A comprehensive phenotypic and genotypic evaluation of Spanish groundnuts from diverse crosses to identify superior and stable donors for fresh seed dormancy.

Breeding high yielding groundnut cultivars with 2-3 weeks of fresh seed dormancy, particularly in Spanish-type cultivars, enhances the sustainability of agriculture in groundnuts. In this context, we conducted a comprehensive phenotypic and genotypic evaluation of advanced breeding lines developed in the genetic background of Spanish types. By employing multi-phenotyping and marker data, we identified PBS 15044, 16004, 16013, 16015, 16016, 16017, 16020, 16021, 16026, 16031, 16035, 16037, 16038, 16039, 16041, and 16042 with 2-3 weeks dormancy (> 90%).The various parametric and non-parametric estimates identified the stable fresh dormant genotypes with one or more superior economic trait. PBS 16021, 15044, 16038, and 16039 identified with high hundred pod weight (HPW) were also reported having high intensity of dormancy (> 90% for up to 3 weeks); PBS 15044, 16016, PBS 16038 and PBS 16039 with high hundred kernel weight (HKW) also reported with up to 3 weeks fresh seed dormancy; and PBS 16013, 16031, and 16038 with up to 3 weeks fresh seed dormancy had high shelling percentage (SP). They can be used to develop lines with the desired level of dormancy, and high yields, by designing appropriate breeding strategies.

The MKK3 MAPK cascade integrates temperature and after-ripening signals to modulate seed germination.

The timing of seed germination is controlled by the combination of internal dormancy and external factors. Temperature is a major environmental factor for seed germination. The permissive temperature range for germination is narrow in dormant seeds and expands during after-ripening (AR) (dormancy release). Quantitative trait loci analyses of preharvest sprouting in cereals have revealed that MKK3, a mitogen-activated protein kinase (MAPK) cascade protein, is a negative regulator of grain dormancy. Here, we show that the MAPKKK19/20-MKK3-MPK1/2/7/14 cascade modulates the germination temperature range in Arabidopsis seeds by elevating the germinability of the seeds at sub- and supraoptimal temperatures. The expression of MAPKKK19 and MAPKKK20 is induced around optimal temperature for germination in after-ripened seeds but repressed in dormant seeds. MPK7 activation depends on the expression levels of MAPKKK19/20, with expression occurring under conditions permissive for germination. Abscisic acid (ABA) and gibberellin (GA) are two major phytohormones which are involved in germination control. Activation of the MKK3 cascade represses ABA biosynthesis enzyme gene expression and induces expression of ABA catabolic enzyme and GA biosynthesis enzyme genes, resulting in expansion of the germinable temperature range. Our data demonstrate that the MKK3 cascade integrates temperature and AR signals to phytohormone metabolism and seed germination.

Genome-wide identification of the sorghum OVATE gene family and revelation of its expression characteristics in sorghum seeds and leaves.

The OVATE gene family plays an important role in regulating the development of plant organs and resisting stress, but its expression characteristics and functions in sorghum have not been revealed. In this study, we identified 26 OVATE genes in the sorghum BTx623 genome, which were divided into four groups and distributed unevenly across 9 chromosomes. Evolutionary analysis showed that after differentiation between sorghum and Arabidopsis, the OVATE gene family may have experienced unique expansion events, and all OVATE family members were negatively selected. Transcriptome sequencing and RT-qPCR results showed that OVATE genes in sorghum showed diverse expression characteristics, such as gene SORBl_3001G468900 and SORBl_3009G173400 were significantly expressed in seeds, while SORBI_3005G042700 and SORBI_3002G417700 were only highly expressed in L1. Meantime, in the promoter region, a large number of hormone-associated cis-acting elements were identified, and these results suggest that members of the OVATE gene family may be involved in regulating specific development of sorghum leaves and seeds. This study improves the understanding of the OVATE gene family of sorghum and provides important clues for further exploration of the function of the OVATE gene family.

Exploring selection signatures in the divergence and evolution of lipid droplet (LD) associated genes in major oilseed crops.

