Unique genetic architecture of prolificacy in 'Sikkim Primitive' maize unraveled through whole-genome resequencing-based DNA polymorphism.

KEY MESSAGE: 'Sikkim Primitive' maize landrace, unique for prolificacy (7-9 ears per plant) possesses unique genomic architecture in branching and inflorescence-related gene(s), and locus Zm00001eb365210 encoding glycosyltransferases was identified as the putative candidate gene underlying QTL (qProl-SP-8.05) for prolificacy. The genotype possesses immense usage in breeding high-yielding baby-corn genotypes. 'Sikkim Primitive' is a native landrace of North Eastern Himalayas, and is characterized by having 7-9 ears per plant compared to 1-2 ears in normal maize. Though 'Sikkim Primitive' was identified in the 1960s, it has not been characterized at a whole-genome scale. Here, we sequenced the entire genome of an inbred (MGUSP101) derived from 'Sikkim Primitive' along with three non-prolific (HKI1128, UMI1200, and HKI1105) and three prolific (CM150Q, CM151Q and HKI323) inbreds. A total of 942,417 SNPs, 24,160 insertions, and 27,600 deletions were identified in 'Sikkim Primitive'. The gene-specific functional mutations in 'Sikkim Primitive' were classified as 10,847 missense (54.36%), 402 non-sense (2.015%), and 8,705 silent (43.625%) mutations. The number of transitions and transversions specific to 'Sikkim Primitive' were 666,021 and 279,950, respectively. Among all base changes, (G to A) was the most frequent (215,772), while (C to G) was the rarest (22,520). Polygalacturonate 4-α-galacturonosyltransferase enzyme involved in pectin biosynthesis, cell-wall organization, nucleotide sugar, and amino-sugar metabolism was found to have unique alleles in 'Sikkim Primitive'. The analysis further revealed the Zm00001eb365210 gene encoding glycosyltransferases as the putative candidate underlying QTL (qProl-SP-8.05) for prolificacy in 'Sikkim Primitive'. High-impact nucleotide variations were found in ramosa3 (Zm00001eb327910) and zeaxanthin epoxidase1 (Zm00001eb081460) genes having a role in branching and inflorescence development in 'Sikkim Primitive'. The information generated unraveled the genetic architecture and identified key genes/alleles unique to the 'Sikkim Primitive' genome. This is the first report of whole-genome characterization of the 'Sikkim Primitive' landrace unique for its high prolificacy.

Dissecting the colocalized GWAS and eQTLs with mediation analysis for high-dimensional exposures and confounders.

To leverage the advancements in genome-wide association studies (GWAS) and quantitative trait loci (QTL) mapping for traits and molecular phenotypes to gain mechanistic understanding of the genetic regulation, biological researchers often investigate the expression QTLs (eQTLs) that colocalize with QTL or GWAS peaks. Our research is inspired by 2 such studies. One aims to identify the causal single nucleotide polymorphisms that are responsible for the phenotypic variation and whose effects can be explained by their impacts at the transcriptomic level in maize. The other study in mouse focuses on uncovering the cis-driver genes that induce phenotypic changes by regulating trans-regulated genes. Both studies can be formulated as mediation problems with potentially high-dimensional exposures, confounders, and mediators that seek to estimate the overall indirect effect (IE) for each exposure. In this paper, we propose MedDiC, a novel procedure to estimate the overall IE based on difference-in-coefficients approach. Our simulation studies find that MedDiC offers valid inference for the IE with higher power, shorter confidence intervals, and faster computing time than competing methods. We apply MedDiC to the 2 aforementioned motivating datasets and find that MedDiC yields reproducible outputs across the analysis of closely related traits, with results supported by external biological evidence. The code and additional information are available on our GitHub page (https://github.com/QiZhangStat/MedDiC).

ZmPILS6 is an auxin efflux carrier required for maize root morphogenesis.

