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Simple Summary: CRM domain proteins play key roles in plant growth and chloroplast development. To explore the role of CRM domain protein in the chloroplast development of rice, we cloned a rice CRM domain protein gene, YL4, and then conducted a tissue-specific expression analysis, subcellular localization, and transcription analysis. The experimental results showed that YL4 was highly expressed in leaves and localized to the chloroplast, and its mutation affected chloroplast-related gene transcription levels and some agronomic traits. These results indicate that the YL4 gene is a pivotal regulatory factor in chloroplast development and rice growth. Chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are a family of plant-specific proteins associated with RNA binding. In this study, we have conducted a detailed characterization of a novel rice CRM gene (LOC_Os04g39060) mutant, yl4, which showed yellow-green leaves at all the stages, had fewer tillers, and had a decreased plant height. Map-based cloning and CRISPR/Cas9 editing techniques all showed that YL4 encoded a CRM domain protein in rice. In addition, subcellular localization revealed that YL4 was in chloroplasts. YL4 transcripts were highly expressed in all leaves and undetectable in roots and stems, and the mutation of YL4 affected the transcription of chloroplast-development-related genes. This study indicated that YL4 is essential for chloroplast development and affects some agronomic traits. [ABSTRACT FROM AUTHOR]

Our study investigates the genetic mechanisms underlying the spotted leaf phenotype in rice, focusing on the spl43 mutant. This mutant is characterized by persistent reddish-brown leaf spots from the seedling stage to maturity, leading to extensive leaf necrosis. Using map-based cloning, we localized the responsible locus to a 330 Kb region on chromosome 2. We identified LOC_Os02g56000, named OsRPT5A, as the causative gene. A point mutation in OsRPT5A, substituting valine for glutamic acid, was identified as the critical factor for the phenotype. Functional complementation and the generation of CRISPR/Cas9-mediated knockout lines in the IR64 background confirmed the central role of OsRPT5A in controlling this trait. The qPCR results from different parts of the rice plant revealed that OsRPT5A is constitutively expressed across various tissues, with its subcellular localization unaffected by the mutation. Notably, we observed an abnormal accumulation of reactive oxygen species (ROS) in spl43 mutants by examining the physiological indexes of leaves, suggesting a disruption in the ROS system. Complementation studies indicated OsRPT5A's involvement in ROS homeostasis and catalase activity regulation. Moreover, the spl43 mutant exhibited enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo), highlighting OsRPT5A's role in rice pathogen resistance mechanisms. Overall, our results suggest that OsRPT5A plays a critical role in regulating ROS homeostasis and enhancing pathogen resistance in rice. [ABSTRACT FROM AUTHOR]

Panicle type is one of the important factors affecting rice (Oryza sativa L.) yield, and the identification of regulatory genes in panicle development can provide significant insights into the molecular network involved. This study identified a large and dense panicle 1 (ldp1) mutant produced from the Wuyunjing 7 (WYJ7) genotype, which displayed significant relative increases in panicle length, number of primary and secondary branches, number of grains per panicle, grain width, and grain yield per plant. Scanning electron microscopy results showed that the shoot apical meristem (SAM) of ldp1 was relatively larger at the bract stage (BM), with a significantly increased number of primary (PBM) and secondary branch (SBM) meristematic centers, indicating that the ldp1 mutation affects early stages in SAM development Comparative RNA-Seq analysis of meristem tissues from WYJ7 and ldp1 at the BM, PBM, and SBM developmental stages indicated that the number of differentially expressed genes (DEGs) were highest (1407) during the BM stage. Weighted gene coexpression network analysis (WGCNA) revealed that genes in one module (turquoise) are associated with the ldp1 phenotype and highly expressed during the BM stage, suggesting their roles in the identity transition and branch differentiation stages of rice inflorescences. Hub genes involved in auxin synthesis and transport pathways, such as OsAUX1, OsAUX4, and OsSAUR25, were identified. Moreover, GO and KEGG analysis of the DEGs in the turquoise module and the 1407 DEGs in the BM stage revealed that a majority of genes involved in tryptophan metabolism and auxin signaling pathway were differentially expressed between WYJ and ldp1. The genetic analysis indicated that the ldp1 phenotype is controlled by a recessive monogene (LDP1), which was mapped to a region between 16.9 and 18.1 Mb on chromosome seven. This study suggests that the ldp1 mutation may affect the expression of key genes in auxin synthesis and signal transduction, enhance the size of SAM, and thus affect panicle development. This study provides insights into the molecular regulatory network underlying rice panicle morphogenesis and lays an important foundation for further understanding the function and molecular mechanism of LDP1 during panicle development. [ABSTRACT FROM AUTHOR]