BACKGROUND: Oil bodies or lipid droplets (LDs) in the cytosol are the subcellular storage compartments of seeds and the sites of lipid metabolism providing energy to the germinating seeds. Major LD-associated proteins are lipoxygenases, phospholipaseD, oleosins, TAG-lipases, steroleosins, caleosins and SEIPINs; involved in facilitating germination and enhancing peroxidation resulting in off-flavours. However, how natural selection is balancing contradictory processes in lipid-rich seeds remains evasive. The present study was aimed at the prediction of selection signatures among orthologous clades in major oilseeds and the correlation of selection effect with gene expression. RESULTS: The LD-associated genes from the major oil-bearing crops were analyzed to predict natural selection signatures in phylogenetically close-knit ortholog clusters to understand adaptive evolution. Positive selection was the major force driving the evolution and diversification of orthologs in a lineage-specific manner. Significant positive selection effects were found in 94 genes particularly in oleosin and TAG-lipases, purifying with excess of non-synonymous substitution in 44 genes while 35 genes were neutral to selection effects. No significant selection impact was noticed in Brassicaceae as against LOX genes of oil palm. A heavy load of deleterious mutations affecting selection signatures was detected in T-lineage oleosins and LOX genes of Arachis hypogaea. The T-lineage oleosin genes were involved in mainly anther, tapetum and anther wall morphogenesis. In Ricinus communis and Sesamum indicum > 85% of PLD genes were under selection whereas selection pressures were low in Brassica juncea and Helianthus annuus. Steroleosin, caleosin and SEIPINs with large roles in lipid droplet organization expressed mostly in seeds and were under considerable positive selection pressures. Expression divergence was evident among paralogs and homeologs with one gene attaining functional superiority compared to the other. The LOX gene Glyma.13g347500 associated with off-flavor was not expressed during germination, rather its paralog Glyma.13g347600 showed expression in Glycine max. PLD-α genes were expressed on all the tissues except the seed,δ genes in seed and meristem while ß and γ genes expressed in the leaf. CONCLUSIONS: The genes involved in seed germination and lipid metabolism were under strong positive selection, although species differences were discernable. The present study identifies suitable candidate genes enhancing seed oil content and germination wherein directional selection can become more fruitful.

CaLAP1 and CaLAP2 orchestrate anthocyanin biosynthesis in the seed coat of Cicer arietinum.

MAIN CONCLUSION: Our findings shed light on the regulation of anthocyanin and proanthocyanidin biosynthesis in chickpea seed coats. Expression of R2R3-MYB transcription factors CaLAP1 and CaLAP2 enhanced the anthocyanins and proanthocyanidins content in chickpea. The seed coat color is a major economic trait in leguminous crop chickpea (Cicer arietinum). Anthocyanins and proanthocyanidins (PAs) are two classes of flavonoids that mainly contribute to the flower, seed coat and color of Desi chickpea cultivars. Throughout the land plant lineage, the accumulation of anthocyanins and PAs is regulated by MYB and bHLH transcription factors (TFs), which form an MBW (MYB, bHLH, and WD40) complex. Here, we report two R2R3-MYB TFs in chickpea belonging to the anthocyanin-specific subgroup-6, CaLAP1 (Legume Anthocyanin Production 1), and CaLAP2 (Legume Anthocyanin Production 2), which are mainly expressed in the flowers and developmental stages of the seeds. CaLAP1 and CaLAP2 interact with TT8-like CabHLH1 and WD40, forming the MBW complex, and bind to the promoter sequences of anthocyanin- and PA biosynthetic genes CaCHS6, CaDFR2, CaANS, and CaANR, leading to anthocyanins and PA accumulation in the seed coat of chickpea. Moreover, these CaLAPs partially complement the anthocyanin-deficient phenotype in the Arabidopsis thaliana sextuple mutant seedlings. Overexpression of CaLAPs in chickpea resulted in significantly higher expression of anthocyanin and PA biosynthetic genes leading to a darker seed coat color with higher accumulation of anthocyanin and PA. Our findings show that CaLAPs positively modulate anthocyanin and PA content in seed coats, which might influence plant development and resistance to various biotic and abiotic stresses.

Unveiling the mysteries of HvANS: a study on anthocyanin biosynthesis in qingke (hordeum vulgare L. var. Nudum hook. f.) seeds.