Plant root systems play a pivotal role in plant physiology and exhibit diverse phenotypic traits. Understanding the genetic mechanisms governing root growth and development in model plants like maize is crucial for enhancing crop resilience to drought and nutrient limitations. This study focused on identifying and characterizing ZmPILS6, an annotated auxin efflux carrier, as a key regulator of various crown root traits in maize. ZmPILS6-modified roots displayed reduced network area and suppressed lateral root formation, which are desirable traits for the "steep, cheap, and deep" ideotype. The research revealed that ZmPILS6 localizes to the endoplasmic reticulum and plays a vital role in controlling the spatial distribution of indole-3-acetic acid (IAA or "auxin") in primary roots. The study also demonstrated that ZmPILS6 can actively efflux IAA when expressed in yeast. Furthermore, the loss of ZmPILS6 resulted in significant proteome remodeling in maize roots, particularly affecting hormone signaling pathways. To identify potential interacting partners of ZmPILS6, a weighted gene coexpression analysis was performed. Altogether, this research contributes to the growing knowledge of essential genetic determinants governing maize root morphogenesis, which is crucial for guiding agricultural improvement strategies.

A spatial transcriptome map of the developing maize ear.

A comprehensive understanding of inflorescence development is crucial for crop genetic improvement, as inflorescence meristems give rise to reproductive organs and determine grain yield. However, dissecting inflorescence development at the cellular level has been challenging owing to a lack of specific marker genes to distinguish among cell types, particularly in different types of meristems that are vital for organ formation. In this study, we used spatial enhanced resolution omics-sequencing (Stereo-seq) to construct a precise spatial transcriptome map of the developing maize ear primordium, identifying 12 cell types, including 4 newly defined cell types found mainly in the inflorescence meristem. By extracting the meristem components for detailed clustering, we identified three subtypes of meristem and validated two MADS-box genes that were specifically expressed at the apex of determinate meristems and involved in stem cell determinacy. Furthermore, by integrating single-cell RNA transcriptomes, we identified a series of spatially specific networks and hub genes that may provide new insights into the formation of different tissues. In summary, this study provides a valuable resource for research on cereal inflorescence development, offering new clues for yield improvement.

Comprehensive identification of maize ZmE2F transcription factors and the positive role of ZmE2F6 in response to drought stress.

BACKGROUND: The early 2 factor (E2F) family is characterized as a kind of transcription factor that plays an important role in cell division, DNA damage repair, and cell size regulation. However, its stress response has not been well revealed. RESULTS: In this study, ZmE2F members were comprehensively identified in the maize genome, and 21 ZmE2F genes were identified, including eight E2F subclade members, seven DEL subfamily genes, and six DP genes. All ZmE2F proteins possessed the DNA-binding domain (DBD) characterized by conserved motif 1 with the RRIYD sequence. The ZmE2F genes were unevenly distributed on eight maize chromosomes, showed diversity in gene structure, expanded by gene duplication, and contained abundant stress-responsive elements in their promoter regions. Subsequently, the ZmE2F6 gene was cloned and functionally verified in drought response. The results showed that the ZmE2F6 protein interacted with ZmPP2C26, localized in the nucleus, and responded to drought treatment. The overexpression of ZmE2F6 enhanced drought tolerance in transgenic Arabidopsis with longer root length, higher survival rate, and biomass by upregulating stress-related gene transcription. CONCLUSIONS: This study provides novel insights into a greater understanding and functional study of the E2F family in the stress response.

Transcription factor ZmDof22 enhances drought tolerance by regulating stomatal movement and antioxidant enzymes activities in maize (Zea mays L.).

