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Jan 04, 2024

Molecular and biochemical characterization of rice developed through conventional integration of nDart1

Scientific Reports volume 13,

Scientific Reports volume 13, Article number: 8139 (2023) Cite this article

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Mutations, the genetic variations in genomic sequences, play an important role in molecular biology and biotechnology. During DNA replication or meiosis, one of the mutations is transposons or jumping genes. An indigenous transposon nDart1-0 was successfully introduced into local indica cultivar Basmati-370 from transposon-tagged line viz., GR-7895 (japonica genotype) through conventional breeding technique, successive backcrossing. Plants from segregating populationsshowed variegated phenotypes were tagged as BM-37 mutants. Blast analysis of the sequence data revealed that the GTP-binding protein, located on the BAC clone OJ1781_H11 of chromosome 5, contained an insertion of DNA transposon nDart1-0. The nDart1-0 has "A" at position 254 bp, whereas nDart1 homologs have "G", which efficiently distinguishes nDart1-0 from its homologs. The histological analysis revealed that the chloroplast of mesophyll cells in BM-37 was disrupted with reduction in size of starch granules and higher number of osmophillic plastoglobuli, which resulted in decreased chlorophyll contents and carotenoids, gas exchange parameters (Pn, g, E, Ci), and reduced expression level of genes associated with chlorophyll biosynthesis, photosynthesis and chloroplast development. Along with the rise of GTP protein, the salicylic acid (SA) and gibberellic acid (GA) and antioxidant contents(SOD) and MDA levels significantly enhanced, while, the cytokinins (CK), ascorbate peroxidase (APX), catalase (CAT), total flavanoid contents (TFC) and total phenolic contents (TPC) significantly reduced in BM-37 mutant plants as compared with WT plants. These results support the notion that GTP-binding proteins influence the process underlying chloroplast formation. Therefore, it is anticipated that to combat biotic or abiotic stress conditions, the nDart1-0 tagged mutant (BM-37) of Basmati-370 would be beneficial.

A monocot semi-aquatic annual grass plant, rice is a member of the Poaceae family and belongs to the genus Oryza1. The Oryza genus has 22 wild taxa, of which two species are highly significant for human consumption2. These species are Oryza sativa L., generally known as Asian rice, and Oryza glaberrima Steud, popularly known as African rice. Several classifications within the Oryza genus include Oryza officinalis, Oryza ridelyi, and Oryza rufipogon. Of the 22 wild species of Oryza, 15 originated in Asia and 7 in sub-Saharan Africa3. These species are beneficial in interspecific rice breeding programs because wild Oryza species have particular traits, including abiotic and biotic stress tolerance4,5. Over half of all humans eat rice, making it the most important cereal crop6.

Moreover, the full genome sequence of rice has been sequenced with great fidelity, and comparatively high number of transposons were found, making it a great model for the study of transposons7,8. An nDart1-0 transposon (pyl-v) was found in a rice virescent rice mutant with light yellow variegated leaves of Taichung-65 (Oryza sativa japonica L.)9. In particular, genotypes, containing aDart1-27 an active autonomous DNA element, carry a transposon gene, the element nDart1 are actively transferred throughout the genome10. The nDart1 elements, which belong to the superfamily hAT, are excised from chromosomal inserted locations and transposed to other sites, despite having a high degree of sequence similarity, nDart1-0 and the closely comparable non-autonomous components nDart1-1 through nDart1-12 display differing transposition frequencies11. Active transposons are frequently employed to tag genes and reveal their functionalities12,13. A tagging method is an effective tool, because nDart1 elements are actively transposed into genome and have a propensity to integrate at genomic locations. The nDart1 elements, especially transposed into locations at the proximal promoter of the genome, which are located 0.5 kb before the putative initiation codons14. The iPCR-based methods and transposon display (TD) methods were also created to efficiently locate inserted sites of nDart1 with in genome15,16.

Both the japonica and indica subspecies of rice have had their whole genomes sequenced, however the experimental research on many functional genes in the rice genome is still ongoing17. Examining how many genes work is now one of the most difficult goals. According to the descriptions of certain specific genes, numerous intermediate components of developmental processes, namely proteins involved in signal transduction pathways, are still substantially conserved. The G protein is a fantastic illustration of these so-called molecular switches18. Through intrinsic activities, these proteins undergo conformational changes brought on by the binding and hydrolysis of GTP14. GTP-binding proteins differ in eukaryotes and prokaryotes and are among the most important proteins for all species19. GTPases or G-proteins are other names for these proteins. It is a molecular switch that is present in all areas of life. It is "activated" by the GTP molecule and "inactivated" by the hydrolysis of the GTP molecule to GDP. It regulates several cellular functions, including the rearrangement of the cell cytoskeleton, signal transduction, translation, transcriptional regulation, vesicle trafficking, and protein transport20,21. The G1, G2, G3, G4, and G5 motifs are present in all G-proteins. These substances are necessary for GTP hydrolysis, GTP conformational change, and GDP/GTP conversion. In the G-protein family, G2 pattern is robustly maintained but has little impact on GDP/GTP exchange activity22,23. The aim of this study was to introduce transposon nDart1-0 into highly growing local Indica rice cultivar "Basmati-370" to obtain mutant through conventional breeding approach and to characterize the ERA-like GTP-binding protein gene and its effects on the activation of phytohormones, which may be helpful to induce biotic/ abiotic stress tolerance in rice.

The required breeding material for the current experiment was collected from two different sources; seed of Indica rice cultivar, Basmati-370 from Rice Research Institute, Kala Shah Kaku, Punjab, Pakistan, and seed of japonica line GR-7895 (consisting nDart1-0 transposon) from Plant Genetic Resources Institute, National Agricultural Research Center (NARC), Islamabad, Pakistan. It has been confirmed that the experimental data collection complied with relevant institutional, national, and international guidelines and legislation with appropriate permissions from Plant Genetic Resources Institute, National Agricultural Research Center (NARC), Islamabad, Pakistan. The data on search experiments revealed with the studies on Basmati-370 have shown that it does not carry any of the aDart elements. The aDart is actually a carrier for nDart1-0 transposon element. For study purposes, mutants were developed for gene tagging by using insertion of the transposon aDart element. A cross was carried out between Basmati-370 and GR-7895 for insertion of aDart element on Basmati-370 from GR-7895 genotype. Dry mature seeds from both lines were surface sterilized for this purpose and raised in controlled environments in pots. To synchronize flowering for crossover, Basmati-370 seeds were cultivated at one-week intervals for four successive weeks. The crossing was carried out by keeping Basmati-370 as a female parent. The F1 seeds were grown under controlled conditions and backcrossed with designated female plants of Basmati-370 to produce BC1F1 and continue this backcrossing to produce BC4F1 (Supplementary material: Fig. S1). The harvested seeds from BC4F1 were grown and self-pollinated to get BC4F2. The seeds from BC4F2 were harvested and further grown under controlled conditions to get segregating population upto BC4F4 of plants to identify the abnormal or albino phenotypes used for gene tagging.

The DNA was extracted from the mutant plants through using a protocol of DNA isolation given by Doyle24. The extracted DNA was then transferred into eppendorf; along with added 7 μl of RNAase, which incubated at 37 ℃ for 60 min. The quality of DNA was verified using 2% agarose gels, while its quantity was measured using Nanodrop. The samples were then diluted for uniform and standard concentration of DNA up to 1 μl of each sample per 50 µl dH2O. The isolated DNA was then quantified by taking a reading of absorbance at 260 nm wavelength keeping using dH2O as a blank sample.

