Thermo-sensitive Genic Male Sterile Line Research Articles

Molecular and cellular mechanisms of photoperiod- and thermo-sensitive genic male sterility in plants.

Photoperiod- and thermo-sensitive genic male sterile (P/TGMS) lines display male sterility under high-temperature/long-day light conditionsand male fertility under low-temperature/short-day light conditions. P/TGMS lines are the fundamental basis for the two-line hybrid breeding, which has notably increased the yield potential and grain quality of rice cultivars. In this review, we focus on the research progress on photoperiod- and thermo-sensitive genic male sterility in plants. The essence of P/TGMS line is their ability to produce viable pollen under varying conditions. We overview the processes involved in anther and pollen development, as well as the molecular, cellular, and genetic mechanisms underlying P/TGMS in Arabidopsis, rice, and other crops. Slow development has been identified as a common mechanism of P/TGMS fertility restoration in both Arabidopsis and rice, while reactive oxygen species homeostasis has been implicated in rice P/TGMS. Furthermore, we discussthe prospective applications of P/TGMS and potential solutions to the challenges in this field. This review deepens the understanding of the mechanisms underlying P/TGMS and its utilization in two-line hybrid breeding across diverse crops.

Development and Trait-Based Molecular Characterization of Thermosensitive Genic Male-Sterile Rice (Oryza sativa L.) Lines at Texas A&M AgriLife Research

This study aimed to develop and genetically characterize thermosensitive genic male-sterility (TGMS) lines for use in hybrid rice (Oryza sativa L.) breeding. Male-sterile F2 to F4 generation lines were screened during the high-temperature summer season, and ratoon crops of selected male-sterile rows were harvested for pure seed. Sixty-six F5 TGMS lines were genotyped using DNA markers controlling 16 traits from the LSU80 QA/QC Rice PlexSeq SNP Panel. Ten TGMS lines with desirable traits that included semidwarf, glabrous, non-aromatic, long-grain, narrow brown leaf spot resistance, and blast resistance genes were selected for further genotypic characterization using markers for low chalkiness (chalk5), wide compatibility (S5-n), cold tolerance (qSCT-11 and qCST-12), and anaerobic germination (AG1 and AG2). TGMS lines TIL21051S and TIL21052S possess favorable alleles for each of the genes evaluated in this study and are desirable parents for two-line hybrid breeding in the southeast United States. TIL21044S, TIL21095S, TIL21060S, and TIL21066S each contain three blast resistance genes and have potential as parental lines. TIL21014S-2, TIL21015S, and TIL21016S-1 include the fgr allele for aroma and can also be used as parental lines for aromatic two-line hybrids.

The E3 ligase OsHel2 impedes readthrough of stalled mRNAs to regulate male fertility in thermo-sensitive genic male sterile rice

Heterosis is extensively utilized in the two-line hybrid breeding system. Photo-/thermo-sensitive genic male sterile (P/TGMS) lines are key components of two-line hybrid rice. TGMS lines containing tms5 have significantly advanced two-line hybrid rice breeding. We cloned the TMS5 gene and found that TMS5 is a tRNA cyclic phosphatase that can remove 2',3'-cyclic phosphate (cP) from cP-ΔCCA-tRNAs for efficient repair to ensure maintenance of mature tRNA levels. tms5 mutation causes increased cP-ΔCCA-tRNAs and reduced mature tRNAs, leading to male sterility at the restrictive temperatures. However, the regulatory network of tms5-mediated TGMS remains to be elucidated. Here, we identified that an E3 ligase OsHel2 cooperates with TMS5 to regulate TGMS at the restrictive temperatures. Consistently, both the accumulation of 2',3'-cP-ΔCCA-tRNAs and insufficiency of mature tRNAs in tms5 mutant were largely recovered in the tms5 oshel2-1 mutant. A lesion in OsHel2 results in partial readthrough of the stalled sequences, thereby evading ribosome-associated protein quality control (RQC) surveillance. Our findings reveal a mechanism by which the OsHel2 impede readthrough of stalled mRNA sequences to regulate male fertility in TGMS rice, thus providing a paradigm for investigating how disorders in the components of the RQC pathway impair cellular functions and lead to diseases or defects in other organisms.