Plant architecture, one of the key factors that determine grain yield in rice, is mainly affected by components such as plant height, tiller number, and panicle morphology. For this paper, we obtained a multi-tillering and lazy mutant from a japonica rice cultivar, Wuyunjing 7 (WYJ7), via treatment with a heavy ion beam. Compared to WYJ7, the mutant showed a significant increase in tiller angle, tiller number, number of primary and secondary branches, and number of grains; however, the plant height and grain thickness of the mutant was significantly decreased. Phenotypic analysis of the F1 hybrids revealed that the multi-tillering and lazy mutant phenotypes were regulated by a recessive gene. The segregation ratio of 1׃3 of the mutant phenotype and the wild-type plant in the F2 population indicated that the former was controlled by a single gene named Multi-Tillering and Lazy 1 (MTL1). Bulked segregant analysis was performed using the individual plants with extremely typical tiller angles in the F2 population. The MTL1 gene was initially mapped within a region of 5.58–17.64 Mb on chromosome 11. By using the F2 segregated population for fine mapping, the MTL1 gene was ultimately fine mapped within the range of 66.67 kb on chromosome 11. The analysis of genes in this region revealed the presence of the previously identified LAZY1 (LA1) gene. Genomic PCR amplification and semi-quantitative RT-PCR assays showed that the LA1 gene could not be amplified and was not expressed, thus indicating that the MTL1 gene might be identical to the LA1 gene. This study suggests that the multi-tillering and lazy mutant phenotypes might be caused by the deletion of LA1 function. This finding can guide further investigations on the functional mechanisms of the LA1 gene, thus enriching the theoretical knowledge of plant architecture in relation to rice. [ABSTRACT FROM AUTHOR]

In pumpkin (Cucurbita moschata), the naked or hull-less seed phenotype has great benefits for breeding this crop for oil or snack use. We previously identified a naked seed mutant in this crop. In this study, we report genetic mapping, identification, and characterization of a candidate gene for this mutation. We showed that the naked seed phenotype is controlled by a single recessive gene (N). The bulked segregant analysis identified a 2.4 Mb region on Chromosome 17 with 15 predicted genes. Multiple lines of evidence suggested that CmoCh17G004790 is the most probable candidate gene for the N locus which encodes a NAC transcription factor WALL THICKENING PROMOTING FACTOR 1 (CmNST1). No nucleotide polymorphism or structural variation was found in the genomic DNA sequences of CmNST1 between the mutant and the wildtype inbred line (hulled seed). However, the cDNA sequence cloned from developing seed coat samples of the naked seed mutant was 112 bp shorter than that from the wildtype which is due to seed coat-specific alternative splicing in the second exon of the mutant CmNST1 transcript. The expression level of CmNST1 in the developing seed coat was higher in the mutant than in the wildtype during early seed coat development which was reversed later. Transcriptomic profiling with RNA-Seq at different stages of seed development in the mutant and wildtype revealed a critical role of CmNST1 as a master regulator for the lignin biosynthesis pathway during seed coat development while other NAC and MYB transcription factors were also involved in forming a regulatory network for the building of secondary cell walls. This work provides a novel mechanism for the well-characterized NST1 transcription factor gene in regulating secondary cell wall development. The cloned gene also provides a useful tool for marker-assisted breeding of hull-less C. moschata varieties. [ABSTRACT FROM AUTHOR]

Chloroplasts are essential sites for plant photosynthesis, and the biogenesis of the photosynthetic complexes involves the interaction of nuclear genes and chloroplast genes. In this study, we identified a rice pale green leaf mutant, crs2. The crs2 mutant showed different degrees of low chlorophyll phenotypes at different growth stages, especially at the seedling stage. Fine mapping and DNA sequencing of crs2 revealed a single nucleotide substitution (G4120A) in the eighth exons of CRS2, causing a G-to-R mutation of the 229th amino acid of CRS2 (G229R). The results of complementation experiments confirmed that this single-base mutation in crs2 is responsible for the phenotype of the crs2 mutant. CRS2 encodes a chloroplast RNA splicing 2 protein localized in the chloroplast. Western blot results revealed an abnormality in the abundance of the photosynthesis-related protein in crs2. However, the mutation of CRS2 leads to the enhancement of antioxidant enzyme activity, which could reduce ROS levels. Meanwhile, with the release of Rubisco activity, the photosynthetic performance of crs2 was improved. In summary, the G229R mutation in CRS2 causes chloroplast protein abnormalities and affects photosystem performance in rice; the above findings facilitate the elucidation of the physiological mechanism of chloroplast proteins affecting photosynthesis. [ABSTRACT FROM AUTHOR]