RNA was isolated from leaves of rice plants from Basmati-370 (WT) and BM-37 (mutant) through the TRIeasyTM Total RNA Extraction Reagent Kit (Yeasen Biotechnology Co., Ltd. Shanghai, China) as per the manufacturer's guidelines. The quality of RNA was verified using 2% agarose gels, while its quantity was measured using Nanodrop. The cDNA was synthesized by an improved qRT-PCR technique derived from the total mRNA25,26. PCR was performed for amplifing of full length cDNA of GTP using specific primers. Oligonucleotide primers (Supplementary material: Table S1) for the amplification of full-length cDNA of OJ1781_H11 were designed based on the reported sequence in rice (Accession No. AC120986). PCR amplified sequence was cloned and sequenced. The sequence comparison and data analysis were performed using blast with the NCBI database.

We examined the expression level of nDart1-0 in Basmati-370 (BM-37) mutant plants, including plumules, third and fifth leaves at the seedlings stage, roots, stems, sheath, young panicles, and flag leaves at the heading stage of plants. Quantitative RT-PCR tests were conducted to measure the amount of nDart1 expression. An additional set of genes (PORA, CAO1, Cab1R, YGL1, CHLD, HEMA, PsbA, RNRS, RbcL, PsaA, RbcS, LhcpII, OsPoLP1, RNRL, FtsZ, Rpl21, RpoB, Rsp20, RpoA, OsRpoTp and Rps7 related of photosynthesis, chloroplast development and chlorophyll biosynthesis were examined for their expression level through qRT-PCR. Primers designed for qRT-PCR are mentioned in Table S2 (Supplementary material). The conditions for gene amplifications are stated in (Supplementary material: Fig. S2).

The genomic DNA from leaves of BM-37 mutant plants was isolated according toDellaporta et al.27. All the mutants were analyzed by n1-0SPiPCR method (Supplementary material; File 1). For PCR, the following conditions were adjusted: an activation phase at 95 °C for 30 s, denaturation with 35 cycles at 60℃ for 30 s, annealing at 72 ℃ for 60 s, and extension at 72 ℃ for 10 min. By designing primers through software application Primer_premier5 from the gene's flanking area around the transposon's insertion site, the transposon fragments were amplified.

The leaf samples from Basmati-370 (WT) and mutant (BM-37) plants devoid of veins were taken from randomly selected seedlings and placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer with pH 7.0 for 5 h, then washed three times with phosphate buffer (0.1 M, pH 7.0). Further, the samples were postfixed with 1% OsO4 for 1 h and washed three times with phosphate buffer (0.1 M, pH 7.0) for 15 min at each wash. Then, samples were dehydrated with graded series of ethanol (30, 50. 70, 80, 90 and 100%, respectively) and doused with concentrated acetone for 20 min, and ultimately imbedded in Spurr's medium before being cut into ultrathin sections. Moreover, the samples after cutting in ultra-thin sections were placed on the copper nets for observation under transmission electron microscope (JEOLTEM-1230EX).

Utilizing a spectrophotometer by making minor adjustments in the methods of Arnon and Wellburn28,29, the carotenoid (Car), total chlorophyll, and chlorophyll (a and b) were assessed. In a nutshell, 0.2 g samples from leaves of seedlings grown under controlled circumstances at the three-leaf stage were collected and homogenized for 18 h in the dark in 5 ml of ethanol:water:acetone (4:1:5) solution. Centrifugation was used to get rid of leftover particles. A UV5100 Spectrophotometer was used to evaluate the supernatants. In addition, the portable photosynthetic system LI-6400 was used to quantify the transpiration rate (E), the concentration of intracellular CO2 (Ci), stomatal conductance (g), and net photosynthetic rate (Pn).

The total phenolic contents were calculated using the Folin-Ciocalteu method, with gallic acid acting as a controlled chemical30. Equivalents of gallic acid per gram of FW-fresh weight were used to indicate the amount of total phenolic compounds. By employing catechin as a reference ingredient, the aluminum trichloride technique was used to measure the flavonoids concentration31. (gE catechin.100 g1 FW) was used to express the overall flavonoid content. Following the manufacturer's recommendations, we used a plant ELISA kit to detect the endogenous salicylic acid (SA), abscisic acid (ABA), gibberellic acid (GA) and cytokinins (CK). To measure the concentrations of SA, ABA, GA and CK in the samples, leaves were pulverized in liquid nitrogen. The hormonal SA, ABA, GA and CK ELISA kits come with a set of calibration standards. The SA, ABA, GA and CK levels in the samples were then measured by fitting the standard curve and generating a comparable trend in the samples.

The leaf samples were homogenized in sodium phosphate buffer (pH 7.8), centrifuged at 13,000 rpm for 20 min at 4 °C. Then superoxide dismutase (SOD) activity was measured spectrophotometrically as described by Zhang et al.32, at 560 nm by assessing the ability of each unit to inhibit 50% photochemical reduction of nitro blue tetrazolium chloride (NBT). Peroxidase (POD) activity was determined according to Zhou and Leul33, at 470 nm and the changes related to guaiacol were normalized with the activity constant (ε = 26.6 mm) and catalase (CAT) activity was determined according to Aebi34. The APX activity was calculated depending on the decrease in absorbance at 290 nm according to Nakano and Asada35 as ascorbate was oxidized. This study measured reactive oxygen species in plant tissues, including hydrogen peroxide (H2O2). Hydrogen peroxide content was measured as described by Velikova et al.36. Malondialdehyde (MDA) content was determined according to Morales and Munné-Bosch,37. The total phenolic content was measured by the Folin Ciocalteu method using gallic acid as a reference compound30. The flavonoids content was determined by the aluminum trichloride method using catechin as reference compound38.

The standard error and mean from three replications are reported for each set of data. Data analysis was carried out using Statistics 8.1, a statistical software programme. The recorded data was analyzed through one way variance analysis and LSD test and Tukey's test to determine pairwise significance at p ˂ 0.05. Origin Pro (version 8.5) was used to create the graphs.

To study gene action, the regulation and characterization of genes have become the most important genetic tool in biotechnology and genetic engineering due to reverse and forward genetic approaches. For the evaluation of genetic potential and variations in the population of crop plants, there is necessary to develop mutants by using irradiations, chemicals, transposons, and induced genetic mutations for tagging genes to carry out functional genomic evaluations of species genomes from narrow to more comprehensive ranges. In our current study, we have described a active, and non-autonomous genomic or DNA transposable element in rice, named non-autonomous DNA-based active rice transposon one (nDart1). The nDart1 is a causative transposable element of allele with spontaneous mutable and virescent nature, the pale-yellow-leaf variegated or pyl-v, which caused pale yellow with dark-green streaks on leaves of rice plants at very early seedling stages usually due to the expression of nDart1 gene in rice. There are multiple transposon elements along with their multiple gene insertional sites, which a crop plant line of genotype has been found in limited numbers because of the reduced requirements for the identification and characterization of specific genes tagging a population genome for achieving maximum saturation of transposable elements. For our current study, we have selected the nDart1 gene to produce rice mutants, which were expressed as pale-yellow-leaf variegated. The nDart1 gene was inserted into the Basmit-370 variety through the conventional backcross breeding method.