The agronomic characteristics and combining ability of glutinous Thermo-sensitive Genic Male Sterile rice (Oryza sativa) lines

Glutinous rice (Oryza sativa), despite its unique culinary qualities, typically yields less than other rice varieties, posing agricultural and economic challenges. The construction of a glutinous thermo-sensitive genic male sterile (TGMS) rice variety could indeed be a way to improve the yield and cultivation efficiency of glutinous rice. Among them, the two-line system operates by crossing a TGMS line, which is sterile at high temperatures but fertile at lower temperatures, with a normal fertile line. In this study, we conducted to access morphological characteristics and combining ability of some glutinous TGMS lines. The parental lines were characterized on 13 agronomic traits including growth duration, plant height, flag leaf width, flag leaf length, panicle length, rachis length, number of primary branches, number of secondary branches, number of panicles per plant, percentage of filled grains, P1000 seeds, grain yield, and amylose content. The obtained results revealed significant variations in growth traits, yield, and amylose content of studied rice lines. Hierarchical clustering based on principal component analysis of rice lines on all measured traits showed three major clusters with high genetic diversity. Furthermore, fifteen glutinous TGMS rice lines and three testers were used in a Line x Tester mating system to produce 45 hybrids. The 45 hybrids were evaluated on 6 traits related to grain yield and morphology in a randomized complete block design experiment with three replications. A line x tester analysis was conducted to estimate the combining ability, genetic variance, and the contribution of parental lines to genetic variation in hybrids. These findings were valuable for rice breeders to orient the strategy for breeding of hybrid glutinous rice varieties with high efficiency.

Rapid generation of fragrant thermo-sensitive genic male sterile rice with enhanced disease resistance via CRISPR/Cas9.

The three, by mutagenesis produced genes OsPi21, OsXa5, and OsBADH2, generated novel lines exhibiting desired fragrance and improved resistance. Elite sterile lines are the basis for hybrid rice breeding, and rice quality and disease resistance become the focus of new sterile lines breeding. Since there are few sterile lines with fragrance and high resistance to blast and bacterial blight at the same time in hybrid rice production, we here integrated the simultaneous mutagenesis of three genes, OsPi21, OsXa5, and OsBADH2, into Zhi 5012S, an elite thermo-sensitive genic male sterile (TGMS) variety, using the CRISPR/Cas9 system, thus eventually generated novel sterile lines would exhibit desired popcorn-like fragrance and improved resistance to blast and bacterial blight but without a loss in major agricultural traits such as yield. Collectively, this study develops valuable germplasm resources for the development of two-line hybrid rice with disease resistance, which provides a way to rapid generation of novel TGMS lines with elite traits.

Transcriptomic Profiling of Two Rice Thermo-Sensitive Genic Male Sterile Lines with Contrasting Seed Storability after Artificial Accelerated Aging Treatment.

Seed storability has a significant impact on seed vitality and is a crucial genetic factor in maintaining seed value during storage. In this study, RNA sequencing was used to analyze the seed transcriptomes of two rice thermo-sensitive genic male sterile (TGMS) lines, S1146S (storage-tolerant) and SD26S (storage-susceptible), with 0 and 7 days of artificial accelerated aging treatment. In total, 2658 and 1523 differentially expressed genes (DEGs) were identified in S1146S and SD26S, respectively. Among these DEGs, 729 (G1) exhibited similar regulation patterns in both lines, while 1924 DEGs (G2) were specific to S1146S, 789 DEGs (G3) were specific to SD26S, and 5 DEGs (G4) were specific to contrary differential expression levels. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that "translation", "ribosome", "oxidative phosphorylation", "ATP-dependent activity", "intracellular protein transport", and "regulation of DNA-templated transcription" were significantly enriched during seed aging. Several genes, like Os01g0971400, Os01g0937200, Os03g0276500, Os05g0328632, and Os07g0214300, associated with seed storability were identified in G4. Core genes Os03g0100100 (OsPMEI12), Os03g0320900 (V2), Os02g0494000, Os02g0152800, and Os03g0710500 (OsBiP2) were identified in protein-protein interaction (PPI) networks. Seed vitality genes, MKKK62 (Os01g0699600), OsFbx352 (Os10g0127900), FSE6 (Os05g0540000), and RAmy3E (Os08g0473600), related to seed storability were identified. Overall, these results provide novel perspectives for studying the molecular response and related genes of different-storability rice TGMS lines under artificial aging conditions. They also provide new ideas for studying the storability of hybrid rice.

Regulation of carbohydrate metabolism during anther development in a thermo-sensitive genic male-sterile wheat line.