In this study, we identified a novel glossy mutant from Chinese cabbage, named SD369, and all wax monomers longer than 26 carbons were significantly decreased. Inheritance analysis revealed that the glossy trait of SD369 was controlled by a single recessive locus, BrWAX3. We fine-mapped the BrWAX3 locus to an interval of 161.82 kb on chromosome A09. According to the annotated genome of Brassica rapa, Bra024749 (BrCER60.A09), encoding a β-ketoacyl-CoA synthase, was identified as the candidate gene. Expression analysis showed that BrCER60.A09 was significantly downregulated in all aerial organs of glossy plants. Subcellular localization indicated that the BrCER60.A09 protein functions in the endoplasmic reticulum. A 5567-bp insertion was identified in exon 1 of BrCER60.A09 in SD369, which lead to a premature stop codon, thus causing a loss of function of the BrCER60.A09 enzyme. Moreover, comparative transcriptome analysis revealed that the 'cutin, suberine, and wax biosynthesis' pathway was significantly enriched, and genes involved in this pathway were almost upregulated in glossy plants. Further, two functional markers, BrWAX3-InDel and BrWAX3-KASP1, were developed and validated. Overall, these results provide a new information for the cuticular wax biosynthesis and provide applicable markers for marker-assisted selection (MAS)-based breeding of Brassica rapa. [ABSTRACT FROM AUTHOR]

Leaf color mutants are common in higher plants that can be used as markers in crop breeding or as an important tool in understanding regulatory mechanisms in chlorophyll biosynthesis and chloroplast development. In virescent leaf mutants, young leaves are yellow in color, which gradually return to normal green when the seedlings grow large. In the present study, we conducted phenotypic characterization and genetic mapping of the cucumber virescent leaf mutant 9110Gt conferred by the v-1 locus. Total chlorophyll and carotenoid content in 9110Gt was reduced by 44% and 21%, respectively, as compared with its wild type parental line 9110G. Electron microscopic investigation revealed fewer chloroplasts per cell and thylakoids per chloroplast in 9110Gt than in 9110G. Fine genetic mapping allowed for the assignment of the v-1 locus to a 50.4 kb genomic DNA region in chromosome 6 with two flanking markers that were 0.14 and 0.16 cM away from v-1, respectively. Multiple lines of evidence supported CsaCNGCs as the only candidate gene for the v-1 locus, which encoded a cyclic-nucleotide-gated ion channel protein. A single nucleotide change in the promoter region of v-1 seemed to be associated with the virescent color change in 9110Gt. Real-time PCR revealed significantly lower expression of CsaCNGCs in the true leaves of 9110Gt than in 9110G. This was the first report that connected the CsaCNGCs gene to virescent leaf color change, which provided a useful tool to establish linkages among virescent leaf color change, chloroplast development, chlorophyll biosynthesis, and the functions of the CsaCNGCs gene. [ABSTRACT FROM AUTHOR]

Abscisic acid plays a pivotal role in the abiotic stress response in plants. Although great progress has been achieved explaining the complexity of the stress and ABA signaling cascade, there are still many questions to answer. Mutants are a valuable tool in the identification of new genes or new alleles of already known genes and in elucidating their role in signaling pathways. We applied a suppressor mutation approach in order to find new components of ABA and abiotic stress signaling in Arabidopsis. Using the abh1 (ABA hypersensitive 1) insertional mutant as a parental line for EMS mutagenesis, we selected several mutants with suppressed hypersensitivity to ABA during seed germination. Here, we present the response to ABA and a wide range of abiotic stresses during the seed germination and young seedling development of two suppressor mutants-- soa2 (suppressor of abh1 hypersensitivity to ABA 2) and soa3 (suppressor of abh1 hypersensitivity to ABA 3). Generally, both mutants displayed a suppression of the hypersensitivity of abh1 to ABA, NaCl and mannitol during germination. Both mutants showed a higher level of tolerance than Columbia-0 (Col-0--the parental line of abh1) in high concentrations of glucose. Additionally, soa2 exhibited better root growth than Col-0 in the presence of high ABA concentrations. soa2 and soa3 were drought tolerant and both had about 50% fewer stomata per mm2 than the wild-type but the same number as their parental line--abh1. Taking into account that suppressor mutants had the same genetic background as their parental line--abh1, it was necessary to backcross abh1 with Landsberg erecta four times for the map-based cloning approach. Mapping populations, derived from the cross of abh1 in the Landsberg erecta background with each suppressor mutant, were created. Map based cloning in order to identify the suppressor genes is in progress. [ABSTRACT FROM AUTHOR]