In our current research study, we have utilized an endogenous and available transposon named nDart1 to develop mutants for tagging genes in the Basmati-370 rice genotype. As we could not find any active nDarts or active autonomous element Darts in local basmati varieties, we introduced japonica-derived aDart and nDart1-0 into the indica rice variety Basmati-370 through backcrossing for mutant genes. Different plants were selected from segregating populations that showed variation and were tagged as mutants (Fig. 1A) from the BC4F4 population. Through genetic analysis, this mutant was surmised to be controlled by a two-component transposon system.

Transposon-induced mutants from segregating population. (A) Phenotypic difference between normal, revertant, and stable albino seedlings; (C, D) phenotypic appearance of third leaves at the seedling stage from WT and BM-37 mutant; (D) segregated mutant plants for tagging of genes.

During growing of rice plants in the field after BC4F4, there were normal green leaves in the WT and variegated types of albino rice plants in BM-37 (Fig. 1B, C), which was introduced with nDart1/aDart1. The lines which showed gene expression were postulated to have a recessive allele that had the nDart1-0 homozygous as well as an active autonomous element as aDart1 with heterozygous allele conditions. It was found that the mutated line produced stable albino progenies (Fig. 1D). The stable mutants indicated that there were not only stable GTP homozygous alleles but also having without an aDart1element with a ratio of 3:1 for F2. It was also found that there was no difference between the WT as well as the mutants in rice seeds, but the difference was found only at the stage of seedling growth, where the normal seeds produced normal green leaves and stem, whereas the mutated seeds produced white shoots and leaves. The results indicated the presence of aDart1 elements as found stable in rice plant phenotypes. It was found that aDart1-dependent nDart1-0 somatic excisions of allele produced a leaf with variegations that developed throughout cell lineage or progenies of mutated individuals of rice plant. However, the white mutated or variegated plants may grow their panicles with fertile grains under extreme conditions.

The frequency of spontaneous mutation under natural growth conditions is extremely low. Transposons are useful for gene tagging and functional analysis to create natural variation, so it is useful to explore active transposon sources. Mutant phenotypes were tagged in the field and DNA was isolated from phenotypically variegated leaves of mutants. DNA was subjected to restriction digestions and self-ligation. Data search revealed that nDart1-0 has thirteen homologs and a high homology above 99%. At position 254 bp, nDart1-0 has "A", whereas the homologs have "G" as shown in Table S3 (Supplementary material). The site "A" was used efficiently to distinguish nDart1-0 from its homologs.

Keeping in view that according to this technique, DNA is restricted with Alu I, self-ligated, and again subject to another restriction digestion with Bmt I and amplification by iPCR using specific primers. Digestion by Alu I, restrict nDart1-0 at position 254 as it carries a unique specific restriction site due to the substitution of a base pair "A" whereas homologs carry "G" at this site during self-ligation, this digested fragment of the nDart1-0 ligates with a fragment of the gene invaded by him (Fig. 2). Self-ligation yields some specific fragments having a part of the nDart1-0 and the gene invaded by him along with other fragments. Again, restriction of this self-ligated DNA by Bmt I cleaves the nDart1-0 as it carries Bmt I site, as a result, we get some fragment of the invaded gene flanked by nDart1-0. These fragments were amplified through iPCR using the specific primers designed for flanking region of nDart1-0. The two rounds or cycles of iPCR (like pre-selective and selective gene amplifications) were carried out for confirmation of results. During the selective type of amplification, the further enhancement and enrichment of transposon elements or their related specific fragments were achieved through using available PCR master mix products like a template to carry out the second transposable element amplification with the nested type of primer, which specifically recognized highly conserved transposable element regions in the genome. Nonspecific genomic rearrangements during iPCR are ruled out because of the primers derived from specific regions of the nDart1-0. On the other hand, the nDart homologues does not carry Alu I site at position 254, they are not digested at this position and consequently are not amplified with specific primers during iPCR (Supplementary material: Fig. S3A). When DNA restricted with Alu I and self-ligated is used as template for iPCR, nDart1-0 and all its homologues are amplified but this amplification is restricted to nDart1-0 if self-ligated DNA is again restricted with Bmt I. For confirmation of fragments obtained either these were transposable elements or not detected transpose element fragments were amplified to study the transposon element flanking sequences, PCR amplified fragments were sequenced and analyzed. The amplification patterns for each fragment were similar i.e., flanked by nDart1-0, except for the difference in genes invaded. So far, we have detected 12 genic regions invaded by nDart1-0 in BM-37 mutant (Supplementary material: Table S3). It was found that there was a mutant amplified fragment which was containing nDart1-0 flanking sequences were detected which were derived due to the insertion of nDart1-0 on to the first exon regions of a putative gene (Supplementary material: Fig. S3B).

The nDart1-0 transposon and its homologues in rice. A Sequence of nDart1-0 transposon in rice. Arrows show primer positionArrows and restriction sites show primer positions are highlighted in red. TSD-target site duplication; TIRs-terminal inverted repeats.

The results from our study have suggested that the nDart1-0 element insertion as well as its excision into the putative gene was as OJ1781_H11 on chromosome 5. Blast analysis of the sequence data revealed that GTP-binding protein locus on the BAC clone OJ1781_H11 of chromosome 5 was found to contain an insertion of DNA transposon nDart1-0. This gene has a nucleotide sequence of 2701 bp and 918 cDNA. The ORF encoded a polypeptide of 305 amino acid sequence and comprises 7 exons interrupted by 6 introns. During the process of gene tagging five revertants were found. These revertant phenotypes were analyzed to find out absence or presence of the transposon. DNA was isolated from the revertant phenotypes and transposon-inserted regions were amplified by the already-designed primers from the flanking regions of the transposon. Amplified fragments (Fig. 3A, B) were cloned and sequenced. Sequence analysis revealed that the transposon moved from the insertion point, leaving behind the footprints.

PCR amplification of nDart1-0 mutants and transcripts of GTP binding protein gene in several tissues of Basmati 370. (A) PCR amplification of nDart1-0 mutants. Lane 1: Basmati 370, Lane2: T-65, Lane3: Nipponbare, Lane4: Mutable whitish-leaf, Lane 5: Stable whitish leaf-1, Lane6: Stable whitish leaf-2, Lane7: Stable whitish leaf-3; (B) transcripts of GTP binding protein gene in several tissues of Basmati 370. Lane 1: Etiolated plant leaf, Lane2: Etiloated plant root, Lane 3: 2-week-old plant leaf, Lane 4: 2-week-old plant root, Lane5: 2-Month plant leaf, Lane6: 2-Month old plant root, Lane 7: 2Month plant stem, Lane8: 2-Month old plant meristem, Lane9: 2-Month old plant glume, Lane 10: 2-Month old plant anther, Lane11-2Month old plant stigma.

To study the expression pattern of transposon nDart1-0, the qRT-PCR was carried out on several tissue levels (3rd and 5th leaves at the seedling stage, plumules, sheath, shoots, roots, young panicles and flag leaves at heading stage)of BM-37 mutant. The highest expression was found in the third leaves stage of seedling, compared to the fifth and flag leaves, across all the tissues analyzed, indicating that early seedling leaves had higher expression than other tissues. The tissues, including stem, sheath, and roots, expressed significantly lower expression level than leaves, while young panicles and plumules showed a minor level of expression for BM-37 (Fig. 4). The findings suggested that nDart1-0 have an influential role in leaf chloroplast development particularly during the early seedling stage.

