YS. A total of 8276 out of 8327 DEGs were mapped using the rice genome annotation X4.2_oryza_sativa.m02. The color scale ranges from −4.5 (down-regulation) to +4.5 (up-regulation).
Figure 5. Transcriptome-based metabolic pathway overview map of rice seedlings showing differentially expressed genes (DEGs) between yellow seedling (YS) and green seedling (GS) phenotypes. Transcriptome data were visualized using MapMan. Each small square corresponds to one DEG assigned to the respective functional bin (e.g., cell wall, lipids, amino acids, etc.), and multiple boxes within a category represent different genes involved in that pathway. The color of each square indicates the log2 fold change between GS and YS: red denotes up-regulation and blue denotes down-regulation in YS. A total of 8276 out of 8327 DEGs were mapped using the rice genome annotation X4.2_oryza_sativa.m02. The color scale ranges from −4.5 (down-regulation) to +4.5 (up-regulation).
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Table 1. Comparison of leaf characteristics of green seedlings (GSs) and yellow seedlings (YSs).
Table 1. Comparison of leaf characteristics of green seedlings (GSs) and yellow seedlings (YSs).
GSsYSs
Stomatal positionAmphistomaticAmphistomatic
Stomata typeDumbbell-shapedDumbbell-shaped
Abaxial side
Stomatal length (μm)26.54 ± 2.7022.74 ± 2.46 ***
Stomatal width (μm)14.53 ± 1.2813.63 ± 1.96 **
Stomatal area (μm2)279.93 ± 38.27241.48 ± 45.63 ***
Stomatal density (counts/0.1 mm2)13.30 ± 1.1627.60 ± 2.01 ***
Papillae density (counts/0.005 mm2)76.70 ± 9.45168.40 ± 12.99 ***
Epicuticular wax typePlateletPlatelet
Adaxial side
Stomatal length (μm)27.51 ± 2.7819.30 ± 1.66 ***
Stomatal width (μm)15.83 ± 2.0810.80 ± 1.46 ***
Stomatal area (μm2)354.46 ± 53.40180.51 ± 28.49 ***
Stomatal density (counts/0.1 mm2)14.50 ± 1.2727.50 ± 3.14 ***
Papillae density (counts/0.005 mm2)104.40 ± 7.68129.90 ± 3.90 ***
Epicuticular wax typePlateletPlatelet
Internal part
Chloroplast arrangementcompactloose
Degree of mesophyll cellshighlow
Note: asterisks with difference on number indicate significant differences determined by Student’s t-test (**; p < 0.01, ***; p < 0.001).
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Jin, B.J.; Park, I.; Park, S.-E.; Jeon, Y.; Eum, A.H.; Song, J.-H.; Shim, K.-C. Chlorophyll Deficiency by an OsCHLI Mutation Reprograms Metabolism and Alters Growth Trade-Offs in Rice Seedlings. Agriculture 2025, 15, 1807. https://doi.org/10.3390/agriculture15171807

AMA Style

Jin BJ, Park I, Park S-E, Jeon Y, Eum AH, Song J-H, Shim K-C. Chlorophyll Deficiency by an OsCHLI Mutation Reprograms Metabolism and Alters Growth Trade-Offs in Rice Seedlings. Agriculture. 2025; 15(17):1807. https://doi.org/10.3390/agriculture15171807

Chicago/Turabian Style

Jin, Byung Jun, Inkyu Park, Sa-Eun Park, Yujin Jeon, Ah Hyeon Eum, Jun-Ho Song, and Kyu-Chan Shim. 2025. "Chlorophyll Deficiency by an OsCHLI Mutation Reprograms Metabolism and Alters Growth Trade-Offs in Rice Seedlings" Agriculture 15, no. 17: 1807. https://doi.org/10.3390/agriculture15171807

APA Style

Jin, B. J., Park, I., Park, S.-E., Jeon, Y., Eum, A. H., Song, J.-H., & Shim, K.-C. (2025). Chlorophyll Deficiency by an OsCHLI Mutation Reprograms Metabolism and Alters Growth Trade-Offs in Rice Seedlings. Agriculture, 15(17), 1807. https://doi.org/10.3390/agriculture15171807

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