BLA1 Affects Leaf Angles by Altering Brassinosteroid Biosynthesis in Rice (Oryza sativa L.).

Brassinosteroids (BRs) are crucial plant hormones influencing diverse developmental processes in rice. While several enzymes in BR biosynthesis have been identified, their regulatory mechanisms remain largely unknown. This study highlights a novel regulatory pathway wherein the CHD3 chromatin remodeler, BLA1, epigenetically modulates the expression of key BR biosynthesis genes, BRD1 and D2. Phenotypic analysis of bla1 mutants revealed significant alterations, such as increased leaf angles and longer mesocotyls, which were alleviated by BR synthesis inhibitors. Moreover, the bla1 mutants showed elevated BR levels that correlated with the significant upregulation of the expression levels of BRD1 and D2, particularly at the lamina joint sites. Mechanistically, the yeast one-hybrid and chromatin immunoprecipitation assays revealed specific binding of BLA1 to the promoter regions of BRD1 and D2, accompanied by a marked enrichment of the transcriptionally active histone modification, H3K4me3, on these loci in the bla1 mutant. Functional assessments of the brd1 and d2 mutants confirmed their reduced sensitivity to BR, further underscoring their critical regulatory roles in BR-mediated developmental processes. Our findings uncovered an epigenetic mechanism that governs BR biosynthesis and orchestrates the expression of BRD1 and D2 to modulate BR levels and influence rice growth and development.

An orchestrated ethylene-gibberellin signaling cascade contributes to mesocotyl elongation and emergence of rice direct seeding.

A mechanized direct seeding of rice with less labor and water usage, has been widely adopted. However, this approach requires varieties that exhibit uniform seedling emergence. Mesocotyl elongation (ME) offers the main drive of fast emergence of rice seedlings from soils; nevertheless, its genetic basis remains unknown. Here, we identify a major rice quantitative trait locus Mesocotyl Elongation1 (qME1), an allele of the Green Revolution gene Semi-Dwarf1 (SD1), encoding GA20-oxidase for gibberellin (GA) biosynthesis. ME1 expression is strongly induced by soil depth and ethylene. When rice grains are direct-seeded in soils, the ethylene core signaling factor OsEIL1 directly promotes ME1 transcription, accelerating bioactive GA biosynthesis. The GAs further degrade the DELLA protein SLENDER RICE 1 (SLR1), alleviating its inhibition of rice PHYTOCHROME-INTERACTING FACTOR-LIKE13 (OsPIL13) to activate the downstream expansion gene OsEXPA4 and ultimately promote rice seedling ME and emergence. The ancient traits of long mesocotyl and strong emergence ability in wild rice and landrace were gradually lost in company with the Green Revolution dwarf breeding process, and an elite ME1-R allele (D349H) is found in some modern Geng varieties (long mesocotyl lengths) in northern China, which can be used in the direct seeding and dwarf breeding of Geng varieties. Furthermore, the ectopic and high expression of ME1 driven by mesocotyl-specific promoters resulted in rice plants that could be direct-seeded without obvious plant architecture or yield penalties. Collectively, we reveal the molecular mechanism of rice ME, and provide useful information for breeding new Green Revolution varieties with long mesocotyl suitable for direct-seeding practice.

Integrating Association Mapping, Linkage Mapping, Fine Mapping with RNA Seq Conferring Seedling Vigor Improvement for Successful Crop Establishment in Deep Sown Direct-Seeded Rice.