Bainong sterility (BNS) is a thermo-sensitive genic male sterile wheat line, characterised by anther fertility transformation in response to low temperature (LT) stress during meiosis, the failure of vacuole decomposition and the absence of starch accumulation in sterile bicellular pollen. Our study demonstrates that the late microspore (LM) stage marks the transition from the anther growth to anther maturation phase, characterised by the changes in anther structure, carbohydrate metabolism and the main transport pathway of sucrose (Suc). Fructan is a main storage polysaccharide in wheat anther, and its synthesis and remobilisation are crucial for anther development. Moreover, the process of pollen amylogenesis and the fate of the large vacuole in pollen are closely intertwined with fructan synthesis and remobilisation. LT disrupts the normal physiological metabolism of BNS anthers during meiosis, particularly affecting carbohydrate metabolism, thus determining the fate of male gametophytes and pollen abortion. Disruption of fructan synthesis and remobilisation regulation serves as a decisive event that results in anther abortion. Sterile pollen exhibits common traits of pollen starvation and impaired starch accumulation due to the inhibition of apoplastic transport starting from the LM stage, which is regulated by cell wall invertase TaIVR1 and Suc transporter TaSUT1.

An E3 ubiquitin ligase CSIT2 controls critical sterility-inducing temperature of thermo-sensitive genic male sterile rice.

Thermo-sensitive genic male sterile (TGMS) lines are the core of two-line hybrid rice (Oryza sativa). However, elevated or unstable critical sterility-inducing temperatures (CSITs) of TGMS lines are bottlenecks that restrict the development of two-line hybrid rice. However, the genes and molecular mechanisms controlling CSIT remain unknown. Here, we report the CRITICAL STERILITY-INDUCING TEMPERATURE 2 (CSIT2) that encodes a really interesting new gene (RING) type E3 ligase, controlling the CSIT of thermo-sensitive male sterility 5 (tms5)-based TGMS lines through ribosome-associated protein quality control (RQC). CSIT2 binds to the large and small ribosomal subunits and ubiquitinates 80S ribosomes for dissociation, and may also ubiquitinate misfolded proteins for degradation. Mutation of CSIT2 inhibits the possible damage to ubiquitin system and protein translation, which allows more proteins such as catalases to accumulate for anther development and inhibits abnormal accumulation of reactive oxygen species (ROS) and premature programmed cell death (PCD) in anthers, partly rescuing male sterility and raised the CSIT of tms5-based TGMS lines. These findings reveal a mechanism controlling CSIT and provide a strategy for solving the elevated or unstable CSITs of tms5-based TGMS lines in two-line hybrid rice.

Estimation of the Thermosensitive Period of the Thermosensitive Genic Male Sterile Rice Line ‘Norin-PL12’

Estimation of the Thermosensitive Period of the Thermosensitive Genic Male Sterile Rice Line ‘Norin-PL12’

The E3 ubiquitin ligase CSIT1 regulates critical sterility-inducing temperature by ribosome-associated quality control to safeguard two-line hybrid breeding in rice

Two-line hybrid breeding can fully utilize heterosis in crops. In thermo-sensitive genic male sterile (TGMS) lines, low critical sterility-inducing temperature (CSIT) is vital to safeguard the production of two-line hybrid seeds in rice (Oryza sativa), but the molecular mechanism determining CSIT is unclear. Here, we report the cloning of CSIT1, which encodes an E3 ubiquitin ligase, and show that CSIT1 modulates the CSIT of thermo-sensitive genic male sterility 5 (tms5)-based TGMS lines through ribosome-associated quality control (RQC). Biochemical assays demonstrated that CSIT1 binds to the 80S ribosomes and ubiquitinates abnormal nascent polypeptides for degradation in the RQC process. Loss of CSIT1 function inhibits the possible damage of tms5 to the ubiquitination system and protein translation, resulting in enhanced accumulation of anther-related proteins such as catalase to suppress abnormal accumulation of reactive oxygen species and premature programmed cell death in the tapetum, thereby leading to a much higher CSIT in the tms5-based TGMS lines. Taken together, our findings reveal a regulatory mechanism of CSIT, providing new insights into RQC and potential targets for future two-line hybrid breeding.

Improvement of Rice Blast Resistance in TGMS Line HD9802S through Optimized Anther Culture and Molecular Marker-Assisted Selection.