KEY MESSAGE: The Dof22 gene encoding a deoxyribonucleic acid binding with one finger in maize, which is associated with its drought tolerance. The identification of drought stress regulatory genes is essential for the genetic improvement of maize yield. Deoxyribonucleic acid binding with one finger (Dof), a plant-specific transcription factor family, is involved in signal transduction, morphogenesis, and environmental stress responses. In present study, by weighted correlation network analysis (WGCNA) and gene co-expression network analysis, 15 putative Dof genes were identified from maize that respond to drought and rewatering. A real-time fluorescence quantitative PCR showed that these 15 genes were strongly induced by drought and ABA treatment, and among them ZmDof22 was highly induced by drought and ABA treatment. Its expression level increased by nearly 200 times after drought stress and more than 50 times after ABA treatment. After the normal conditions were restored, the expression levels were nearly 100 times and 40 times of those before treatment, respectively. The Gal4-LexA/UAS system and transcriptional activation analysis indicate that ZmDof22 is a transcriptional activator regulating drought tolerance and recovery ability in maize. Further, overexpressed transgenic and mutant plants of ZmDof22 by CRISPR/Cas9, indicates that the ZmDof22, improves maize drought tolerance by promoting stomatal closure, reduces water loss, and enhances antioxidant enzyme activity by participating in the ABA pathways. Taken together, our findings laid a foundation for further functional studies of the ZmDof gene family and provided insights into the role of the ZmDof22 regulatory network in controlling drought tolerance and recovery ability of maize.

Regulatory role of AGC genes in heat stress adaptation in maize (Zea mays).

Heat stress represents a significant environmental challenge that restricts maize (Zea mays ) growth and yield on a global scale. Within the plant kingdom, the AGC gene family, encoding a group of protein kinases, has emerged as crucial players in various stress responses. Nevertheless, a comprehensive understanding of AGC genes in Z. mays under heat-stress conditions remains elusive. A genome-wide analysis was done using bioinformatics techniques to identify 39 AGC genes in Z. mays , categorising them into three subfamilies based on their conserved domains. We investigated their phylogenetic relationships, gene structures (including intron-exon configurations), and expression patterns. These genes are likely involved in diverse signalling pathways, fulfilling distinct roles when exposed to heat stress conditions. Notably, most ZmAGC1.5, ZmAGC1.9, ZmNDR3, ZmNDR5 and ZmIRE3 exhibited significant changes in expression levels under heat stress, featuring a high G-box ratio. Furthermore, we pinpointed a subset of AGC genes displaying highly coordinated expression, implying their potential involvement in the heat stress response pathway. Our study offers valuable insights into the contribution of AGC genes to Z. mays 's heat stress response, thus facilitating the development of heat-tolerant Z. mays varieties.

Genome-wide identification and expression analysis of Ubiquitin-specific protease gene family in maize (Zea mays L.).

BACKGROUND: Ubiquitin-specific proteases (UBPs) are a large family of deubiquitinating enzymes (DUBs). They are widespread in plants and are critical for plant growth, development, and response to external stresses. However, there are few studies on the functional characteristics of the UBP gene family in the important staple crop, maize (Zea mays L.). RESULTS: In this study, we performed a bioinformatic analysis of the entire maize genome and identified 45 UBP genes. Phylogenetic analysis indicated that 45 ZmUBP genes can be divided into 15 subfamilies. Analysis of evolutionary patterns and divergence levels indicated that ZmUBP genes were present before the isolation of dicotyledons, were highly conserved and subjected to purifying selection during evolution. Most ZmUBP genes exhibited different expression levels in different tissues and developmental stages. Based on transcriptome data and promoter element analysis, we selected eight ZmUBP genes whose promoters contained a large number of plant hormones and stress response elements and were up-regulated under different abiotic stresses for RT-qPCR analysis, results showed that these genes responded to abiotic stresses and phytohormones to varying degrees, indicating that they play important roles in plant growth and stress response. CONCLUSIONS: In this study, the structure, location and evolutionary relationship of maize UBP gene family members were analyzed for the first time, and the ZmUBP genes that may be involved in stress response and plant growth were identified by combining promoter element analysis, transcriptome data and RT-qPCR analysis. This study informs research on the involvement of maize deubiquitination in stress response.

Maize miRNAs and their putative target genes involved in chilling stress response in 5-day old seedlings.