Transcript levels for expression study of different tissues in BM-37 mutant. Plumules; 3rd-third leaves at the seedling stage; 5th-fifth leaves at the seedling stage; YR-young roots; Stem, Sheath, FL-flag leaves and YP-young panicle. All values represent the mean ± SD of five replicates. Different letters above error bars indicate the significant differences between treatments at p ≤ 0.05.

Using TEM, the upper part of leaves at the tillering stage were studied to examine the ultra-structural alterations in the chloroplast of WT and BM-37 mutant plants. In contrast to the BM-37 chloroplasts, which showed well-developed lamella structures, the grana lamella stacks in WT plants were sparse and sparsely populated. This suggests that the BM-37 mutation had a significant impact on biogenesis. The outcome also demonstrated that starch granules were difficult to locate in the chloroplast of the BM-37 mutant (Fig. 5A, B), although they were present normally in the WT. In the BM-37 mutant, there were more osmophilic plastoglobuli (OP) than in the WT, indicating that the chlorophyll metabolism was substantially compromised.

TEM images of chloroplasts in plants of WT and BM-37 mutant (A and B). G, grana stacks; OP, osmophilic plastglobuli; SG, starch granules.

At the seedling and heading phases of the WT and BM-37 plants, the carotenoid and chlorophyll contents were examined to ascertain the impact of the nDart1-0 transposon on photosynthesis pigment metabolism. The chlorophyll (a, b and a + b) contents and carotenoid levels were drastically decreased in BM-37 at seedling stage as compared with WT; however, the chlorophyll contents and carotenoids showed non-significant results at the heading stage in both WT and BM-37 mutant plants (Fig. 6A, B). Additionally, the gas exchange parameters were measured to compare the photosynthetic characteristics of WT and BM-37 mutant plants. As compared to WT, the BM-37 mutant dramatically lowered the rates of photosynthetic activity (Pn), stomatal conductance (g), intercellular CO2 concentration (Ci), and transpiration (E) (Fig. 6C–F).

Chlorophyll pigment contents, carotenoid contents, and photosynthetic parameters analysis. (A) Chlorophyll and carotenoid contents in leaves at seedling stage of BM-37 mutant and WT; (B) chlorophyll and carotenoid contents in leaves at heading stage of BM-37 mutant and WT (C) net photosynthetic rate (Pn); (D) stomatal conductance (g); (E) intercellular CO2 concentration (Ci); (F) transpiration rate (E). All values represent the mean ± SD of five replicates. According to Tukey's test, asterisks indicate the statistical significance levels (*p < 0.05).

In WT plants and BM-37 mutant plants, the expression levels for different genes related to chloroplast development, photosynthesis, and chlorophyll biosynthesis were evaluated at the seedling stage. The results showed that genes associated with biosynthesis of chlorophyll; comprising PORA, CHLD, YGLI, CAO1 and HEMA and the photosynthesis related genes such as RbcS, Cab1R, PsaA, PsbA, RbcL and LhcpII39,40, were significantly down regulated in pyl-v37 mutant plants than the WT plants (Fig. 7A, B).

Quantitative expression analysis of genes related to (A) chlorophyll biosynthesis, (B) photosynthesis, and (C) chloroplast development in the BM-37 mutant and WT plants.The expression level of each gene was analyzed by qPCR.

We also studied the expression level of different genes related to chloroplast development, including RpoA, RpoB, OsRpoTp, Rps7, Rps20, RNRL, RNRS, OsPolP1, FtsZ and Rpl2141,42,43,44,45,46. Relative expression levels of all genes associated with chloroplast development were significantly down regulated in the BM-37 mutant as comparable to WT plants (Fig. 7C). Under abiotic stress, it's possible that these important genes' aberrant expression caused the mutant phenotype. The findings showed that the BM-37 mutation had a significant impact on the metabolism of chlorophyll, photosynthesis and chloroplasts development in BM-37 mutant cells.

The BM-37 mutant and WT leaves at seedling stage were used to assess the levels of the most prevalent growth-related phytohormones, such as SA, CK, GA and ABA in the plant. According to the findings, CK levels were substantially lower and GA and SA contents were significantly higher in the BM-37 mutant than in the WT, while ABA levels did not differ significantly from those of the WT (Fig. 8A–D). According to the findings, the BM-37 mutant displayed efficient hormonal signaling for chloroplast formation.

Endegenous phytohormones, enzymatic antioxidants, low molecular weight antioxidants (LMWAs), and reactive oxygen species (ROS) in BM-37 mutant and WT. (A) CK-cytokinins; (B) ABA-abscisic acid; (C) SA-salicylic acid and (D) GA-gibberellic acid; (E) SOD-superoxide dismutase; (G) APX-ascorbate peroxidase; (I) POD-peroxidase; (K) CAT-catalase; (F) TFC-total flavonoid contents; (H) TPC-total phenolics content; (J) H2O2-hydrogen peroxide and (L) MDA-malonaldehyde content. All values are the mean ± SD of three biological replications. * p < 0.05 by Tukey's test.

We also evaluated ROS, such as H2O2 production and MDA contents, and their scavenger antioxidants, such as lower molecular weight antioxidants and enzymatic antioxidants, in order to study the activities of antioxidants for comparison between WT and BM-37 mutant (Fig. 8E–L). Enzymatic antioxidants like CAT and APX, which are significantly lower in leaves of BM-37 while SOD is considerably higher in leaves of the WT, might scavenge the oxidative compounds (Fig. 8E, G, K). MDA levels were substantially enhanced in BM-37 plants than in the WT plants (Fig. 8L), showing that mutant leaves had more oxidative damage. The results also showed that there are no appreciable changes between BM-37 and WT values for H2O2 and POD (Fig. 8I, J). The results also depicted that the values of TFC and TPC also significantly reduced in BM-37 mutant leaves as compared with WT leaves (Fig. 8F, H). The study found that BM-37 had inadequate antioxidant defense because it was less effective than the WT to scavenge ROS generated by defective chloroplasts.

For gene-tagging, insertional mutagenesis by T-DNA, Tos17 or maize transposons, Ac/Ds and En/dSpm is very useful, but main disadvantage is the frequent presence of somaclonal variation. The frequency of gene tagging by endogenous retrotransposon Tos17 is very low47,48. Mutations by inserting endogenous DNA TEs are desirable to produce transposon-tagged mutant resources. Only the disadvantage with an endogenous DNA TE is to generate stable mutants induced by footprints. In this case, it is difficult to identify the mutant gene. Previously efforts were made to reactivate mPing, a tourist-like thhe tourist-like MITE family member, in the rice genome through tissue culture techniques49,50 and gamma-ray irradiations51,52,53. Insertion of mPing induced duplications of targeted site (TSDs) of TTA or TAA, and mPing usually tend to insert into the non-coding region or intron, so mPing was not considered a good transposon-tagging tool. On the other hand, DNA TEs, nDat1-0 were discovered in rice9. Structurally, nDart1-0 is non-autonomous active DNA transposon with a small size (607-bp), and highly rich GC (about 72%), and tends to transpose into gene regions, it carries a 19-bp perfect segment of terminal inverted repeats (TIRs), which usually has 8-bp high GC targeted site duplications and belongs to hAT super family54,55. The nDart1-0 inserted in OsClpP5 was also found to invade two other genes, indicating its high efficiency for gene-tagging9. The blast search revealed that Nipponbare and indica variety 9311 carries 13 and 7 nDart1 homologs, respectively. The efficient nDart1-0 utilization for gene-tagging usually depends upon aDart presence or occurrence in that rice variety. It was observed that Nipponbare carries 63 candidates of autonomous element aDart56. These specific features distinguish nDart1-0 from most of the last and previously studies and discoveries of non-autonomous transposable elements in DNA.