The spikelet developmental processes that control structure and floral organ identity play critical roles in rice grain yield formation. In this study, we characterized a novel rice mutant, SLL1-ZH, which exhibits a variety of defective agronomic characters, including semi-dwarf, rolling leaf, deformed panicles, and reduced grains production. Morphological analysis also revealed that the SLL1-ZH mutant shows numerous defects of floral organs, such as cracked glumes, hooked and thin lemmas, shrunken but thickened paleas, an indeterminate number of stamens and stigmas, and heterotopic ovaries. Map-based cloning identified a single nucleotide substitution (C to G) in the first exon of LOC_Os09g23200 that is responsible for the SLL1-ZH phenotype. In addition, qPCR analysis showed a significant change in the relative expression of SLL1-ZH in the mutant during inflorescence differentiation and in the different floral organs. Transcription of rice floral organ development-related factors also changed significantly in the mutant. Therefore, our results suggested that SLL1-ZH plays a great role in plant growth, spikelet development, and grain yield in rice. [ABSTRACT FROM AUTHOR]

A forward genetic approach is a powerful tool for identifying the genes underlying the phenotypes of interest. However, the conventional map-based cloning method is lengthy, requires a large mapping population and confirmation of many candidate genes in a broad genetic region to clone the causal variant. The whole-genome sequencing method clones the variants with a certain failure probability for multiple reasons, especially for heterozygotes, and could not be used to clone the mutation of epigenetic modifications. Here, we applied the highly complementary characteristics of these two methods and developed a sequencing-based mapping method (SBM) for identifying the location of plant variants effectively with a small population and low cost, which is very user-friendly for most popular laboratories. This method used the whole-genome sequencing data of two pooled populations to screen out enough markers. These markers were used to identify and narrow the candidate region by analyzing the marker-indexes and recombinants. Finally, the possible mutational sites were identified using the whole-genome sequencing data and verified in individual mutants. To elaborate the new method, we displayed the cloned processes in one Arabidopsis heterozygous mutant and two rice homozygous mutants. Thus, the sequencing-based mapping method could clone effectively different types of plant mutations and was a powerful tool for studying the functions of plant genes in the species with known genomic sequences. [ABSTRACT FROM AUTHOR]

Seed coat color is an important agronomic trait of edible seed pumpkin in Cucurbita maxima. In this study, the development pattern of seed coat was detected in yellow and white seed coat accessions Wuminglv and Agol. Genetic analysis suggested that a single recessive gene white seed coat (wsc) is involved in seed coat color regulation in Cucurbita maxima. An F2 segregating population including 2798 plants was used for fine mapping and a candidate region containing nine genes was identified. Analysis of 54 inbred accessions revealed four main Insertion/Deletion sites in the promoter of CmaCh15G005270 encoding an MYB transcription factor were co-segregated with the phenotype of seed coat color. RNA-seq analysis and qRT-PCR revealed that some genes involved in phenylpropanoid/flavonoid metabolism pathway displayed remarkable distinction in Wuminglv and Agol during the seed coat development. The flanking InDel marker S1548 was developed to predict the seed coat color in the MAS breeding with an accuracy of 100%. The results may provide valuable information for further studies in seed coat color formation and structure development in Cucurbitaceae crops and help the molecular breeding of Cucurbita maxima. [ABSTRACT FROM AUTHOR]

Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape. [ABSTRACT FROM AUTHOR]

The leaf blade is the main photosynthetic organ and its morphology is related to light energy capture and conversion efficiency. We isolated a novel rice Dynamic Narrow-Rolled Leaf 1 (dnrl1) mutant showing reduced width of leaf blades, rolled leaves and lower chlorophyll content. The narrow-rolled leaf phenotype resulted from the reduced number of small longitudinal veins per leaf, smaller size and irregular arrangement of bulliform cells compared with the wild-type. DNRL1 was mapped to chromosome 7 and encoded a putative 3-deoxy-7-phosphoheptulonate synthase (DAHPS) which catalyzes the conversion of phosphoenolpyruvate and D-erythrose 4-phosphate to DAHP and phosphate. Sequence analysis revealed that a single base substitution (T–A) was detected in dnrl1, leading to a single amino acid change (L376H) in the coding protein. The mutation led to a lower expression level of DNRL1 as well as the lower activity of DAHPS in the mutant compared with the wild type. Genetic complementation and over-expression of DNRL1 could rescue the narrow-rolled phenotype. DNRL1 was constitutively expressed in all tested organs and exhibited different expression patterns from other narrow-rolled leaf genes. DNRL1-GFP located to chloroplasts. The lower level of chlorophyll in dnrl1 was associated with the downregulation of the genes responsible for chlorophyll biosynthesis and photosynthesis. Furthermore, dnrl1 showed significantly reduced levels of aromatic amino acids including Trp, Phe and Tyr. We conclude that OsDAHPS, encoded by DNRL1, plays a critical role in leaf morphogenesis by mediating the biosynthesis of amino acids in rice. [ABSTRACT FROM AUTHOR]