BACKGROUND: Ongoing large-scale shift towards direct seeded rice (DSR) necessitates a convergence of breeding and genetic approaches for its sustenance and harnessing natural resources and environmental benefits. Improving seedling vigour remains key objective for breeders working with DSR. The present study aims to understand the genetic control of seedling vigour in deep sown DSR. Combined genome-wide association mapping, linkage mapping, fine mapping, RNA-sequencing to identify candidate genes and validation of putative candidate genes were performed in the present study. RESULTS: Significant phenotypic variations were observed among genotypes in both F3:4:5 and BC2F2:3 populations. The mesocotyl length showed significant positive correlation with %germination, root and shoot length. The 881 kb region on chromosome 7 reported to be associated with mesocotyl elongation. RNA-seq data and RT-PCR results identified and validated seven potential candidate genes. The four promising introgression lines free from linkage drag and with longer mesocotyl length, longer root length, semi-dwarf plant height have been identified. CONCLUSION: The study will provide rice breeders (1) the pre breeding material in the form of anticipated DSR adapted introgression lines possessing useful traits and alleles improving germination under deep sown DSR field conditions (2) the base for the studies involving functional characterization of candidate genes. The development and utilization of improved introgression lines and molecular markers may play an important role in genomics-assisted breeding (GAB) during the pyramiding of valuable genes providing adaptation to rice under DSR. Our results offer a robust and reliable package that can contribute towards enhancing genetic gains in direct seeded rice breeding programs.

ZmCOP1 Regulates Maize Mesocotyl Length and Plant Height through the Phytohormone Pathways.

The morphogenesis of crops is critical to their yield performance. COP1 (constitutively photomorphogenic1) is one of the core regulators in plant morphogenesis and has been deeply studied in Arabidopsis thaliana. However, the function of COP1 in maize is still unclear. Here, we found that the mesocotyl lengths of zmcop1 loss-of-function mutants were shorter than those of wild-type B73 in darkness, while the mesocotyl lengths of lines with ZmCOP1 overexpression were longer than those of wild-type B104. The plant height with zmcop1 was shorter than that of B73 in both short- and long-day photoperiods. Using transcriptome RNA sequencing technology, we identified 33 DEGs (differentially expressed genes) between B73's etiolated seedlings and those featuring zmcop1, both in darkness. The DEGs were mainly enriched in the plant phytohormone pathways. Our results provide direct evidence that ZmCOP1 functions in the elongation of etiolated seedlings in darkness and affects plant height in light. Our data can be applied in the improvement of maize plant architecture.

Identification of Genetic Loci for Rice Seedling Mesocotyl Elongation in Both Natural and Artificial Segregating Populations.

Mesocotyl elongation of rice seedlings is a key trait for deep sowing tolerance and well seedling establishment in dry direct sowing rice (DDSR) production. Subsets of the Rice Diversity Panel 1 (RDP1, 294 accessions) and Hanyou 73 (HY73) recombinant inbred line (RIL) population (312 lines) were screened for mesocotyl length (ML) via dark germination. Six RDP1 accessions (Phudugey, Kasalath, CA902B21, Surjamkuhi, Djimoron, and Goria) had an ML longer than 10 cm, with the other 19 accessions being over 4 cm. A GWAS in RDP1 detected 118 associated SNPs on all 12 chromosomes using a threshold of FDR-adjusted p < 0.05, including 11 SNPs on chromosomes 1, 4, 5, 7, 10, and 12 declared by -log10(P) > 5.868 as the Bonferroni-corrected threshold. Using phenotypic data of three successive trials and a high-density bin map from resequencing genotypic data, four to six QTLs were detected on chromosomes 1, 2, 5, 6, and 10, including three loci repeatedly mapped for ML from two or three replicated trials. Candidate genes were predicted from the chromosomal regions covered by the associated LD blocks and the confidence intervals (CIs) of QTLs and partially validated by the dynamic RNA-seq data in the mesocotyl along different periods of light exposure. Potential strategies of donor parent selection for seedling establishment in DDSR breeding were discussed.

Natural variation of OsML1, a mitochondrial transcription termination factor, contributes to mesocotyl length variation in rice.

