Sucrose-associated SnRK1a1-mediated phosphorylation of Opaque2 modulates endosperm filling in maize.

During maize endosperm filling, sucrose not only serves as a source of carbon skeletons for storage-reserve synthesis but also acts as a stimulus to promote this process. However, the molecular mechanisms underlying sucrose and endosperm filling are poorly understood. In this study, we found that sucrose promotes the expression of endosperm-filling hub gene Opaque2 (O2), coordinating with storage-reserve accumulation. We showed that the protein kinase SnRK1a1 can attenuate O2-mediated transactivation, but sucrose can release this suppression. Biochemical assays revealed that SnRK1a1 phosphorylates O2 at serine 41 (S41), negatively affecting its protein stability and transactivation ability. We observed that mutation of SnRK1a1 results in larger seeds with increased kernel weight and storage reserves, while overexpression of SnRK1a1 causes the opposite effect. Overexpression of the native O2 (O2-OE), phospho-dead (O2-SA), and phospho-mimetic (O2-SD) variants all increased 100-kernel weight. Although O2-SA seeds exhibit smaller kernel size, they have higher accumulation of starch and proteins, resulting in larger vitreous endosperm and increased test weight. O2-SD seeds display larger kernel size but unchanged levels of storage reserves and test weight. O2-OE seeds show elevated kernel dimensions and nutrient storage, like a mixture of O2-SA and O2-SD seeds. Collectively, our study discovers a novel regulatory mechanism of maize endosperm filling. Identification of S41 as a SnRK1-mediated phosphorylation site in O2 offers a potential engineering target for enhancing storage-reserve accumulation and yield in maize.

An ARF gene mutation creates flint kernel architecture in dent maize.

Dent and flint kernel architectures are important characteristics that affect the physical properties of maize kernels and their grain end uses. The genes controlling these traits are unknown, so it is difficult to combine the advantageous kernel traits of both. We found mutation of ARFTF17 in a dent genetic background reduces IAA content in the seed pericarp, creating a flint-like kernel phenotype. ARFTF17 is highly expressed in the pericarp and encodes a protein that interacts with and inhibits MYB40, a transcription factor with the dual functions of repressing PIN1 expression and transactivating genes for flavonoid biosynthesis. Enhanced flavonoid biosynthesis could reduce the metabolic flux responsible for auxin biosynthesis. The decreased IAA content of the dent pericarp appears to reduce cell division and expansion, creating a shorter, denser kernel. Introgression of the ARFTF17 mutation into dent inbreds and hybrids improved their kernel texture, integrity, and desiccation, without affecting yield.

Maize DDK1 encoding an Importin-4 ß protein is essential for seed development and grain filling by mediating nuclear exporting of eIF1A.

Nuclear-cytoplasmic trafficking is crucial for protein synthesis in eukaryotic cells due to the spatial separation of transcription and translation by the nuclear envelope. However, the mechanism underlying this process remains largely unknown in plants. In this study, we isolated a maize (Zea mays) mutant designated developmentally delayed kernel 1 (ddk1), which exhibits delayed seed development and slower filling. Ddk1 encodes a plant-specific protein known as Importin-4 ß, and its mutation results in reduced 80S monosomes and suppressed protein synthesis. Through our investigations, we found that DDK1 interacts with eIF1A proteins in vivo. However, in vitro experiments revealed that this interaction exhibits low affinity in the absence of RanGTP. Additionally, while the eIF1A protein primarily localizes to the cytoplasm in the wild-type, it remains significantly retained within the nuclei of ddk1 mutants. These observations suggest that DDK1 functions as an exportin and collaborates with RanGTP to facilitate the nuclear export of eIF1A, consequently regulating endosperm development at the translational level. Importantly, both DDK1 and eIF1A are conserved among various plant species, implying the preservation of this regulatory module across diverse plants.

Spatial transcriptomics uncover sucrose post-phloem transport during maize kernel development.