BACKGROUND: Based on our previous research, a full-length cDNA sequence of HvANS gene was isolated from purple and white Qingke. The open reading frame (ORF) in the purple variety Nierumuzha was 1320 base pairs (bp), encoding 439 amino acids, while the ORF in the white variety Kunlun 10 was 1197 bp, encoding 398 amino acids. A nonsynonymous mutation was found at the position of 1195 bp (T/C) in the coding sequence (CDS) of the HvANS gene. We carried out a series of studies to further clarify the relationship between the HvANS gene and anthocyanin synthesis in Qingke. RESULTS: The conservative structural domain prediction results showed that the encoded protein belonged to the PLN03178 superfamily. Multiple comparisons showed that this protein had the highest homology with Hordeum vulgare, at 88.61%. The approximately 2000 bp promoter sequence of the HvANS gene was identical in both varieties. The real-time fluorescence PCR (qRT-PCR) results revealed that HvANS expression was either absent or very low in the roots, stems, leaves, and awns of Nierumuzha. In contrast, the HvANS expression was high in the seed coats and seeds of Nierumuzha. Likewise, in Kunlun 10, HvANS expression was either absent or very low, indicating a tissue-specific and variety-specific pattern for HvANS expression. The subcellular localization results indicated that HvANS was in the cell membrane. Metabolomic results indicated that the HvANS gene is closely related to the synthesis of three anthocyanin substances (Idaein chloride, Kinetin 9-riboside, and Cyanidin O-syringic acid). Yeast single hybridization experiments showed that the HvANS promoter interacted with HvANT1, which is the key anthocyanin regulatory protein. In a yeast two-hybrid experiment, we obtained two significantly different proteins (ZWY2020 and POMGNT2-like) and verified the results by qRT-PCR. CONCLUSIONS: These results provide a basis for further studies on the regulatory mechanism of HvANS in the synthesis of anthocyanins in Qingke purple grains.

Rapid improvement of grain appearance in three-line hybrid rice via CRISPR/Cas9 editing of grain size genes.

KEY MESSAGE: Genetic editing of grain size genes quickly improves three-line hybrid rice parents to increase the appearance quality and yield of hybrid rice. Grain size affects rice yield and quality. In this study, we used CRISPR/Cas9 to edit the grain size gene GW8 in the maintainer line WaitaiB (WTB) and restorer line Guanghui998 (GH998). The new slender sterile line WTEA (gw8) was obtained in the BC2F1 generation by transferring the grain mutation of the maintainer plant to the corresponding sterile line WantaiA (WTA, GW8) in the T1 generation. Two slender restorer lines, GH998E1 (gw8(II)) and GH998E2 (gw8(I)), were obtained in T1 generation. In the early stage, new sterile and restorer lines in grain mutations were created by targeted editing of GS3, TGW3, and GW8 genes. These parental lines were mated to detect the impact of grain-type mutations on hybrid rice yield and quality. Mutations in gs3, gw8, and tgw3 had a minimal impact on agronomic traits except the grain size and thousand-grain weight. The decrease in grain width in the combination mainly came from gw8/gw8, gs3/gs3 increased the grain length, gs3/gs3-gw8/gw8 had a more significant effect on the grain length, and gs3/gs3-gw8/gw8(I) contributed more to grain length than gs3/gs3-gw8/gw8(II). The heterozygous TGW3/tgw3 may not significantly increase grain length. Electron microscopy revealed that the low-chalky slender-grain variety had a cylindrical grain shape, a uniform distribution of endosperm cells, and tightly arranged starch grains. Quantitative fluorescence analysis of endospermdevelopment-related genes showed that the combination of slender grain hybrid rice caused by gs3 and gw8 mutations promoted endosperm development and improved appearance quality. An appropriate grain size mutation resulted in hybrid rice varieties with high yield and quality.

Identifying critical growth stage and resilient genotypes in cowpea under drought stress contributes to enhancing crop tolerance for improvement and adaptation in Cameroon.