BACKGROUND: In the context of early sowing of maize as a promising adaptation strategy that could significantly reduce the negative effects of climate change, an in-depth understanding of mechanisms underlying plant response to low-temperature stress is demanded. Although microRNAs (miRNAs) have been recognized as key regulators of plant stress response, research on their role in chilling tolerance of maize during early seedling stages is scarce. Therefore, it is of great significance to explore chilling-responsive miRNAs, reveal their expression patterns and associated target genes, as well as to examine the possible functions of the conserved and novel miRNAs. In this study, the role of miRNAs was examined in 5d-old maize seedlings of one tolerant and one sensitive inbred line exposed to chilling (10/8 °C) stress for 6 h and 24 h, by applying high throughput sequencing. RESULTS: A total of 145 annotated known miRNAs belonging to 30 families and 876 potentially novel miRNAs were identified. Differential expression (DE) analysis between control and stress conditions identified 98 common miRNAs for both genotypes at one time point and eight miRNAs at both time points. Target prediction and enrichment analysis showed that the DE zma-miR396, zma-miR156, zma-miR319, and zma-miR159 miRNAs modulate growth and development. Furthermore, it was found that several other DE miRNAs were involved in abiotic stress response: antioxidative mechanisms (zma-miR398), signal transduction (zma-miR156, zma-miR167, zma-miR169) and regulation of water content (zma-miR164, zma-miR394, zma-miR396). The results underline the zma-miRNAs involvement in the modulation of their target genes expression as an important aspect of the plant's survival strategy and acclimation to chilling stress conditions. CONCLUSIONS: To our understanding, this is the first study on miRNAs in 5-d old seedlings' response to chilling stress, providing data on the role of known and novel miRNAs post-transcriptional regulation of expressed genes and contributing a possible platform for further network and functional analysis.

Sweet corn association panel and genome-wide association analysis reveal loci for chilling-tolerant germination.

Sweet corn is highly susceptible to the deleterious effects of low temperatures during the initial stages of growth and development. Employing a 56K chip, high-throughput single-nucleotide polymorphism (SNP) sequencing was conducted on 100 sweet corn inbred lines. Subsequently, six germination indicators-germination rate, germination index, germination time, relative germination rate, relative germination index, and relative germination time-were utilized for genome-wide association analysis. Candidate genes were identified via comparative analysis of homologous genes in Arabidopsis and rice, and their functions were validated using quantitative real-time polymerase chain reaction (qRT-PCR). The results revealed 35,430 high-quality SNPs, 16 of which were significantly correlated. Within 50 kb upstream and downstream of the identified SNPs, 46 associated genes were identified, of which six were confirmed as candidate genes. Their expression patterns indicated that Zm11ΒHSDL5 and Zm2OGO likely play negative and positive regulatory roles, respectively, in the low-temperature germination of sweet corn. Thus, we determined that these two genes are responsible for regulating the low-temperature germination of sweet corn. This study contributes valuable theoretical support for improving sweet corn breeding and may aid in the creation of specific germplasm resources geared toward enhancing low-temperature tolerance in sweet corn.

The ZmbHLH47-ZmSnRK2.9 Module Promotes Drought Tolerance in Maize.

Drought stress globally poses a significant threat to maize (Zea mays L.) productivity and the underlying molecular mechanisms of drought tolerance remain elusive. In this study, we characterized ZmbHLH47, a basic helix-loop-helix (bHLH) transcription factor, as a positive regulator of drought tolerance in maize. ZmbHLH47 expression was notably induced by both drought stress and abscisic acid (ABA). Transgenic plants overexpressing ZmbHLH47 displayed elevated drought tolerance and ABA responsiveness, while the zmbhlh47 mutant exhibited increased drought sensitivity and reduced ABA sensitivity. Mechanistically, it was revealed that ZmbHLH47 could directly bind to the promoter of ZmSnRK2.9 gene, a member of the subgroup III SnRK2 kinases, activating its expression. Furthermore, ZmSnRK2.9-overexpressing plants exhibited enhanced ABA sensitivity and drought tolerance, whereas the zmsnrk2.9 mutant displayed a decreased sensitivity to both. Notably, overexpressing ZmbHLH47 in the zmsnrk2.9 mutant closely resembled the zmsnrk2.9 mutant, indicating the importance of the ZmbHLH47-ZmSnRK2.9 module in ABA response and drought tolerance. These findings provided valuable insights and a potential genetic resource for enhancing the environmental adaptability of maize.

Fine mapping of major QTL qshgd1 for spontaneous haploid genome doubling in maize (Zea mays L.).