Maize kernels are complex biological systems composed of three genetic sources, namely maternal tissues, progeny embryos, and progeny endosperms. The lack of gene expression profiles with spatial information has limited the understanding of the specific functions of each cell population, and hindered the exploration of superior genes in kernels. In our study, we conduct microscopic sectioning and spatial transcriptomics analysis during the grain filling stage of maize kernels. This enables us to visualize the expression patterns of all genes through electronical RNA in situ hybridization, and identify 11 cell populations and 332 molecular marker genes. Furthermore, we systematically elucidate the spatial storage mechanisms of the three major substances in maize kernels: starch, protein, and oil. These findings provide valuable insights into the functional genes that control agronomic traits in maize kernels.

Characterization of Dof Transcription Factors and the Heat-Tolerant Function of PeDof-11 in Passion Fruit (Passiflora edulis).

Abiotic stress is the focus of passion fruit research since it harms the industry, in which high temperature is an important influencing factor. Dof transcription factors (TFs) act as essential regulators in stress conditions. TFs can protect against abiotic stress via a variety of biological processes. There is yet to be published a systematic study of the Dof (PeDof) family of passion fruit. This study discovered 13 PeDof family members by using high-quality genomes, and the members of this characterization were identified by bioinformatics. Transcriptome sequencing and qRT-PCR were used to analyze the induced expression of PeDofs under high-temperature stress during three periods, in which PeDof-11 was significantly induced with high expression. PeDof-11 was then chosen and converted into yeast, tobacco, and Arabidopsis, with the findings demonstrating that PeDof-11 could significantly respond to high-temperature stress. This research lays the groundwork for a better understanding of PeDof gene regulation under high-temperature stress.

THP9 enhances seed protein content and nitrogen-use efficiency in maize.

Teosinte, the wild ancestor of maize (Zea mays subsp. mays), has three times the seed protein content of most modern inbreds and hybrids, but the mechanisms that are responsible for this trait are unknown1,2. Here we use trio binning to create a contiguous haplotype DNA sequence of a teosinte (Zea mays subsp. parviglumis) and, through map-based cloning, identify a major high-protein quantitative trait locus, TEOSINTE HIGH PROTEIN 9 (THP9), on chromosome 9. THP9 encodes an asparagine synthetase 4 enzyme that is highly expressed in teosinte, but not in the B73 inbred, in which a deletion in the tenth intron of THP9-B73 causes incorrect splicing of THP9-B73 transcripts. Transgenic expression of THP9-teosinte in B73 significantly increased the seed protein content. Introgression of THP9-teosinte into modern maize inbreds and hybrids greatly enhanced the accumulation of free amino acids, especially asparagine, throughout the plant, and increased seed protein content without affecting yield. THP9-teosinte seems to increase nitrogen-use efficiency, which is important for promoting a high yield under low-nitrogen conditions.

First Report of Fruit rot Caused by Phytophthora nicotianae on Passion Fruit in Guangxi Province, China.