Drought stress following climate change is likely a scenario that will have to face crop growers in tropical regions. In mitigating this constraint, the best option should be the selection and use of resilient varieties that can withstand drought threats. Therefore, a pot experiment was conducted under greenhouse conditions at the Research and Teaching Farm of the Faculty of Agronomy and Agricultural Sciences of the University of Dschang. The objectives are to identify sensitive growth stage, to identify drought-tolerant genotypes with the help of yield-based selection indices and to identify suitable selection indices that are associated with yield under non-stress and stress circumstances. Eighty-eight cowpea genotypes from the sahelian and western regions of Cameroon were subjected to drought stress at vegetative (VDS) and flowering (FDS) stages by withholding water for 28 days, using a split plot design with two factors and three replications. Seed yields under stress (Ys) and non-stress (Yp) conditions were recorded. Fifteen drought indices were calculated for the two drought stress levels against the yield from non-stress plants. Drought Intensity Index (DII) under VDS and FDS were 0.71 and 0.84 respectively, indicating severe drought stress for both stages. However, flowering stage was significantly more sensitive to drought stress compared to vegetative stage. Based on PCA and correlation analysis, Stress Tolerance Index (STI), Relative Efficiency Index (REI), Geometric Mean Productivity (GMP), Mean Productivity (MP), Yield Index (YI) and Harmonic Mean (HM) correlated strongly with yield under stress and non-stress conditions and are therefore suitable to discriminate high-yielding and tolerant genotypes under both stress and non-stress conditions. Either under VDS and FDS, CP-016 exhibited an outstanding performance under drought stress and was revealed as the most drought tolerant genotype as shown by ranking, PCA and cluster analysis. Taking into account all indices, the top five genotypes namely CP-016, CP-021, MTA-22, CP-056 and CP-060 were identified as the most drought-tolerant genotypes under VDS. For stress activated at flowering stage (FDS), CP-016, CP-056, CP-021, CP-028 and MTA-22 were the top five most drought-tolerant genotypes. Several genotypes with insignificant Ys and irrelevant rank among which CP-037, NDT-001, CP-036, CP-034, NDT-002, CP-031, NDT-011 were identified as highly drought sensitive with low yield stability. This study identified the most sensitive stage and drought tolerant genotypes that are proposed for genetic improvement of cowpea.

Central Roles of ZmNAC128 and ZmNAC130 in Nutrient Uptake and Storage during Maize Grain Filling.

Grain filling is critical for determining yield and quality, raising the question of whether central coordinators exist to facilitate the uptake and storage of various substances from maternal to filial tissues. The duplicate NAC transcription factors ZmNAC128 and ZmNAC130 could potentially serve as central coordinators. By analyzing differentially expressed genes from zmnac128 zmnac130 mutants across different genetic backgrounds and growing years, we identified 243 highly and differentially expressed genes (hdEGs) as the core target genes. These 243 hdEGs were associated with storage metabolism and transporters. ZmNAC128 and ZmNAC130 play vital roles in storage metabolism, and this study revealed two additional starch metabolism-related genes, sugary enhancer1 and hexokinase1, as their direct targets. A key finding of this study was the inclusion of 17 transporter genes within the 243 hdEGs, with significant alterations in the levels of more than 10 elements/substances in mutant kernels. Among them, six out of the nine upregulated transporter genes were linked to the transport of heavy metals and metalloids (HMMs), which was consistent with the enrichment of cadmium, lead, and arsenic observed in mutant kernels. Interestingly, the levels of Mg and Zn, minerals important to biofortification efforts, were reduced in mutant kernels. In addition to their direct involvement in sugar transport, ZmNAC128 and ZmNAC130 also activate the expression of the endosperm-preferential nitrogen and phosphate transporters ZmNPF1.1 and ZmPHO1;2. This coordinated regulation limits the intake of HMMs, enhances biofortification, and facilitates the uptake and storage of essential nutrients.

Genome Size Variation in Sesamum indicum L. Germplasm from Niger.

Sesamum indicum L. (Pedaliaceae) is one of the most economically important oil crops in the world, thanks to the high oil content of its seeds and its nutritional value. It is cultivated all over the world, mainly in Asia and Africa. Well adapted to arid environments, sesame offers a good opportunity as an alternative subsistence crop for farmers in Africa, particularly Niger, to cope with climate change. For the first time, the variation in genome size among 75 accessions of the Nigerien germplasm was studied. The sample was collected throughout Niger, revealing various morphological, biochemical and phenological traits. For comparison, an additional accession from Thailand was evaluated as an available Asian representative. In the Niger sample, the 2C DNA value ranged from 0.77 to 1 pg (753 to 978 Mbp), with an average of 0.85 ± 0.037 pg (831 Mbp). Statistical analysis showed a significant difference in 2C DNA values among 58 pairs of Niger accessions (p-value < 0.05). This significant variation indicates the likely genetic diversity of sesame germplasm, offering valuable insights into its possible potential for climate-resilient agriculture. Our results therefore raise a fundamental question: is intraspecific variability in the genome size of Nigerien sesame correlated with specific morphological and physiological traits?