The transposable elements caused variegation or albino plants, when these elements expressed in rice varieties57. In maize and Antirrhinum majus, the transposon tagging genes have been reported, which have been successfully isolated along with different ranges of transposable genes or elements58,59,60. Therefore, we developed a strategy to tag genes mutated by the invasion of nDart1-0. The newly developed strategy using iPCR (n1-0SPiPCR) is useful for our nDart1-0-specific tagging system. By this system, the transposons elements are usually tagged through an inverse PCR by restriction-ligation-restriction-mediated, which starts from specific sequences of transposon, and amplifies the part of flanking sequences to a specific site for restriction. The resulting PCR products may be used to clone, analyze or sequenced61,62,63. The previous studies elaborated that both aDart and nDart1-0 have been found useful tool for gene-tagging in japonica and indica rice species. We have found that the utilization of a well-established systematic and computational approach for isolation of active transposon element nDart1-0 from the host cell genome, may help to understand the pattern of inheritance of transposable elements in rice64,65. However, this technique not only the fast method for detecting the nDart1-0, while it also has its utilization in gene tagging for various genes in rice genome.

The albino or variegated mutant plants provide important and valuable information regarding plastid growth and development; their genetic analysis has been difficult because of their higher lethal phenotypic effects66. From the present study, the muted allele was successfully identified by utilizing the recently developed procedure named as nDart1-0-iPCR. It has been found from tagging for nDart1/aDart1 line that it contained the most frequent and repeated transposable element nDart1-0 while the other line had nDart1 transposable elements67,68. It has been found that nDart1 transposable elements showed their behavior to integrate within the promoter regions of genes69. Insertion of nDart1-0 also was found to be a cause for mutation in GTP gene at 13 bp downstream from the initiation ATG codon of GTP gene. There was a frameshift mutation in GTP gene due to nDart1-0 insertion downstream of GTP gene transcriptional initiation sites. The results from previous research experiments have also revealed that the nDart1 insertion at the transcriptional initiation sites caused effects on the expression levels in downstream gene sites. The OsClp5 has shown its effects which were disrupted due to insertion on 5’- UTR position of nDart1 transposon in pyl-v mutant70. It was found that mutant rice line exhibited an increased and enhanced inflorescence, indicating that the nDart1-0 insertion may cause up-regulated level of gene expression from downstream sites of gene names such as Aberrant Panicle Organization1 in rice71,72,73.

From various research studies, the GTP protein is required in higher amounts for the biogenesis of chloroplast while lower amount for etioplast biogenesis. Translation efficiency of GTP has been found to be activated from etioplast during chloroplast development. The cell division of plastids mostly occurred during P0 up to the very early P4 growth stages, while the activation for photosynthetic apparatus has been found during P4 growth stage of rice liens74,75,76. Our current study suggests that the GTP protein functions during the development of thylakoid membranes of chloroplasts due to a well-established genetic system of plastids. While in the non-green plant tissues, it has also been found that the GTP protein abundance and accumulation during chloroplast development was regulated at the post-transcriptional level.

The BM-37 mutant, with insertion of nDart1-0 have lower contents of carotenoid and chlorophyll at seedling stage and a major reduction in pigment would probably affect the chloroplast development and ultimately photosynthesis process (Fig. 6A). Transmission electron microscopy confirmed ultrastructural changes in BM-37 due to mutation of nDart1-0 gene (Fig. 5). More number of plastoglobuli observed in BM-37 that is consistent with the previous research that plastoglobuli number increased when mutants have defect in thylakoid membrane biogenesis and their formation prevent thylakoid from oxidative damage77. In BM-37, the chloroplast contained smaller starch granule, indicating that starch formation was disturbed, which affect the energy supply for chloroplast development78. Moreover, the reduction in chlorophyll contents of BM-37 mutant have decreased the key photosynthetic parameters, such as the net photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO2 concentration (Fig. 6C–F) which was also observed in mutant where gas exchange parameters (Pn, Tr, g and Ci) decreased with reduction in chlorophyll contents79. In addition, lower transcript level of genes involved in chloroplast development, chlorophyll biosynthesis and photosynthesis of BM-37 mutant (Fig. 7A–C) have provided the evidence that mutation in nDart1-0 has disturbed plastid-to-nucleus retrograde signaling which is consistent with the previous finding that chloroplast retrograde signaling was also disrupted in spc1 mutant of Arabidopsis80. Taken together, these results indicated that mutation in BM-37 was associated with chloroplast development and photosynthetic efficiency.

The BM-37 mutant plants at seedling stage lead to substantial reduction of chlorophyll and carotenoids contents resulted in impaired chloroplast development that could accumulate higher ROS. Carotenoids provide protection against photo oxidative damage by detoxification of excessive free radicals and reactive oxygen species (ROS)81. For instance, impaired chloroplast biogenesis in leaf variegated rice zebra225 as well as mutation in ACD2 encoding red chlorophyll catabolite reductase82 resulted in excessive accumulation of ROS. Plant produce anti oxidative enzymes to detoxify ROS such as SOD to cope with superoxides while CAT, POD and APX to scavenge H2O2. Moreover, it has been reported that non-enzymatic antioxidants can be oxidized to monodehydroascorbate under higher ROS level83. Accordingly, our results showed lower contents of non-enzymatic antioxidants (Fig. 8F, H) in BM-37 mutant plants. Hence, the results inferred that malfunctioning of ROS scavenging enzymes and accumulation of H2O2 and MDA due to impaired chloroplast development has introduced the yellow leaf color in the BM-37 mutant at seedling stage.

Under various abiotic stresses, the reactive oxygen species (ROS) and lipid peroxidation produced in crop plants which caused damages in cells, and lead to the death of crop plants. The reduction in growth results from cellular and subcellular abnormalities in plant cells84,85,86. Each crop plant cell has the ability to produce antioxidants (SOD, POD, CAT and APX) to induce the self-defense mechanism in cell, especially for chloroplast and mitochondria87,88. The reactive oxygen species (H2O2) mostly affects the membranes of chloroplast and mitochondria, which ultimately caused death of cell. The production of peroxidase (POD), catalase (CAT), guaiacol peroxidase (GPOD), hydrogen peroxide (H2O2), superoxide dismutase (SOD), lipoxygenase (LOX), catalase (CAT), ascorbate peroxidase (APOD) and glutathion-S-transferase (GST), glutathione, proline, ascorbic acid, etc. are bioorganic compounds produced in cells89,90. The GTP proteins provide an environment for these antioxidants to work under various conditions. The GTP proteins are localized in the cells where there is a need to combat stress conditions. Exploration of various studies on plant hormonal regulation and G-protein signaling reveals several intriguing similarities in their functional roles. In addition, Arabidopsis G-protein mutants display altered phenotypes in response to different plant hormones91,92, and transcriptome data reveals a significant change in the expression of G-protein genes under different hormonal treatments93.From these studies, it is concluded that transposon nDart1-0 is a useful system to develop mutants for functional gene analysis, and n1-0 SPI PCR is a powerful tool to analyze mutants developed by the insertion of transposon nDart1-0. The transcripts of GTP binding protein were observed in various tissues of plants, usually in the early stages of plants. Era-like GTP binding protein is a new member of an old family, and it is vital for chloroplast biosynthesis and plant survival.