Rice blast caused by Magnaporthe oryzae is one of the most serious rice diseases worldwide. The early indica rice thermosensitive genic male sterile (TGMS) line HD9802S has the characteristics of stable fertility, reproducibility, a high outcrossing rate, excellent rice quality, and strong combining ability. However, this line exhibits poor blast resistance and is highly susceptible to leaf and neck blasts. In this study, backcross introduction, molecular marker-assisted selection, gene chipping, anther culture, and resistance identification in the field were used to introduce the broad-spectrum blast-resistance gene R6 into HD9802S to improve its rice blast resistance. Six induction media were prepared by varying the content of each component in the culture medium. Murashige and Skoog's medium with 3 mg/L 2,4-dichlorophenoxyacetic acid, 2 mg/L 1-naphthaleneacetic acid, and 1 mg/L kinetin and N6 medium with 800 mg/L casein hydrolysate, 600 mg/L proline, and 500 mg/L glutamine could improve the callus induction rate and have a higher green seedling rate and a lower white seedling rate. Compared to HD9802S, two doubled haploid lines containing R6 with stable fertility showed significantly enhanced resistance to rice blast and no significant difference in spikelet number per panicle, 1000-grain weight, or grain shape. Our findings highlight a rapid and effective method for improving rice blast resistance in TGMS lines.

Low temperature compensates for defective tapetum initiation to restore the fertility of the novel TGMS line ostms15.

In rice breeding, thermosensitive genic male sterility (TGMS) lines based on the tms5 locus have been extensively employed. Here, we reported a novel rice TGMS line ostms15 (Oryza sativa ssp. japonica ZH11) which show male sterility under high temperature and fertility under low temperature. Field evaluation from 2018 to 2021 revealed that its sterility under high temperature is more stable than that of tms5 (ZH11), even with occasional low temperature periods, indicating its considerable value for rice breeding. OsTMS15 encodes an LRR-RLK protein MULTIPLE SPOROCYTE1 (MSP1) which was reported to interact with its ligand to initiate tapetum development for pollen formation. In ostms15, a point mutation from GTA (Val) to GAA (Glu) in its TIR motif of the LRR region led to the TGMS phenotype. Cellular observation and gene expression analysis showed that the tapetum is still present in ostms15, while its function was substantially impaired under high temperature. However, its tapetum function was restored under low temperature. The interaction between mOsTMS15 and its ligand was reduced while this interaction was partially restored under low temperature. Slow development was reported to be a general mechanism of P/TGMS fertility restoration. We propose that the recovered protein interaction together with slow development under low temperature compensates for the defective tapetum initiation, which further restores ostms15 fertility. We used base editing to create a number of TGMS lines with different base substitutions based on the OsTMS15 locus. This work may also facilitate the mechanistic investigation and breeding of other crops.

Microspore expressed SCULP1 is required for p-coumaroylation of sporopollenin, exine integrity, and pollen development in wheat.

Sporopollenin is one of the most structurally sophisticated and chemically recalcitrant biopolymers. In higher plants, sporopollenin is the dominant component of exine, the outer wall of pollen grains, and contains covalently linked phenolics that protect the male gametes from harsh environments. Although much has been learned about the biosynthesis of sporopollenin precursors in the tapetum, the nutritive cell layer surrounding developing microspores, little is known about how the biopolymer is assembled on the microspore surface. We identified SCULP1 (SKS clade universal in pollen) as a seed plant conserved clade of the multicopper oxidase family. We showed that SCULP1 in common wheat (Triticum aestivum) is specifically expressed in the microspore when sporopollenin assembly takes place, localized to the developing exine, and binds p-coumaric acid in vitro. Through genetic, biochemical, and 3D reconstruction analyses, we demonstrated that SCULP1 is required for p-coumaroylation of sporopollenin, exine integrity, and pollen viability. Moreover, we found that SCULP1 accumulation is compromised in thermo-sensitive genic male sterile wheat lines and its expression partially restored exine integrity and male fertility. These findings identified a key microspore protein in autonomous sporopollenin polymer assembly, thereby laying the foundation for elucidating and engineering sporopollenin biosynthesis.

Pyramiding of Multiple Genes to Improve Rice Blast Resistance of Photo-Thermo Sensitive Male Sterile Line, without Yield Penalty in Hybrid Rice Production.