Mesocotyl length (ML) is a crucial factor in determining the establishment and yield of rice planted through dry direct seeding, a practice that is increasingly popular in rice production worldwide. ML is determined by the endogenous and external environments, and inherits as a complex trait. To date, only a few genes have been cloned, and the mechanisms underlying mesocotyl elongation remain largely unknown. Here, through a genome-wide association study using sequenced germplasm, we reveal that natural allelic variations in a mitochondrial transcription termination factor, OsML1, predominantly determined the natural variation of ML in rice. Natural variants in the coding regions of OsML1 resulted in five major haplotypes with a clear differentiation between subspecies and subpopulations in cultivated rice. The much-reduced genetic diversity of cultivated rice compared to the common wild rice suggested that OsML1 underwent selection during domestication. Transgenic experiments and molecular analysis demonstrated that OsML1 contributes to ML by influencing cell elongation primarily determined by H2 O2 homeostasis. Overexpression of OsML1 promoted mesocotyl elongation and thus improved the emergence rate under deep direct seeding. Taken together, our results suggested that OsML1 is a key positive regulator of ML, and is useful in developing varieties for deep direct seeding by conventional and transgenic approaches.

Maize WRKY28 interacts with the DELLA protein D8 to affect skotomorphogenesis and participates in the regulation of shade avoidance and plant architecture.

Competition for light from neighboring vegetation can trigger the shade-avoidance response (SAR) in plants, which is detrimental to their yield. The molecular mechanisms regulating SAR are well established in Arabidopsis, and some regulators of skotomorphogenesis have been found to be involved in the regulation of the SAR and plant architecture. However, the role of WRKY transcription factors in this process has rarely been reported, especially in maize (Zea mays). Here, we report that maize Zmwrky28 mutants exhibit shorter mesocotyls in etiolated seedlings. Molecular and biochemical analyses demonstrate that ZmWRKY28 directly binds to the promoter regions of the Small Auxin Up RNA (SAUR) gene ZmSAUR54 and the Phytochrome-Interacting Factor (PIF) gene ZmPIF4.1 to activate their expression. In addition, the maize DELLA protein Dwarf Plant8 (D8) interacts with ZmWRKY28 in the nucleus to inhibit its transcriptional activation activity. We also show that ZmWRKY28 participates in the regulation of the SAR, plant height, and leaf rolling and erectness in maize. Taken together, our results reveal that ZmWRKY28 is involved in GA-mediated skotomorphogenic development and can be used as a potential target to regulate SAR for breeding of high-density-tolerant cultivars.

Screening germplasm and detecting QTLs for mesocotyl elongation trait in rice (Oryza sativa L.) by association mapping.

BACKGROUND: Rice is one of the most important food crops in the world and mainly cultivated in paddy field by transplanting seedlings. However, increasing water scarcity due to climate change, labor cost for transplanting, and competition from urbanization is making this traditional method of rice production unsustainable in the long term. In the present study, we mined favorable alleles for mesocotyl elongation length (MEL) by combining the phenotypic data of 543 rice accessions with genotypic data of 262 SSR markers through association mapping method. RESULTS: Among the 543 rice accessions studied, we found 130 accessions could elongate mesocotyl length under dark germination condition. A marker-trait association analysis based on a mixed linear model revealed eleven SSR markers were associated with MEL trait with p-value less than 0.01. Among the 11 association loci, seven were novel. In total, 30 favorable marker alleles for MEL were mined, and RM265-140 bp showed the highest phenotypic effect value of 1.8 cm with Yuedao46 as the carrier accession. The long MEL group of rice accessions had higher seedling emergence rate than the short MEL group in the field. The correlation coefficient (r GCC-FSC = 0.485**) between growth chamber condition (GCC) and field soil condition (FSC) showed positive relationship and highly significant (P < 0.01) indicating that the result obtained in GCC could basically represent that obtained under FSC. CONCLUSION: Not every genotype of the rice possesses the ability to elongate its mesocotyl length under dark or deep sowing condition. Mesocotyl elongation length is a quantitative trait controlled by many gene loci, and can be improved by pyramiding favorable alleles dispersed at different loci in different germplasm into a single genotype.

Assay system for mesocotyl elongation and hydrotropism of maize primary root in response to low moisture gradient.

We designed and validated a test system that simulates a growth environment for Zea mays L. maize seedlings under conditions of low moisture gradient in darkness. This system allowed us to simultaneously measure mesocotyl elongation and the primary root hydrotropic response in seedlings before the emergence phase in a collection of maize hybrids. We found great variation in these two traits with statistically significant reduction of their elongations under the low moisture gradient condition that indicate the richness of maize genetic diversity. Hence, the objective of designing a new test system that evaluates the association between these underground traits with the potential use to measure other traits in maize seedlings related to early vigor was achieved.