KEY MESSAGE: A large-effect QTL was fine mapped, which revealed 79 gene models, with 10 promising candidate genes, along with a novel inversion. In commercial maize breeding, doubled haploid (DH) technology is arguably the most efficient resource for rapidly developing novel, completely homozygous lines. However, the DH strategy, using in vivo haploid induction, currently requires the use of mutagenic agents which can be not only hazardous, but laborious. This study focuses on an alternative approach to develop DH lines-spontaneous haploid genome duplication (SHGD) via naturally restored haploid male fertility (HMF). Inbred lines A427 and Wf9, the former with high HMF and the latter with low HMF, were selected to fine-map a large-effect QTL associated with SHGD-qshgd1. SHGD alleles were derived from A427, with novel haploid recombinant groups having varying levels of the A427 chromosomal region recovered. The chromosomal region of interest is composed of 45 megabases (Mb) of genetic information on chromosome 5. Significant differences between haploid recombinant groups for HMF were identified, signaling the possibility of mapping the QTL more closely. Due to suppression of recombination from the proximity of the centromere, and a newly discovered inversion region, the associated QTL was only confined to a 25 Mb region, within which only a single recombinant was observed among ca. 9,000 BC1 individuals. Nevertheless, 79 gene models were identified within this 25 Mb region. Additionally, 10 promising candidate genes, based on RNA-seq data, are described for future evaluation, while the narrowed down genome region is accessible for straightforward introgression into elite germplasm by BC methods.

Integrative transcriptome analysis uncovers common components containing CPS2 regulated by maize lncRNA GARR2 in gibberellin response.

MAIN CONCLUSION: The combined transcriptome outcome provides an important clue to the regulatory cascade centering on lncRNA GARR2 and CPS2 gene in GA response. Long non-coding RNAs (lncRNAs) serve as regulatory components in transcriptional hierarchy governing multiple aspects of biological processes. Dissecting regulatory mechanisms underpinning tetracyclic diterpenoid gibberellin (GA) cascade holds both theoretical and applied significance. However, roles of lncRNAs in transcriptional modulation of GA pathway remain largely elusive. Gypsy retrotransposon-derived GIBBERELLIN RESPONSIVE lncRNA2 (GARR2) has been reported as GA-responsive maize lncRNA. Here a novel GARR2-edited line garr2-1 was identified, characteristic of GA-induced phenotype of increased seedling height and elongated leaf sheath. Transcriptome analysis indicated that transcriptional abundance of five genes [ent-copalyl diphosphate synthase2 (CPS2), ent-kaurene synthase4 (KS4), ent-kaurene synthase6 (KS6), ent-kaurene oxidase2 (KO2), and ent-kaurenoic acid oxidase1/Dwarf3 (KAO1/D3)] was elevated in garr2-1 for early steps of GA biosynthesis. Five GA biosynthetic genes as hub regulators were interlaced to shape regulatory network of GA response. Different transcriptome resources were integrated to discover common differentially expressed genes (DEGs) in the independent GARR2-edited lines GARR2KO and garr2-1. A total of 320 common DEGs were retrieved. These common DEGs were enriched in diterpenoid biosynthetic pathway. Integrative transcriptome analysis revealed the common CPS2 encoding the CPS enzyme that catalyzes the conversion of the precursor trans-geranylgeranyl diphosphate to ent-copalyl diphosphate. The up-regulated CPS2 supported the GA-induced phenotype of slender seedlings observed in the independent GARR2-edited lines GARR2KO and garr2-1. Our integrative transcriptome analysis uncovers common components of the GA pathway regulated by lncRNA GARR2. These common components, especially for the GA biosynthetic gene CPS2, provide a valuable resource for further delineating the underlying mechanisms of lncRNA GARR2 in GA response.

Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Assessing genotypes of buffel grass (Cenchrus ciliaris) as an alternative to maize silage for sheep nutrition.