Passion fruit (Passiflora edulis) is an economically important fruit crop in many tropical and subtropical regions worldwide. In recent years, passion fruit was widely cultivated in Guangxi Province. In 2020, a rot disease occurred on immature fruit of passion fruit in several commercial orchards of Nanning, Guangxi, caused about 50% incidence. The first appeared as small, irregular, water-soaked, brown lesions on immature fruit. As the disease progressed, the lesions rapidly enlarged, causing fruit rot. A layer of sparse white mycelia appeared on the lesions at high humidity. The disease first developed in June, its peak periods from August to September. Five diseased fruits were collected from five different orchards. The edges of symptomatic fleshy mesocarp tissue were cut into pieces (5 mm × 5 mm), surface-sterilized in 75% ethanol solution for 60 s, rinsed three times with sterilized distilled water, and plated on potato dextrose agar (PDA). Plates were incubated at 25°C in the dark. After 5 days, similar white colonies with abundant aerial mycelia developed from all plated tissue samples. Five isolates were obtained, and they were identified as Phytophthora nicotianae based on morphological characteristics and DNA analysis. Spherical hyphal swellings were commonly produced. Numerous sporangia were formed in sterile soil extract. Sporangia were ovoid or obpyriform, papillate, and measured 25 to 58 µm (average 41 µm) × 21 to 45 µm (average 29 µm). Chlamydospores were spherical and 19 to 43 µm in diameter (average 30 µm) (Erwin and Ribeiro 1996). The genomic DNA of a representative isolate Seg2-5 was extracted from mycelia through modified CTAB method (Murray and Thompson 1980). The rDNA internal transcribed spacer (ITS) region, ypt1, and coxII were amplified and sequenced with primers ITS1/ITS4 (White et al., 1990), Yph1F/Yph2R (Schena et al. 2008), and FM75F/FM78R (Villa et al. 2006), respectively. BLAST searches of the ITS, ypt1, and coxII sequences (Accession No. MW470847, MW770870, and MW770871) showed 99 to 100% identity with sequences of P. nicotianae (Accession No. JF792540, MK058408, and MH551183). Based on morphological characteristics and phylogenetic analysis, isolate Seg2-5 was identified as P. nicotianae. To confirm pathogenicity, asymptomatic and immature fruits 'Mantianxing' of passion fruit were previously disinfested in 0.5% sodium hypochlorite. Mycelial plugs of isolate Seg2-5 were placed onto the surface of fruits by nonwounded and pin-prick inoculation. Blank plugs were used as negative controls. Each treatment had five replicates and the test was repeated twice. Fruits were maintained in plastic boxes at 28°C and the initial disease spots appeared at 3 dpi or 5 dpi with wounded or non-wounded inoculation. After 7 to 10 days, all inoculated fruits showed similar symptoms as observed initially in the field, whereas control fruits remained healthy. P. nicotianae was successfully reisolated and identified from the inoculated fruits based on morphological characters and ITS sequence, thus confirming Koch's postulates. P. nicotianae had been previously isolated from passion fruit in South Africa (Van and Huller 1970), Vietnam (Nguyen et al. 2015), and Fujian Province of China (Luo et al. 1993). To our knowledge, this is the first report of P. nicotianae infecting passion fruit in Guangxi Province, China.

Carotenoids modulate kernel texture in maize by influencing amyloplast envelope integrity.

The mechanism that creates vitreous endosperm in the mature maize kernel is poorly understood. We identified Vitreous endosperm 1 (Ven1) as a major QTL influencing this process. Ven1 encodes ß-carotene hydroxylase 3, an enzyme that modulates carotenoid composition in the amyloplast envelope. The A619 inbred contains a nonfunctional Ven1 allele, leading to a decrease in polar and an increase in non-polar carotenoids in the amyloplast. Coincidently, the stability of amyloplast membranes is increased during kernel desiccation. The lipid composition in endosperm cells in A619 is altered, giving rise to a persistent amyloplast envelope. These changes impede the gathering of protein bodies and prevent them from interacting with starch grains, creating air spaces that cause an opaque kernel phenotype. Genetic modifiers were identified that alter the effect of Ven1A619, while maintaining a high ß-carotene level. These studies provide insight for breeding vitreous kernel varieties and high vitamin A content in maize.

Long-read sequencing reveals genomic structural variations that underlie creation of quality protein maize.

Mutation of o2 doubles maize endosperm lysine content, but it causes an inferior kernel phenotype. Developing quality protein maize (QPM) by introgressing o2 modifiers (Mo2s) into the o2 mutant benefits millions of people in developing countries where maize is a primary protein source. Here, we report genome sequence and annotation of a South African QPM line K0326Y, which is assembled from single-molecule, real-time shotgun sequencing reads collinear with an optical map. We achieve a N50 contig length of 7.7 million bases (Mb) directly from long-read assembly, compared to those of 1.04 Mb for B73 and 1.48 Mb for Mo17. To characterize Mo2s, we map QTLs to chromosomes 1, 6, 7, and 9 using an F2 population derived from crossing K0326Y and W64Ao2. RNA-seq analysis of QPM and o2 endosperms reveals a group of differentially expressed genes that coincide with Mo2 QTLs, suggesting a potential role in vitreous endosperm formation.

High frequency DNA rearrangement at qγ27 creates a novel allele for Quality Protein Maize breeding.