Primary Root and Mesocotyl Elongation in Maize Seedlings: Two Organs with Antagonistic Growth below the Soil Surface.

Maize illustrates one of the most complex cases of embryogenesis in higher plants that results in the development of early embryo with distinctive organs such as the mesocotyl, seminal and primary roots, coleoptile, and plumule. After seed germination, the elongation of root and mesocotyl follows opposite directions in response to specific tropisms (positive and negative gravitropism and hydrotropism). Tropisms represent the differential growth of an organ directed toward several stimuli. Although the life cycle of roots and mesocotyl takes place in darkness, their growth and functions are controlled by different mechanisms. Roots ramify through the soil following the direction of the gravity vector, spreading their tips into new territories looking for water; when water availability is low, the root hydrotropic response is triggered toward the zone with higher moisture. Nonetheless, there is a high range of hydrotropic curvatures (angles) in maize. The processes that control root hydrotropism and mesocotyl elongation remain unclear; however, they are influenced by genetic and environmental cues to guide their growth for optimizing early seedling vigor. Roots and mesocotyls are crucial for the establishment, growth, and development of the plant since both help to forage water in the soil. Mesocotyl elongation is associated with an ancient agriculture practice known as deep planting. This tradition takes advantage of residual soil humidity and continues to be used in semiarid regions of Mexico and USA. Due to the genetic diversity of maize, some lines have developed long mesocotyls capable of deep planting while others are unable to do it. Hence, the genetic and phenetic interaction of maize lines with a robust hydrotropic response and higher mesocotyl elongation in response to water scarcity in time of global heating might be used for developing more resilient maize plants.

Targeted mutagenesis of POLYAMINE OXIDASE 5 that negatively regulates mesocotyl elongation enables the generation of direct-seeding rice with improved grain yield.

Under conditions of labor or resource scarcity, direct seeding, rather than transplantation, is the preferred mode of rice (Oryza sativa) cultivation. This approach requires varieties that exhibit uniform seedling emergence. Mesocotyl elongation (ME), the main driver of rapid emergence of rice seedlings from soil, is enhanced by darkness and inhibited by light. Plant polyamine oxidases (PAOs) oxidize polyamines (PAs) and release H2O2. Here, we established that OsPAO5 expression in rice seedlings is increased in the presence of light and inhibited by darkness. To determine its role in ME, we created OsPAO5 mutants using CRISPR/Cas9. Compared with the wild type, pao5 mutants had longer mesocotyls, released less H2O2, and synthesized more ethylene. The mutant seedlings emerged at a higher and more uniform rate, indicating their potential for use in direct seeding. Nucleotide polymorphism analysis revealed that an SNP (PAO5-578G/A) located 578 bp upstream of the OsPAO5 start codon alters its expression, and was selected during rice mesocotyl domestication. The PAO5-578G genotype conferring a long mesocotyl mainly exists in wild rice, most Aus accessions, and some Geng (Japonica) accessions. Intriguingly, knocking out OsPAO5 can remarkably increase the grain weight, grain number, and yield potential. In summary, we developed a novel strategy to obtain elite rice with higher emergence vigor and yield potential, which can be conveniently and widely used to breed varieties of direct-seeding rice.

Uncovering novel loci for mesocotyl elongation and shoot length in indica rice through genome-wide association mapping.