These studies conclude that transposon nDart1-0 is a useful system for developinga useful system for developing mutants for gene functional analysis and n1-0 SPI PCR is a powerful tool to analyze mutants developed by the insertion of transposon nDart1-0. The transcripts of GTP binding protein were observed in various plant tissues, usually in the early stages of plants. Era-like GTP binding protein is a new member of an old family, and it vital for chloroplast biosynthesis and plant survival. The protocol described may be helpful as a potential method for identifying transposable elements from other organisms, including plants like maize. However, it might have been hoped that this technique described not only the fast method for detecting the nDart1-0 but also its utilization in gene tagging for various genes in the rice genome.

All of the data generated or analyzed has been provided in the manuscript and supplementary files.

Maurya, N., Singh, O. P., Singh, S. N., Gautam, P. & Kumar, A. Manuscript effect of temperature on morpho-physiological traits with respect of grain yield of basmati rice. J. Pharmaco. Phytochem. 7(5), 412–416 (2018).

Google Scholar

Yin, Y. et al. Distinctive structure and assembly of phyllosphere microbial communities between wild and cultivated rice. Microbiol. Spectrum. 2023, e04371-22 (2023).

Article Google Scholar

Wambugu, P. W., Ndjiondjop, M.-N. & Henry, R. Advances in molecular genetics and genomics of African rice (Oryza glaberrima Steud). Plants 8, 376 (2019).

Article CAS PubMed PubMed Central Google Scholar

Shaibu, A. A. et al. Screening African rice (Oryza glaberrima) for tolerance to abiotic stresses: II. Lowland drought. Crop Sci. 58(1), 133–142 (2018).

Article Google Scholar

Brasileiro, A. C. et al. Ectopic expression of an expansin-like B gene from wild Arachis enhances tolerance to both abiotic and biotic stresses. The Plant J. 107(6), 1681–1696 (2021).

Article CAS PubMed Google Scholar

Srivastava, S., Pathare, V. S., Sounderajan, S. & Suprasanna, F. Nitrogen supply influences arsenic accumulation and stress responses of rice (Oryza sativa L.) seedlings. J. Hazard. Mater. 367, 599–606 (2019).

Article CAS PubMed Google Scholar

International Rice Genome Sequencing Project. The map-based sequence of the rice genome. Nature 436, 793–800 (2005).

Article Google Scholar

Maekawa, M., Tsugane, K. & Iida, S. Effective contribution of the nDart1 transposon-tagging system to rice functional genomics in Advances in Genetics Research (ed. Urbano, K.V.). Nova Sci. 2011, 259–272 (2011).

Google Scholar

Tsugane, K. et al. An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice. Plant J. 45(1), 46–57 (2006).

Article CAS PubMed Google Scholar

Shimatani, Z. et al. Characterization of autonomous Dart1 transposons belonging to the hAT superfamily in rice. Mol. Genet. Genom. 281(3), 329–344 (2009).

Article CAS Google Scholar

Johzuka-Hisatomi, Y. et al. Homologous recombination-dependent gene targeting and an active DNA transposon nDart-promoted gene tagging for rice functional genomics. In Rice Biology in the Genomics Era 81–94 (Springer, 2008).

Ramakrishnan, M. et al. Transposable elements in plants: Recent advancements, tools and prospects. Plant Mol. Biol. Rep. 40(4), 628–645 (2022).

Article CAS Google Scholar

Fang, J. et al. A transposon insertion in the OsUBC12 promoter enhances cold tolerance during germination in japonica rice (Oryza sativa). https://doi.org/10.21203/rs.3.rs-2541033/v1 (2023).

Takagi, K., Maekawa, M., Tsugane, K. & Iida, S. Transposition and target preferences of an active nonautonomous DNA transposon nDart1 and its relatives belonging to the hAT superfamily in rice. Mol. Genet. Genom. 284, 343–355 (2010).

Article CAS Google Scholar

Takagi, K., Ishikawa, N., Maekawa, M., Tsugane, K. & Iida, S. Transposon display for active DNA transposons in rice. Genes Genet. Syst. 82, 109–122 (2007).

Article CAS PubMed Google Scholar

Hayashi-Tsugane, M. et al. A mutable albino allele in rice reveals that formation of thylakoid membranes requires the snow-white leaf1 gene. Plant Cell Physiol. 55(1), 3–15 (2014).

Article CAS PubMed Google Scholar

Zhao, Q. et al. Pan-genome analysis highlights the extent of genomic variation in cultivated and wild rice. Nat. Genet. 50(2), 278–284 (2018).

Article CAS PubMed Google Scholar

Qiao, L. et al. A circuit for secretion-coupled cellular autonomy in multicellular eukaryotic cells. Mol. Syst. Biol. 2023, e11127 (2023).

Article Google Scholar

Jimah, J. R. & Hinshaw, J. E. Structural insights into the mechanism of dynamin superfamily proteins. Trend Cell Biol. 29(3), 257–273 (2019).

Article CAS Google Scholar

Meena, L. S. Cloning and characterization of engA, a GTP-binding protein from Mycobacterium tuberculosis H37Rv. Biologicals 39(2), 94–99 (2011).

Article CAS PubMed Google Scholar

Vernoud, V., Horton, A. C., Yang, Z. & Nielsen, E. Analysis of the small GTPase gene superfamily of Arabidopsis. Plant Physiol. 131(3), 1191–1208 (2003).

Article CAS PubMed PubMed Central Google Scholar

Shan, S. O. ATPase and GTPase tangos drive intracellular protein transport. Trends Biochem. Sci. 41(12), 1050–1060 (2016).

Article CAS PubMed PubMed Central Google Scholar

Kotyada, C., Chandra, M., Tripathi, A., Narooka, A. R. & Verma, A. Atypical Switch-I Arginine plays a catalytic role in GTP hydrolysis by Rab21 from Entamoeba histolytica. Biochem. Biophys. Res. Commun. 506(3), 660–667 (2018).

Article CAS PubMed Google Scholar

Doyle, J. J. Isolation of plant DNA from fresh tissue. Focus 12, 13–15 (1990).

Google Scholar

Jahan, P. et al. mRNA extraction, cDNA synthesis and tillering specific gene isolation from BLB resistant Binashail rice. Asian J. Med. Biol. Res. 1(2), 265–270 (2015).

Article Google Scholar

Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25(4), 402–408 (2001).

Article CAS PubMed Google Scholar

Dellaporta, S. L., Wood, J. & Hicks, J. B. A plant DNA minipreparation: Version II. Plant Mol. Biol. Rep. 1(4), 19–21 (1983).

Article CAS Google Scholar

Arnon, D. I. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24(1), 1 (1949).

Article CAS PubMed PubMed Central Google Scholar

Wellburn, A. R. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 144(3), 307–313 (1994).

Article CAS Google Scholar

Singleton, V. L. & Rossi, J. A. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–158 (1965).

Article CAS Google Scholar

Zhishen, J., Mengcheng, T. & Jianming, W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64(4), 555–559 (1999).