Nutritive value of five Cenchrus ciliaris (buffel grass) genotypes (IG96-50, IG96-96, IG96-358, IG96-401 and IG96-403) weredetermined. Their sugar contents (>70 mg/g of dry matter) and ensiling potential were evaluated using in vitro batch culture and in vivo studies. Research indicated significant differences (P < 0.05) in the dry matter, organic matter, ether extract, neutral detergent fiber, acid detergent fiber, cellulose and lignin contents of the C. ciliaris genotypes tested. Genotypes also differed (P < 0.05) in total carbohydrates, structural carbohydrates, non-structural carbohydrates and protein fractions. Genotype IG96-96 had the lowest total digestible nutrients, digestible energy and metabolizable energy contents (377.2 g/kg, 6.95 and 5.71 MJ/kg of dry matter, respectively), and net energy values for lactation, maintenance and growth. After 45 days of ensiling, C. ciliaris silages differed (P < 0.05) in dry matter, pH, and lactic acid contents, and their values ranged between 255-339, 4.06-5.17 g/kg of dry matter and 10.8-28.0 g/kg of dry matter, respectively. Maize silage had higher (P < 0.05) Organic Matter (919.5g/kg of dry matter), ether extract (20.4g/kg of dry matter) and hemi-cellulose (272.3 g/kg of dry matter) than IG96-401 and IG96-96 silages. The total carbohydrates and non-structural carbohydrates of maize silage were higher (P < 0.05), while structural carbohydrates were comparable (P < 0.05) with C. ciliaris silages. Sheep on maize silage had (P < 0.05) higher metabolizable energy, lower crude protein, and digestible crude protein intake (g/kg of dry matter) than those on C. ciliaris silage diets. Nitrogen intake and urinary-N excretion were higher (P < 0.05) on genotype IG96-96 silage diet. Overall, this study suggested that certain C. ciliaris genotypes, notably IG96-401 and IG96-96, exhibited nutritive values comparable to maize silage in sheep studies, offering a promising avenue for future exploration as potential alternatives in diversified and sustainable livestock nutrition programs.

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Regulatory mechanisms used by ZmMYB39 to enhance drought tolerance in maize (Zea mays) seedlings.

Drought is a significant abiotic stressor that limits maize (Zea mays L.) growth and development. Thus, enhancing drought tolerance is critical for promoting maize production. Our findings demonstrated that ZmMYB39 is an MYB transcription factor with transcriptional activation activity. Drought stress experiments involving ZmMYB39 overexpression and knockout lines indicated that ZmMYB39 positively regulated drought stress tolerance in maize. DAP-Seq, EMSA, dual-LUC, and RT-qPCR provided initial insights into the molecular regulatory mechanisms by which ZmMYB39 enhances drought tolerance in maize. ZmMYB39 directly promoted the expression of ZmP5CS1, ZmPOX1, ZmSOD2, ZmRD22, ZmNAC49, and ZmDREB2A, which are involved in stress resistance. ZmMYB39 enhanced drought tolerance by interacting with and promoting the expression of ZmFNR1, ZmHSP20, and ZmDOF6. Our study offers a theoretical basis for understanding the molecular regulatory networks involved in maize drought stress response. Furthermore, ZmMYB39 serves as a valuable genetic resource for breeding drought-resistant maize.

ACMGA: a reference-free multiple-genome alignment pipeline for plant species.

BACKGROUND: The short-read whole-genome sequencing (WGS) approach has been widely applied to investigate the genomic variation in the natural populations of many plant species. With the rapid advancements in long-read sequencing and genome assembly technologies, high-quality genome sequences are available for a group of varieties for many plant species. These genome sequences are expected to help researchers comprehensively investigate any type of genomic variants that are missed by the WGS technology. However, multiple genome alignment (MGA) tools designed by the human genome research community might be unsuitable for plant genomes. RESULTS: To fill this gap, we developed the AnchorWave-Cactus Multiple Genome Alignment (ACMGA) pipeline, which improved the alignment of repeat elements and could identify long (> 50 bp) deletions or insertions (INDELs). We conducted MGA using ACMGA and Cactus for 8 Arabidopsis (Arabidopsis thaliana) and 26 Maize (Zea mays) de novo assembled genome sequences and compared them with the previously published short-read variant calling results. MGA identified more single nucleotide variants (SNVs) and long INDELs than did previously published WGS variant callings. Additionally, ACMGA detected significantly more SNVs and long INDELs in repetitive regions and the whole genome than did Cactus. Compared with the results of Cactus, the results of ACMGA were more similar to the previously published variants called using short-read. These two MGA pipelines identified numerous multi-allelic variants that were missed by the WGS variant calling pipeline. CONCLUSIONS: Aligning de novo assembled genome sequences could identify more SNVs and INDELs than mapping short-read. ACMGA combines the advantages of AnchorWave and Cactus and offers a practical solution for plant MGA by integrating global alignment, a 2-piece-affine-gap cost strategy, and the progressive MGA algorithm.