MAIN CONCLUSION: Totally, 23 loci were detected, and 383 candidate genes were identified, and four of these candidate genes, Os01g0392100, Os04g0630000, Os01g0904700 and Os07g0615000, were regarded as promising targets. Direct-seeding cultivation is becoming popular in rice (Oryza sativa L.)-planting countries because it is labor- and time-efficient. However, low seedling establishment and slow seedling emergence have restricted the application and popularity of the technique. Mesocotyl elongation and shoot length are two important traits that can enhance rice seedling emergence. A single nucleotide polymorphism (SNP) is a genome sequence variation caused by a single base within a population, and SNPs evenly distributed throughout the genomes of plant species. In this study, a genome-wide association study (GWAS), based on 4136 SNPs, was performed using a compressed mixed linear model that accounted for population structure and relative kinship to detect novel loci for the two traits. Totally, 23 loci were identified, including five loci located known QTLs region. For the mesocotyl elongation, 17 major loci were identified, explaining ~19.31 % of the phenotypic variation. For the shoot length, six major loci were detected, explaining ~ 39.79 % of the phenotypic variation. In total, 383 candidate genes were included in a 200-kb genomic region (± 100 kb of each locus). Additionally, 32 SNPs were identified in 30 candidate genes. Relative expression level analyses indicated that four candidate genes containing SNP variations, Os01g0392100, Os04g0630000, Os01g0904700 and Os07g0615000, represented promising targets. Finally, eight elite accessions with long mesocotyl and shoot lengths were chosen as breeding donors for further rice direct-seeding variety modifications.

The tillering phenotype of the rice plastid terminal oxidase (PTOX) loss-of-function mutant is associated with strigolactone deficiency.

The significance of plastid terminal oxidase (PTOX) in phytoene desaturation and chloroplast function has been demonstrated using PTOX-deficient mutants, particularly in Arabidopsis. However, studies on its role in monocots are lacking. Here, we report cloning and characterization of the rice (Oryza sativa) PTOX1 gene. Using Ecotype Targeting Induced Local Lesions IN Genomes (EcoTILLING) and TILLING as forward genetic tools, we identified the causative mutation of an EMS mutant characterized by excessive tillering, semi-dwarfism and leaf variegation that corresponded to the PTOX1 gene. The tillering and semi-dwarf phenotypes of the ptox1 mutant are similar to phenotypes of known strigolactone (SL)-related rice mutants, and both phenotypic traits could be rescued by application of the synthetic SL GR24. The ptox1 mutant accumulated phytoene in white leaf sectors with a corresponding deficiency in ß-carotene, consistent with the expected function of PTOX1 in promoting phytoene desaturase activity. There was also no accumulation of the carotenoid-derived SL ent-2'-epi-5-deoxystrigol in root exudates. Elevated concentrations of auxin were detected in the mutant, supporting previous observations that SL interaction with auxin is important in shoot branching control. Our results demonstrate that PTOX1 is required for both carotenoid and SL synthesis resulting in SL-deficient phenotypes in rice.

Mapping and characterization of quantitative trait loci for mesocotyl elongation in rice (Oryza sativa L.).

Mesocotyl elongation is an important trait for seedling emergence in direct-seeding cultivation in rice. In this study, a backcross inbred line (BIL) population from a cross between Kasalath and Nipponbare was employed to map quantitative trait loci (QTLs) for mesocotyl elongation. A total of 5 QTLs for mesocotyl length were identified on chromosomes 1, 3, 7, 9, and 12 in 2 independent experiments. At all QTL, the Kasalath alleles contributed to an increase in mesocotyl length. Two QTLs (qMel-1 and qMel-3) on chromosomes 1 and 3 were consistently detected in both experiments. To fine map the QTLs, a cross was made between 2 chromosome segment substitution lines (CSSL-6 and CSSL-15), each harboring the Kasalath allele across the qMel-1 and qMel-3 regions, and an F2:3 population was developed. A two-way ANOVA indicated that no epistatic interaction was detected between the 2 QTLs in the F2 population (P = 0.31). Moreover, analysis of two F3 near-isogenic lines (NILs) derived from the same cross, indicated that the 2 QTLs act additively in distinct or complementary pathways in controlling mesocotyl elongation. Substitution mapping indicated that the qMel-1 QTL was located between the 2 SSR markers RM5448 and RM5310, which are 3,799-kb apart, and that the qMel-3 QTL was located between the 2 SSR markers RM3513 and RM1238, which are 6,964-kb apart. To our knowledge, this is the first report to fine-map QTLs for mesocotyl elongation and to analyze their interaction.