QTL Analysis of ß-Glucan Content and Other Grain Traits in a Recombinant Population of Spring Barley.

Barley with high grain ß-glucan content is valuable for functional foods. The identification of loci for high ß-glucan content is, thus, of great importance for barley breeding. Segregation mapping for the content in ß-glucan and other barley grain components (starch, protein, lipid, ash, phosphorous, calcium, sodium) was performed using the progeny of the cross between Glacier AC38, a mutant with high amylose, and CDC Fibar, a high ß-glucan waxy cultivar. The offspring of this cross showed transgressive segregation for ß-glucan content. Linkage analysis based on single-nucleotide polymorphism (SNP) molecular markers was used for the genotyping of the parents and recombinant inbred lines (RILs). Two Quantitative Trait Loci (QTL) for ß-glucan content and several QTL for other grain components were found. The former ones, located on chromosomes 1H and 7H, explained 27.9% and 27.4% of the phenotypic variance, respectively. Glacier AC38 provided the allele for high ß-glucan content at the QTL on chromosome 1H, whereas CDC Fibar contributed the allele at the QTL on chromosome 7H. Their recombination resulted in a novel haplotype with higher ß-glucan content, up to 18.4%. Candidate genes are proposed for these two QTL: HvCslF9, involved in ß-glucan biosynthesis, for the QTL on chromosome 1H; Horvu_PLANET_7H01G069300, a gene encoding an ATP-Binding Cassette (ABC) transporter, for the QTL on chromosome 7H.

Enhancing Erucic Acid and Wax Ester Production in Brassica carinata through Metabolic Engineering for Industrial Applications.

Metabolic engineering enables oilseed crops to be more competitive by having more attractive properties for oleochemical industrial applications. The aim of this study was to increase the erucic acid level and to produce wax ester (WE) in seed oil by genetic transformation to enhance the industrial applications of B. carinata. Six transgenic lines for high erucic acid and fifteen transgenic lines for wax esters were obtained. The integration of the target genes for high erucic acid (BnFAE1 and LdPLAAT) and for WEs (ScWS and ScFAR) in the genome of B. carinata cv. 'Derash' was confirmed by PCR analysis. The qRT-PCR results showed overexpression of BnFAE1 and LdPLAAT and downregulation of RNAi-BcFAD2 in the seeds of the transgenic lines. The fatty acid profile and WE content and profile in the seed oil of the transgenic lines and wild type grown in biotron were analyzed using gas chromatography and nanoelectrospray coupled with tandem mass spectrometry. A significant increase in erucic acid was observed in some transgenic lines ranging from 19% to 29% in relation to the wild type, with a level of erucic acid reaching up to 52.7%. Likewise, the transgenic lines harboring ScFAR and ScWS genes produced up to 25% WE content, and the most abundant WE species were 22:1/20:1 and 22:1/22:1. This study demonstrated that metabolic engineering is an effective biotechnological approach for developing B. carinata into an industrial crop.

MassARRAY and SABER Analyses of SNPs in Embryo DNA Reveal the Abscission of Self-Fertilised Progeny during Fruit Development of Macadamia (Macadamia integrifolia Maiden & Betche).

Yield in many crops is affected by abscission during the early stages of fruitlet development. The reasons for fruitlet abscission are often unclear but they may include genetic factors because, in some crops, self-pollinated fruitlets are more likely to abscise than cross-pollinated fruitlets. Pollen parentage can also affect final fruit size and fruit quality. Here, we aimed to understand the effects of pollen parentage on fruitlet retention and nut quality in orchards of macadamia (Macadamia integrifolia Maiden & Betche). We identified the pollen parent of macadamia 'cultivar '816' embryos by analysing single nucleotide polymorphisms (SNPs) in their DNA using customised MassARRAY and Single Allele Base Extension Reaction (SABER) methods. This allowed us to determine the proportions of self-fertilised and cross-fertilised progeny during premature fruit drop at 6 weeks and 10 weeks after peak anthesis, as well as at nut maturity. We determined how pollen parentage affected nut-in-shell (NIS) mass, kernel mass, kernel recovery, and oil concentration. Macadamia trees retained cross-fertilised fruitlets rather than self-fertilised fruitlets. The percentage of progeny that were cross-fertilised increased from 6% at 6 weeks after peak anthesis to 97% at nut maturity, with each tree producing on average 22 self-fertilised nuts and 881 cross-fertilised nuts. Three of the four cross-pollen parents provided fruit with significantly higher NIS mass, kernel mass, or kernel recovery than the few remaining self-fertilised fruit. Fruit that were cross-fertilised by '842', 'A4', or 'A203' had 16-29% higher NIS mass and 24-44% higher kernel mass than self-fertilised fruit. Nuts that were cross-fertilised by 'A4' or 'A203' also had 5% or 6% higher kernel recovery, worth approximately $US460-540 more per ton for growers than self-fertilised nuts. The highly selective abscission of self-fertilised fruitlets and the lower nut quality of self-fertilised fruit highlight the critical importance of cross-pollination for macadamia productivity.