Rice blast caused by pathogenic fungus Magnaporthe oryzae is one of the most serious diseases in rice. The pyramiding of effective resistance genes into rice varieties is a potential approach to reduce the damage of blast disease. In this study, combinations of three resistance genes, Pigm, Pi48 and Pi49, were introduced into a thermo-sensitive genic male sterile (PTGMS) line Chuang5S through marker-assisted selection. The results showed that the blast resistance of improved lines increased significantly compared with Chuang5S, and the three gene pyramiding lines (Pigm + Pi48 + Pi49) had higher rice blast resistance level than monogenic line and digenic lines (Pigm +Pi48, Pigm + Pi49). The genetic backgrounds of the improved lines were highly similar (>90%) to the recurrent parent Chuang5S by using the RICE10K SNP chip. In addition, agronomic traits evaluation also showed pyramiding lines with two or three genes similar to Chuang5S. The yields of the hybrids developed from improved PTGMS lines and Chuang5S are not significantly different. The newly developed PTGMS lines can be practically used for the breeding of parental lines and hybrid varieties with broad spectrum blast resistance.

Comparative transcriptome analysis provides insight into the important pathways and key genes related to the pollen abortion in the thermo-sensitive genic male sterile line 373S in Brassica napus L.

The thermo-sensitive genic male sterility (TGMS) system plays a key role in the production of two-line hybrids in rapeseed (Brassica napus). To uncover key cellular events and genetic regulation associated with TGMS, a combined study using cytological methods and RNA-sequencing analysis was conducted for the rapeseed TGMS line 373S. Cytological studies showed that microspore cytoplasm of 373S plants was condensed, the microspore nucleus was degraded at an early stage, the exine was irregular, and the tapetum developed abnormally, eventually leading to male sterility. RNA-sequencing analysis identified 430 differentially expressed genes (298 upregulated and 132 downregulated) between the fertile and sterile samples. Gene ontology analysis demonstrated that the most highly represented biological processes included sporopollenin biosynthetic process, pollen exine formation, and extracellular matrix assembly. Kyoto encyclopedia of genes and genomes analysis indicated that the enriched pathways included amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Moreover, 26 transcript factors were identified, which may be associated with abnormal tapetum degeneration and exine formation. Subsequently, 19 key genes were selected, which are considered to regulate pollen development and even participate in pollen exine formation. Our results will provide important insight into the molecular mechanisms underlying TGMS in rapeseed.

Development of Two Novel Photoperiod and Thermo-Sensitive Genic Male Sterile Lines in Rice (Oryza sativa L.)

Development of Two Novel Photoperiod and Thermo-Sensitive Genic Male Sterile Lines in Rice (Oryza sativa L.)

Determining Type of Sterility, Thresholds, Seeding Date and Rates of Arkansas Male Sterile Lines for Hybrid Seed Production

Two-line hybrid rice production requires environmental genetic male-sterile (EGMS) lines that become sterile in certain environmental conditions. In this study, we aimed to identify the genetic type(s), sterility thresholds, optimum planting date, and efficient seeding density of eight male-sterile lines, including four lines developed at the University of Arkansas, Rice Research and Extension Center (RREC) and four at the Dale Bumpers National Rice Research Center (DBNRRC) both located in Stuttgart, AR, USA. The lines were screened in growth chambers using different temperatures and day lengths to identify the type of EGMS line and sterility thresholds. A single location was used in testing the first year of the study and three locations used in the second year of the planting date study. All experiments were arranged in randomized blocks with three replications to identify the optimum planting date and seeding rate. Three planting dates were tested each year for the planting date experiment, and three seeding densities were used in the seed density study. The growth chamber study showed that all RREC lines were thermosensitive genic male-sterile lines, and the sterility threshold temperature for one of these lines was >32.2 °C while that of others was >29.4 °C. The type of sterility and threshold of DBNRRC lines were inconclusive due to their genetic heterogenicity and environmental response inconsistency. In Arkansas, April 25 was the optimum planting date for sterile conditions, and a 431 seed/m2 seeding rate was required to produce an adequate number of panicles for hybrid seed production while avoiding wasting expensive male-sterile seeds.