Article CAS Google Scholar

Zhang, W. F. et al. Effects of 5-aminolevulinic acid on oilseed rape seedling growth under herbicide toxicity stress. J. Plant Growth Regul. 27, 159–169 (2008).

Article Google Scholar

Zhou, W. & Leul, M. Uniconazole-induced tolerance of rape plants to heat stress in relation to changes in hormonal levels, enzyme activities and lipid peroxidation. Plant Growth Regul. 27, 99–104 (1999).

Article CAS Google Scholar

Aebi, H. Catalase in vitro. Meth. Enzymol. 105, 121–126 (1984).

Article CAS Google Scholar

Nakano, Y. & Asada, K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880 (1981).

CAS Google Scholar

Velikova, V., Yordanov, I. & Edreva, A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: Protective role of exogenous polyamines. Plant Sci. 151, 59–66 (2000).

Article CAS Google Scholar

Morales, M. & Munné-Bosch, S. Malondialdehyde: Facts and artifacts. Plant Physiol. 180, 1246–1250 (2019).

Article CAS PubMed PubMed Central Google Scholar

Zhishen, J., Mengcheng, T. & Jianming, W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64, 555–559 (1999).

Article CAS Google Scholar

Wu, Z. et al. A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis. Plant Physiol. 145(1), 29–40 (2007).

Article CAS PubMed PubMed Central Google Scholar

Kyozuka, J. et al. Light-regulated and cell-specific expression of tomato rbcS-gusA and rice rbcS-gusA fusion genes in transgenic rice. Plant Physiol. 102(3), 991–1000 (1993).

Article CAS PubMed PubMed Central Google Scholar

Kusumi, K. et al. A plastid protein NUS1 is essential for build-up of the genetic system for early chloroplast development under cold stress conditions. Plant J. 68(6), 1039–1050 (2011).

Article CAS PubMed Google Scholar

Takeuchi, R., Kimura, S., Saotome, A. & Sakaguchi, K. Biochemical properties of a plastidial DNA polymerase of rice. Plant Mol. Biol. 64(5), 601–611 (2007).

Article CAS PubMed Google Scholar

Vitha, S., McAndrew, R. S. & Osteryoung, K. W. FtsZ ring formation at the chloroplast division site in plants. J. Cell Biol. 153(1), 111–120 (2001).

Article CAS PubMed PubMed Central Google Scholar

Yoo, S. C. et al. Rice virescent3 and stripe1 encoding the large and small subunits of ribonucleotide reductase are required for chloroplast biogenesis during early leaf development. Plant Physiol. 150(1), 388–401 (2009).

Article CAS PubMed PubMed Central Google Scholar

Gong, X. et al. Disruption of the rice plastid ribosomal protein S20 leads to chloroplast developmental defects and seedling lethality. G3 Genes Genom. Genet. 3(10), 1769–1777 (2013).

Google Scholar

Hiratsuka, J. et al. The complete sequence of the rice (Oryza sativa) chloroplast genome: Intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol. Gen. Genet. 217(2), 185–194 (1989).

Article CAS PubMed Google Scholar

Hirochika, H. Contribution of the Tos17 retrotransposon to rice functional genomics. Curr. Opin. Plant Biol. 4(2), 118–122 (2001).

Article CAS PubMed Google Scholar

Hirochika, H. et al. Rice mutant resources for gene discovery. Plant Mol. Biol. 54(3), 325–334 (2004).

Article CAS PubMed Google Scholar

Kikuchi, K., Terauchi, K., Wada, M. & Hirano, H. Y. The plant MITE mPing is mobilized in anther culture. Nature 421(6919), 167–170 (2003).

Article ADS CAS PubMed Google Scholar

Liu, D., Zhang, S., Fauquet, C. & Crawford, N. M. The Arabidopsis transposon Tag1 is active in rice, undergoing germinal transposition and restricted, late somatic excision. Mol. Gen. Genet. 262(3), 413–420 (1999).

Article CAS PubMed Google Scholar

Goff, S. A. Rice as a model for cereal genomics. Curr. Opin. Plant Biol. 2(2), 86–89 (1999).

Article CAS PubMed Google Scholar

Jiang, N. et al. An active DNA transposon family in rice. Nature 421(6919), 163–167 (2003).

Article ADS CAS PubMed Google Scholar

Nakazaki, T. et al. Mobilization of a transposon in the rice genome. Nature 421(6919), 170–172 (2003).

Article ADS CAS PubMed Google Scholar

Kumar, A. & Hirochika, H. Applications of retrotransposons as genetic tools in plant biology. Trend. Plant Sci. 6(3), 127–134 (2001).

Article CAS Google Scholar

Koonin, E. & Galperin, M. Y. Sequence-Evolution-Function: Computational Approaches in Comparative Genomics (Springer, 2002).

Google Scholar

Komatsu, M., Shimamoto, K. & Kyozuka, J. Two-step regulation and continuous retrotransposition of the rice LINE-type retrotransposon Karma. Plant Cell 15(8), 1934–1944 (2003).

Article CAS PubMed PubMed Central Google Scholar

Huang, Y., Shukla, H. & Lee, Y. C. G. Species-specific chromatin landscape determines how transposable elements shape genome evolution. Elife 11, e81567 (2022).

Article CAS PubMed PubMed Central Google Scholar

Khush, G. S. Origin, dispersal, cultivation and variation of rice. Plant Mol. Biol. 35(1), 25–34 (1997).

Article CAS PubMed Google Scholar

Lin, X. et al. In planta mobilization of mPing and its putative autonomous element Pong in rice by hydrostatic pressurization. J. Exp. Bot. 57(10), 2313–2323 (2006).

Article CAS PubMed Google Scholar

Walbot, V. Saturation mutagenesis using maize transposons. Curr. Opin. Plant Biol. 3(2), 103–107 (2000).

Article CAS PubMed Google Scholar

Harris, S. B. Virtual rice. EMBO Rep. 3(6), 511–513 (2002).

Article CAS PubMed PubMed Central Google Scholar

Hamer, L., DeZwaan, T. M., Montenegro-Chamorro, M. V., Frank, S. A. & Hamer, J. E. Recent advances in large-scale transposon mutagenesis. Curr. Opin. Chem. Biol. 5(1), 67–73 (2001).

Article CAS PubMed Google Scholar

Kempin, S. A. et al. Targeted disruption in Arabidopsis. Nature 389(6653), 802–803 (1997).

Article ADS CAS PubMed Google Scholar

Goff, S. A. et al. A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296(5565), 92–100 (2002).

Article ADS CAS PubMed Google Scholar

Gale, M. D. & Devos, K. M. Comparative genetics in the grasses. Proc. Natl. Acad. Sci. 95(5), 1971–1974 (1998).

Article ADS CAS PubMed PubMed Central Google Scholar

Yang, S., Overlander, M. & Fiedler, J. Genetic analysis of the barley variegation mutant, grandpa1. BMC Plant Biol. 21(1), 1–11 (2021).

Article Google Scholar

Miyao, A. et al. Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell 15(8), 1771–1780 (2003).

Article PubMed PubMed Central Google Scholar

Takagi, K., Ishikawa, N., Maekawa, M., Tsugane, K. & Iida, S. Transposon display for active DNA transposons in rice. Gene. Genet. Syst. 82(2), 109–122 (2007).