Modulation of histone acetylation enables fully mechanized hybrid rice breeding.

Hybrid rice has achieved high grain yield and greatly contributes to food security, but the manual-labour-intensive hybrid seed production process limits fully mechanized hybrid rice breeding. For next-generation hybrid seed production, the use of small-grain male sterile lines to mechanically separate small hybrid seeds from mixed harvest is promising. However, it is difficult to find ideal grain-size genes for breeding ideal small-grain male sterile lines without penalties in the number of hybrid seeds and hybrid rice yield. Here we report that the use of small-grain alleles of the ideal grain-size gene GSE3 in male sterile lines enables fully mechanized hybrid seed production and dramatically increases hybrid seed number in three-line and two-line hybrid rice systems. The GSE3 gene encodes a histone acetyltransferase that binds histones and influences histone acetylation levels. GSE3 is recruited by the transcription factor GS2 to the promoters of their co-regulated grain-size genes and influences the histone acetylation status of their co-regulated genes. Field trials demonstrate that genome editing of GSE3 can be used to immediately improve current elite male sterile lines of hybrid rice for fully mechanized hybrid rice breeding, providing a new perspective for mechanized hybrid breeding in other crops.

The GA and ABA signaling is required for hydrogen-mediated seed germination in wax gourd.

BACKGROUND: Hydrogen gas (H2), a novel and beneficial gaseous molecule, plays a significant role in plant growth and development processes. Hydrogen-rich water (HRW) is regarded as a safe and easily available way to study the physiological effects of H2 on plants. Several recent research has shown that HRW attenuates stress-induced seed germination inhibition; however, the underlying modes of HRW on seed germination remain obscure under non-stress condition. RESULTS: In this current study, we investigated the possible roles of gibberellin (GA) and abscisic acid (ABA) in HRW-regulated seed germination in wax gourd (Benincasa hispida) through pharmacological, physiological, and transcriptome approaches. The results showed that HRW application at an optimal dose (50% HRW) significantly promoted seed germination and shortened the average germination time (AGT). Subsequent results suggested that 50% HRW treatment stimulated GA production by regulating GA biosynthesis genes (BhiGA3ox, BhiGA2ox, and BhiKAO), whereas it had no effect on the content of ABA and the expression of its biosynthesis (BhiNCED6) and catabolism genes (BhiCYP707A2) but decreased the expression of ABA receptor gene (BhiPYL). In addition, inhibition of GA production by paclobutrazol (PAC) could block the HRW-mediated germination. Treatment with ABA could hinder HRW-mediated seed germination and the ABA biosynthesis inhibitor sodium tungstate (ST) could recover the function of HRW. Furthermore, RNA-seq analysis revealed that, in the presence of GA or ABA, an abundance of genes involved in GA, ABA, and ethylene signal sensing and transduction might involve in HRW-regulated germination. CONCLUSIONS: This study portrays insights into the mechanism of HRW-mediated seed germination, suggesting that HRW can regulate the balance between GA and ABA to mediate seed germination through ethylene signals in wax gourd.

Generation of New ß-Conglycinin-Deficient Soybean Lines by Editing the lincRNA lincCG1 Using the CRISPR/Cas9 System.

Soybean ß-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in ß-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean ß-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the ß-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and ß-subunits of soybean ß-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the ß-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and ß-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein ß-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the ß-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

SUnSeT: spectral unmixing of hyperspectral images for phenotyping soybean seed traits.