A MYB-related transcription factor ZmMYBR29 is involved in grain filling.

BACKGROUND: The endosperm serves as the primary source of nutrients for maize (Zea mays L.) kernel embryo development and germination. Positioned at the base of the endosperm, the transfer cells (TCs) of the basal endosperm transfer layer (BETL) generate cell wall ingrowths, which enhance the connectivity between the maternal plant and the developing kernels. These TCs play a crucial role in nutrient transport and defense against pathogens. The molecular mechanism underlying BETL development in maize remains unraveled. RESULTS: This study demonstrated that the MYB-related transcription factor ZmMYBR29, exhibited specific expression in the basal cellularized endosperm, as evidenced by in situ hybridization analysis. Utilizing the CRISPR/Cas9 system, we successfully generated a loss-of-function homozygous zmmybr29 mutant, which presented with smaller kernel size. Observation of histological sections revealed abnormal development and disrupted morphology of the cell wall ingrowths in the BETL. The average grain filling rate decreased significantly by 26.7% in zmmybr29 mutant in comparison to the wild type, which impacted the dry matter accumulation within the kernels and ultimately led to a decrease in grain weight. Analysis of RNA-seq data revealed downregulated expression of genes associated with starch synthesis and carbohydrate metabolism in the mutant. Furthermore, transcriptomic profiling identified 23 genes that expressed specifically in BETL, and the majority of these genes exhibited altered expression patterns in zmmybr29 mutant. CONCLUSIONS: In summary, ZmMYBR29 encodes a MYB-related transcription factor that is expressed specifically in BETL, resulting in the downregulation of genes associated with kernel development. Furthermore, ZmMYBR29 influences kernels weight by affecting the grain filling rate, providing a new perspective for the complementation of the molecular regulatory network in maize endosperm development.

ZmmiR398b negatively regulates maize resistance to sugarcane mosaic virus infection by targeting ZmCSD2/4/9.

MicroRNAs (miRNAs) are widely involved in various biological processes of plants and contribute to plant resistance against various pathogens. In this study, upon sugarcane mosaic virus (SCMV) infection, the accumulation of maize (Zea mays) miR398b (ZmmiR398b) was significantly reduced in resistant inbred line Chang7-2, while it was increased in susceptible inbred line Mo17. Degradome sequencing analysis coupled with transient co-expression assays revealed that ZmmiR398b can target Cu/Zn-superoxidase dismutase2 (ZmCSD2), ZmCSD4, and ZmCSD9 in vivo, of which the expression levels were all upregulated by SCMV infection in Chang7-2 and Mo17. Moreover, overexpressing ZmmiR398b (OE398b) exhibited increased susceptibility to SCMV infection, probably by increasing reactive oxygen species (ROS) accumulation, which were consistent with ZmCSD2/4/9-silenced maize plants. By contrast, silencing ZmmiR398b (STTM398b) through short tandem target mimic (STTM) technology enhanced maize resistance to SCMV infection and decreased ROS levels. Interestingly, copper (Cu)-gradient hydroponic experiments demonstrated that Cu deficiency promoted SCMV infection while Cu sufficiency inhibited SCMV infection by regulating accumulations of ZmmiR398b and ZmCSD2/4/9 in maize. These results revealed that manipulating the ZmmiR398b-ZmCSD2/4/9-ROS module provides a prospective strategy for developing SCMV-tolerant maize varieties.