Article CAS Google Scholar

May, B. P. & Martienssen, R. A. Transposon mutagenesis in the study of plant development. Crit. Rev. Plant Sci. 22(1), 1–35 (2003).

Article CAS Google Scholar

Sato, Y. et al. Loss-of-function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants. EMBO J. 18(4), 992–1002 (1999).

Article CAS PubMed PubMed Central Google Scholar

Sasaki, T. & Burr, B. International Rice Genome Sequencing Project: The effort to completely sequence the rice genome. Curr. Opin. Plant Biol. 3(2), 138–142 (2000).

Article CAS PubMed Google Scholar

Murai, N., Li, Z., Kawagoe, Y. & Hayashimoto, A. Transposition of the maize activator element in transgenic rice plants. Nucleic Acids Res. 19(3), 617–622 (1991).

Article CAS PubMed PubMed Central Google Scholar

Takano, M. et al. Isolation and characterization of rice phytochrome A mutants. Plant Cell 13(3), 521–534 (2001).

Article CAS PubMed PubMed Central Google Scholar

Agrawal, G. K. et al. Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion. Tagging of a zeaxanthin epoxidase gene and a novel OsTATC gene. Plant Physiol. 125(3), 1248–1257 (2001).

Article CAS PubMed PubMed Central Google Scholar

Tyagi, A. K. & Mohanty, A. Rice transformation for crop improvement and functional genomics. Plant Sci. 158(1–2), 1–18 (2000).

Article CAS PubMed Google Scholar

Yu, J. et al. A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296(5565), 79–92 (2002).

Article ADS CAS PubMed Google Scholar

Bédard, J. et al. Suppressors of the chloroplast protein import mutant reveal a genetic link between protein import and thylakoid biogenesis. Plant Cell 29, 1726 (2017).

Article PubMed PubMed Central Google Scholar

Deng, L. et al. Characterization and fine-mapping of a novel premature leaf senescence mutant yellow leaf and dwarf 1 in rice. Plant Physiol. Biochem. 111, 50 (2017).

Article CAS PubMed Google Scholar

Gang, H. et al. Loss of GLK1 transcription factor function reveals new insights in chlorophyll biosynthesis and chloroplast development. J. Exp. Bot. 70, 3125 (2019).

Article CAS PubMed Google Scholar

Dong, H. et al. The Arabidopsis spontaneous cell death1 gene, encoding a ζ-carotene desaturase essential for carotenoid biosynthesis, is involved in chloroplast development, photoprotection and retrograde signaling. Cell Res. 17, 458 (2007).

Article CAS PubMed Google Scholar

Howitt, C. A. & Pogson, B. J. Carotenoid accumulation and function in seeds and non-green tissues. Plant Cell Environ. 29, 435 (2006).

Article CAS PubMed Google Scholar

Yao, N. & Greenberg, J. T. Arabidopsis accelerated cell death2 modulates programmed cell death. Plant Cell 18, 397 (2006).

Article CAS PubMed PubMed Central Google Scholar

Apel, K. & Hirt, H. Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373 (2004).

Article CAS PubMed Google Scholar

Jan, S. et al. 24-Epibrassinolide alleviates the injurious effects of Cr (VI) toxicity in tomato plants: Insights into growth, physio-biochemical attributes, antioxidant activity and regulation of Ascorbate–glutathione and Glyoxalase cycles. J. Plant. Growth Regul. 39, 1587–1604 (2020).

Article CAS Google Scholar

Ahmad, R. et al. Hydrogen sulfide alleviates chromium stress on cauliflower by restricting its uptake and enhancing antioxidative system. Physiol. Plant 168, 289–300 (2020).

CAS PubMed Google Scholar

Lei, K. et al. Seed soaking with melatonin promotes seed germination under chromium stress via enhancing reserve mobilization and antioxidant metabolism in wheat. Ecotoxicol. Environ. Saf. 220, 112241 (2021).

Article CAS PubMed Google Scholar

Wakeel, A., Xu, M. & Gan, Y. Chromium-induced reactive oxygen species accumulation by altering the enzymatic antioxidant system and associated cytotoxic, genotoxic, ultrastructural, and photosynthetic changes in plants. Int. J. Mol. Sci. 21, 728 (2020).

Article CAS PubMed PubMed Central Google Scholar

Fan, W.-J., Feng, Y.-X., Li, Y.-H., Lin, Y.-J. & Yu, X.-Z. Unraveling genes promoting ROS metabolism in subcellular organelles of Oryza sativa in response to trivalent and hexavalent chromium. Sci. Total Environ. 744, 140951 (2020).

Article ADS CAS PubMed Google Scholar

Wang, M., Zhang, S. & Ding, F. Melatonin mitigates chilling-induced oxidative stress and photosynthesis inhibition in tomato plants. Antioxidants 9, 218 (2020).

Article PubMed PubMed Central Google Scholar

Dreyer, A. & Dietz, K.-J. Reactive oxygen species and the redox-regulatory network in cold stress acclimation. Antioxidants. 7, 169 (2018).

Article PubMed PubMed Central Google Scholar

Xu, D. B. et al. A G-protein β subunit, AGB1, negatively regulates the ABA response and drought tolerance by down-regulating AtMPK6-related pathway in Arabidopsis. PLoS ONE 10(1), e0116385 (2015).

Article PubMed PubMed Central Google Scholar

Roy Choudhury, S. et al. Flexible functional interactions between G-protein subunits contribute to the specificity of plant responses. Plant J. 102(2), 207–221 (2020).

Article CAS PubMed Google Scholar

Jose, J. & Choudhury, S. R. Heterotrimeric G-proteins mediated hormonal responses in plants. Cell. Signal. 76, 109799 (2020).

Article CAS PubMed Google Scholar

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This work was supported by the Science and Technology Office of Zhejiang Province, China (project no. 2021C02063-6) and supported by the DOE Office of Science, Office of Biological and Environmental Research (BER), United States, grant Nos. DE-SC0006634 and DE-SC0012379

Department of Agronomy, Zhejiang University, Hangzhou, 310058, Zhejiang, China

Sanaullah Jalil, Asad Ullah Khan, Muhammad Mudassir Nazir, Sharafat Ali & Xiaoli Jin

Crop Sciences Institute, National Agricultural Research Center, Islamabad, 44000, Pakistan

Sanaullah Jalil

Department of Plant Breeding and Genetics, Faculty of Agricultural Sciences, University of the Punjab, Lahore, 54590, Pakistan

Qurban Ali & Muhammad Arshad Javed

Department of Horticultural Sciences, Faculty of Agriculture and Environment, The Islamia University of Bahawalpur, Bahawalpur, 63100, Pakistan

Faisal Zulfiqar

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S.J., conceptualization, investigation, writing original draft; Q.A., conceptualization, investigation; A.U.K., F.Z., S.A., M.A.J., and M.M.N., reviewing, editing. Formal analysis; X.J., supervising, writing, reviewing and editing. All authors reviewed the manuscript.

Correspondence to Xiaoli Jin.

The authors declare no competing interests.

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Jalil, S., Ali, Q., Khan, A.U. et al. Molecular and biochemical characterization of rice developed through conventional integration of nDart1-0 transposon gene. Sci Rep 13, 8139 (2023). https://doi.org/10.1038/s41598-023-35095-7

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Received: 21 July 2022

Accepted: 12 May 2023

Published: 19 May 2023

DOI: https://doi.org/10.1038/s41598-023-35095-7

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