Copy number variation (CNV) is a major source of genetic variation and often contributes to phenotypic variation in maize. The duplication at the 27-kDa γ-zein locus (qγ27) is essential to convert soft endosperm into hard endosperm in quality protein maize (QPM). This duplication is unstable and generally produces CNV at this locus. We conducted genetic experiments designed to directly measure DNA rearrangement frequencies occurring in males and females of different genetic backgrounds. The average frequency with which the duplication rearranges to single copies is 1.27 × 10-3 and varies among different lines. A triplication of γ27 gene was screened and showed a better potential than the duplication for the future QPM breeding. Our results highlight a novel approach to directly determine the frequency of DNA rearrangements, in this case resulting in CNV at the qγ27 locus. Furthermore, this provides a highly effective way to test suitable parents in QPM breeding.

Maize VKS1 Regulates Mitosis and Cytokinesis During Early Endosperm Development.

Cell number is a critical factor that determines kernel size in maize (Zea mays). Rapid mitotic divisions in early endosperm development produce most of the cells that make up the starchy endosperm; however, the mechanisms underlying early endosperm development remain largely unknown. We isolated a maize mutant that shows a varied-kernel-size phenotype (vks1). Vks1 encodes ZmKIN11, which belongs to the kinesin-14 subfamily and is predominantly expressed in early endosperm development. VKS1 dynamically localizes to the nucleus and microtubules and plays key roles in the migration of free nuclei in the coenocyte as well as in mitosis and cytokinesis in early mitotic divisions. Absence of VKS1 has relatively minor effects on plants but causes deformities in spindle assembly, sister chromatid separation, and phragmoplast formation in early endosperm development, thereby resulting in reduced cell proliferation. Severities of aberrant mitosis and cytokinesis within individual vks1 endosperms differ, thereby resulting in varied kernel sizes. Our discovery highlights VKS1 as a central regulator of mitosis in early maize endosperm development and provides a potential approach for future yield improvement.

Maize Oxalyl-CoA Decarboxylase1 Degrades Oxalate and Affects the Seed Metabolome and Nutritional Quality.

The organic acid oxalate occurs in microbes, animals, and plants; however, excessive oxalate accumulation in vivo is toxic to cell growth and decreases the nutritional quality of certain vegetables. However, the enzymes and functions required for oxalate degradation in plants remain largely unknown. Here, we report the cloning of a maize (Zea mays) opaque endosperm mutant that encodes oxalyl-CoA decarboxylase1 (EC4.1.1.8; OCD1). Ocd1 is generally expressed and is specifically induced by oxalate. The ocd1 mutant seeds contain a significantly higher level of oxalate than the wild type, indicating that the ocd1 mutants have a defect in oxalate catabolism. The maize classic mutant opaque7 (o7) was initially cloned for its high lysine trait, although the gene function was not understood until its homolog in Arabidopsis thaliana was found to encode an oxalyl-CoA synthetase (EC 6.2.1.8), which ligates oxalate and CoA to form oxalyl-CoA. Our enzymatic analysis showed that ZmOCD1 catalyzes oxalyl-CoA, the product of O7, into formyl-CoA and CO2 for degradation. Mutations in ocd1 caused dramatic alterations in the metabolome in the endosperm. Our findings demonstrate that ZmOCD1 acts downstream of O7 in oxalate degradation and affects endosperm development, the metabolome, and nutritional quality in maize seeds.

The genetic architecture of amylose biosynthesis in maize kernel.

Starch is the most abundant storage carbohydrate in maize kernel. The content of amylose and amylopectin confers unique properties in food processing and industrial application. Thus, the resurgent interest has been switched to the study of individual amylose or amylopectin rather than total starch, whereas the enzymatic machinery for amylose synthesis remains elusive. We took advantage of the phenotype of amylose content and the genotype of 9,007,194 single nucleotide polymorphisms from 464 inbred maize lines. The genome-wide association study identified 27 associated loci involving 39 candidate genes that were linked to amylose content including transcription factors, glycosyltransferases, glycosidases, as well as hydrolases. Except the waxy gene that encodes the granule-bound starch synthase, the remaining candidate genes were located in the upstream pathway of amylose synthesis, while the downstream members were already known from prior studies. The linked candidate genes could be transferred to manipulate amylose content and thus add value to maize kernel in the breeding programme.