Identification and Functional Analysis of MicroRNAs and Their Target Genes in Reverse Thermosensitive Genic Male Sterility of Eggplant

Thermosensitive genic male sterile (TGMS) lines are the core of two-line hybrid systems. MicroRNAs (miRNAs) play critical roles in plant growth and development. However, knowledge of regulation of anther development by miRNAs in TGMS eggplant (Solanum melongena) is largely unexplored. To investigate the mechanism underlying miRNA regulation of male sterility, we employed high-throughput small RNA sequencing in anther samples from the reverse TGMS line 05ms and the temperature-insensitive line S63 in eggplant, under high temperature and low temperature conditions. The 05ms line is sterile at low temperature and fertile at high temperature. A total of 166,273,427 raw reads were obtained, 143 known miRNAs from 42 miRNA families and 104 novel miRNAs were detected. Further, six differentially expressed miRNAs (DEMs) were identified, including three known (miR168b-3p, miR397–5p, and miR408) and three novel miRNAs (Novel_116, Novel_119, and Novel_97), which might be related to anther development. Moreover, the six DEMs were validated by quantitative real-time polymerase chain reaction and 892 target genes of which were predicted. Gene Ontology analysis of target genes revealed significant enrichment in the “copper ion binding,” “oxidation-reduction process,” and “oxidoreductase activity” terms. Kyoto Encyclopedia of Genes and Genomes analysis revealed that “plant hormone signal transduction” and “other glycan degradation” were enriched. In addition, we constructed regulatory networks comprising miRNAs, target genes, and important terms/pathways and found the miR397-5p was the most linked miRNA, down-regulated under low temperature. Our findings contribute to understanding of the roles of miRNA during anther development and provide the theoretical foundation for two-line hybrid breeding of eggplant.

CRISPR/Cas9-Induced Mutagenesis of TMS5 Confers Thermosensitive Genic Male Sterility by Influencing Protein Expression in Rice (Oryza sativa L.)

The development of thermosensitive genic male sterile (TGMS) lines is the key to breeding two-line hybrid rice, which has been widely applied in China to increase grain yield. CRISPR/Cas9 has been widely used in genome editing to create novel mutants in rice. In the present study, a super grain quality line, GXU 47, was used to generate a new TGMS line with specific mutations in a major TGMS gene tms5 generated with CRISPR/Cas9-mediated genome editing in order to improve the rice quality of two-line hybrids. A mutagenesis efficiency level of 75% was achieved, and three homozygous T-DNA-free mutant lines were screened out. The mutants exhibited excellent thermosensitive male fertility transformation characteristics with complete male sterility at ≥24 °C and desirable male fertility at around 21 °C. Proteomic analysis based on isobaric tags for relative and absolute quantification (iTRAQ) was performed to unveil the subsequent proteomic changes. A total of 192 differentially expressed proteins (DEPs), including 35 upregulated and 157 downregulated, were found. Gene ontology (GO) analysis revealed that the DEPs were involved in a single-organism biosynthetic process, a single-organism metabolic process, oxidoreductase activity, and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEPs were involved in ubiquinone and other terpenoid quinone biosynthesis, the biosynthesis of secondary metabolites, metabolic pathways, and phenylpropanoid biosynthesis. Our study shows that high mutation efficiency was achieved in both target sites, and T-DNA-free mutant lines were obtained in the T1 generation. The present study results prove that it is feasible and efficient to generate an excellent mutant line with CRISPR/Cas9, which provides a novel molecular mechanism of male sterility caused by the mutation of tms5.

Cia Zeaxanthin Biosynthesis, OsZEP and OsVDE Regulate Striped Leaves Occurring in Response to Deep Transplanting of Rice.

The rice leaf color mutant B03S was previously generated from the photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice line Efeng 1S, of which male sterility manifests by photoperiod and temperature but exhibits mainly temperature-sensitive characteristics. After these plants were deeply transplanted, the new leaves manifested typical zebra stripe patterns. Here, B03S was subjected to deep and shallow transplanting, shading with soil and aluminum foil, and control conditions in situ to determine the cause of the striped-leaf trait. The direct cause of striped leaves is the base of the leaf sheath being under darkness during deep transplanting, of which the critical shading range reached or exceeds 4 cm above the base. Moreover, typical striped leaves were analyzed based on the targeted metabolome method by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) combined with transcriptome and real-time quantitative PCR (qPCR)-based verification to clarify the metabolic pathways and transcriptional regulation involved. Carotenoids enter the xanthophyll cycle, and the metabolites that differentially accumulate in the striped leaves include zeaxanthin and its derivatives for photooxidative stress protection, driven by the upregulated expression of OsZEP. These findings improve the understanding of the physiological and metabolic mechanisms underlying the leaf color mutation in rice plants, enrich the theoretical foundation of the nonuniform leaf color phenomenon widely found in nature and highlight key advancements concerning rice production involving the transplanting of seedlings or direct broadcasting of seeds.