KEY MESSAGE: Hyperspectral features enable accurate classification of soybean seeds using linear discriminant analysis and GWAS for novel seed trait genes. Evaluating crop seed traits such as size, shape, and color is crucial for assessing seed quality and improving agricultural productivity. The introduction of the SUnSet toolbox, which employs hyperspectral sensor-derived image analysis, addresses this necessity. In a validation test involving 420 seed accessions from the Korean Soybean Core Collections, the pixel purity index algorithm identified seed- specific hyperspectral endmembers to facilitate segmentation. Various metrics extracted from ventral and lateral side images facilitated the categorization of seeds into three size groups and four shape groups. Additionally, quantitative RGB triplets representing seven seed coat colors, averaged reflectance spectra, and pigment indices were acquired. Machine learning models, trained on a dataset comprising 420 accession seeds and 199 predictors encompassing seed size, shape, and reflectance spectra, achieved accuracy rates of 95.8% for linear discriminant analysis model. Furthermore, a genome-wide association study utilizing hyperspectral features uncovered associations between seed traits and genes governing seed pigmentation and shapes. This comprehensive approach underscores the effectiveness of SUnSet in advancing precision agriculture through meticulous seed trait analysis.

Two MADS-box proteins, AGL9 and AGL15, recruit the FIS-PRC2 complex to trigger the phase transition from endosperm proliferation to embryo development in Arabidopsis.

Spatiotemporal regulation of gene expression by polycomb repressive complex 2 (PRC2) is critical for animal and plant development. The Arabidopsis fertilization independent seed (FIS)-PRC2 complex functions specifically during plant reproduction from gametogenesis to seed development. After a double fertilization event, triploid endosperm proliferates early, followed by the growth of a diploid embryo, which replaces the endosperm in Arabidopsis and many dicots. Key genes critical for endosperm proliferation such as IKU2 and MINI3 are activated after fertilization. Here we report that two MADS-box AGAMOUS-LIKE (AGL) proteins associate with the key endosperm proliferation loci and recruit the FIS-PRC2 repressive complex at 4-5 days after pollination (DAP). Interestingly, AGL9 and AGL15 only accumulate toward the end of endosperm proliferation at 4-5 DAP and promote the deposition of H3K27me3 marks at key endosperm proliferation loci. Disruption of AGL9 and AGL15 or overexpression of AGL9 or AGL15 significantly influence endosperm proliferation and cellularization. Genome-wide analysis with cleavage Under Targets and tagmentation (CUT&Tag) sequencing and RNA sequencing revealed the landscape of endosperm H3K27me3 marks and gene expression profiles in Col-0 and agl9 agl15. CUT&Tag qPCR also demonstrated the occupancy of the two MADS-box proteins and FIS-PRC2 on a few representative target loci. Our studies suggest that MADS-box proteins could potentially recruit PRC2 to regulate many other developmental processes in plants or even in fungi and animals.

Genome-wide characterization of Related to ABI3/VP1 transcription factors among U's triangle Brassica species reveals a negative role for BnaA06.RAV3L in seed size.

The transcription factors Related to ABI3/VP1 (RAV) are crucial for various plant processes and stress responses. Although the U's triangle Brassica species genomes have been released, the knowledge regarding the RAV family is still limited. In this study, we identified 123 putative RAV genes across the six U's triangle Brassica species (Brassica rapa, 14; Brassica oleracea, 14; Brassica nigra, 13; Brassica carinata, 27; Brassica juncea, 28; Brassica napus, 27). Phylogenetic analysis categorized them into three groups. The RAV genes exhibited diversity in both functional and structural aspects, particularly in gene structure and cis-acting elements within their promoters. The expression analysis revealed that BnaRAV genes in Group 1/2 exhibited diverse expression patterns across various tissues, while those in Group 3 did not show expression except for BnaRAV3L-2 and BnaRAV3L-6, which were exclusively expressed in seeds. Furthermore, the seed-specific expression of BnaA06. RAV3L (BnaRAV3L-2) was confirmed through promoter-GUS staining. Subcellular localization studies demonstrated that BnaA06.RAV3L is localized to the nucleus. The overexpression of BnaA06. RAV3L in Arabidopsis led to a remarkable inhibition of seed-specific traits such as seed width, seed length, seed area, and seed weight. This study provides insights into the functional evolution of the RAV gene family in U triangle Brassica species. It establishes a foundation for uncovering the molecular mechanisms underlying the negative role of RAV3L in seed development.