Short petiole is a valuable trait for the improvement of plant canopy of ideotypes with high yield. Here, we identified a soybean mutant line derived short petiole (dsp) with extremely short petiole in the field, which is obviously different from most short-petiole lines identified previously. Genetic analysis on 941 F2 individuals and subsequent segregation analysis of 184 F2:3 and 172 F3:4 families revealed that the dsp mutant was controlled by two recessive genes, named as dsp1 and dsp2. Map-based cloning showed that these two recessive genes were located on two nonhomologous regions of chromosome 07 and chromosome 11, of which the dsp1 locus was mapped at a physical interval of 550.5-Kb on chromosome 07 near to centromere with flanking markers as BARCSOYSSR_07_0787 and BARCSOYSSR_07_0808; whereas, the dsp2 locus was mapped to a 263.3-Kb region on chromosome 11 with BARCSOYSSR_11_0037 and BARCSOYSSR_11_0043 as flanking markers. A total of 36 and 33 gene models were located within the physical genomic interval of dsp1 and dsp2 loci, respectively. In conclusion, the present study identified markers linked with genomic regions responsible for short-petiole phenotype of soybean, which can be effectively used to develop ideal soybean cultivars through marker-assisted breeding. [ABSTRACT FROM AUTHOR]

Light is one of the most important environmental factors that affect many aspects of plant growth, including chlorophyll (Chl) synthesis and flowering time. Here, we identified a rice mutant, yellow leaf and early flowering (ye1), and characterized the gene YE1 by using a map-based cloning method. YE1 encodes a heme oxygenase, which is localized to the chloroplasts. YE1 is expressed in various green tissues, especially in leaves, with a diurnal-rhythmic expression pattern, and its transcripts is also induced by light during leaf-greening. The mutant displays decreased Chl contents with less and disorderly thylakoid lamellar layers in chloroplasts, which reduced the photosynthesis rate. The early flowering phenotype of ye1 was not photoperiod-sensitive. Furthermore, the expression levels of Chl biosynthetic genes were downregulated in ye1 seedlings during de-etiolation responses to light. We also found that rhythmic expression patterns of genes involved in photoperiodic flowering were altered in the mutant. Based on these results, we infer that YE1 plays an important role in light-dependent Chl biogenesis as well as photoperiodic flowering pathway in rice. [ABSTRACT FROM AUTHOR]

Leaf color mutants are common in higher plants that can be used as markers in crop breeding or as an important tool in understanding regulatory mechanisms in chlorophyll biosynthesis and chloroplast development. In virescent leaf mutants, young leaves are yellow in color, which gradually return to normal green when the seedlings grow large. In the present study, we conducted phenotypic characterization and genetic mapping of the cucumber virescent leaf mutant 9110Gt conferred by the v-1 locus. Total chlorophyll and carotenoid content in 9110Gt was reduced by 44% and 21%, respectively, as compared with its wild type parental line 9110G. Electron microscopic investigation revealed fewer chloroplasts per cell and thylakoids per chloroplast in 9110Gt than in 9110G. Fine genetic mapping allowed for the assignment of the v-1 locus to a 50.4 kb genomic DNA region in chromosome 6 with two flanking markers that were 0.14 and 0.16 cM away from v-1, respectively. Multiple lines of evidence supported CsaCNGCs as the only candidate gene for the v-1 locus, which encoded a cyclic-nucleotide-gated ion channel protein. A single nucleotide change in the promoter region of v-1 seemed to be associated with the virescent color change in 9110Gt. Real-time PCR revealed significantly lower expression of CsaCNGCs in the true leaves of 9110Gt than in 9110G. This was the first report that connected the CsaCNGCs gene to virescent leaf color change, which provided a useful tool to establish linkages among virescent leaf color change, chloroplast development, chlorophyll biosynthesis, and the functions of the CsaCNGCs gene., Competing Interests: The authors declare no conflict of interest.