Mitochondrial VOLTAGE-DEPENDENT ANION CHANNEL 3 regulates stomatal closure by abscisic acid signaling.

In Arabidopsis (Arabidopsis thaliana), stomatal closure mediated by abscisic acid (ABA) is redundantly controlled by ABA receptor family proteins (PYRABACTIN RESISTANCE 1 [PYR1]/PYR1-LIKE [PYLs]) and subclass III SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2 (SnRK2s). Among these proteins, the roles of PYR1, PYL2, and SnRK2.6 are more dominant. A recent discovery showed that ABA-induced accumulation of reactive oxygen species (ROS) in mitochondria promotes stomatal closure. By analyzing stomatal movements in an array of single and higher order mutants, we revealed that the mitochondrial protein VOLTAGE-DEPENDENT ANION CHANNEL 3 (VDAC3) jointly regulates ABA-mediated stomatal closure with a specialized set of PYLs and SnRK2s by affecting cellular and mitochondrial ROS accumulation. VDAC3 interacted with 9 PYLs and all 3 subclass III SnRK2s. Single mutation in VDAC3, PYLs (except PYR1 and PYL2), or SnRK2.2/2.3 had little effect on ABA-mediated stomatal closure. However, knocking out PYR1, PYL1/2/4/8, or SnRK2.2/2.3 in vdac3 mutants resulted in significantly delayed or attenuated ABA-mediated stomatal closure, despite the presence of other PYLs or SnRK2s conferring redundant functions. We found that cellular and mitochondrial accumulation of ROS induced by ABA was altered in vdac3pyl1 mutants. Moreover, H2O2 treatment restored ABA-induced stomatal closure in mutants with decreased stomatal sensitivity to ABA. Our work reveals that VDAC3 ensures redundant control of ABA-mediated stomatal closure by canonical ABA signaling components.

Genetic dissection of maize (Zea mays L.) chlorophyll content using multi-locus genome-wide association studies.

BACKGROUND: The chlorophyll content (CC) is a key factor affecting maize photosynthetic efficiency and the final yield. However, its genetic basis remains unclear. The development of statistical methods has enabled researchers to design and apply various GWAS models, including MLM, MLMM, SUPER, FarmCPU, BLINK and 3VmrMLM. Comparative analysis of their results can lead to more effective mining of key genes. RESULTS: The heritability of CC was 0.86. Six statistical models (MLM, BLINK, MLMM, FarmCPU, SUPER, and 3VmrMLM) and 1.25 million SNPs were used for the GWAS. A total of 140 quantitative trait nucleotides (QTNs) were detected, with 3VmrMLM and MLM detecting the most (118) and fewest (3) QTNs, respectively. The QTNs were associated with 481 genes and explained 0.29-10.28% of the phenotypic variation. Additionally, 10 co-located QTNs were detected by at least two different models or methods, three co-located QTNs were identified in at least two different environments, and six co-located QTNs were detected by different models or methods in different environments. Moreover, 69 candidate genes within or near these stable QTNs were screened based on the B73 (RefGen_v2) genome. GRMZM2G110408 (ZmCCS3) was identified by multiple models and in multiple environments. The functional characterization of this gene indicated the encoded protein likely contributes to chlorophyll biosynthesis. In addition, the CC differed significantly between the haplotypes of the significant QTN in this gene, and CC was higher for haplotype 1. CONCLUSION: This study's results broaden our understanding of the genetic basis of CC, mining key genes related to CC and may be relevant for the ideotype-based breeding of new maize varieties with high photosynthetic efficiency.

The genetic architecture of prolificacy in maize revealed by association mapping and bulk segregant analysis.

KEY MESSAGE: Here, we revealed maize prolificacy highly correlated with domestication and identified a causal gene ZmEN1 located in one novel QTL qGEN261 that regulating maize prolificacy by using multiple-mapping methods. The development of maize prolificacy (EN) is crucial for enhancing yield and breeding specialty varieties. To achieve this goal, we employed a genome-wide association study (GWAS) to analyze the genetic architecture of EN in maize. Using 492 inbred lines with a wide range of EN variability, our results demonstrated significant differences in genetic, environmental, and interaction effects. The broad-sense heritability (H2) of EN was 0.60. Through GWAS, we identified 527 significant single nucleotide polymorphisms (SNPs), involved 290 quantitative trait loci (QTL) and 806 genes. Of these SNPs, 18 and 509 were classified as major effect loci and minor loci, respectively. In addition, we performed a bulk segregant analysis (BSA) in an F2 population constructed by a few-ears line Zheng58 and a multi-ears line 647. Our BSA results identified one significant QTL, qBEN1. Importantly, combining the GWAS and BSA, four co-located QTL, involving six genes, were identified. Three of them were expressed in vegetative meristem, shoot tip, internode and tip of ear primordium, with ZmEN1, encodes an unknown auxin-like protein, having the highest expression level in these tissues. It suggested that ZmEN1 plays a crucial role in promoting axillary bud and tillering to encourage the formation of prolificacy. Haplotype analysis of ZmEN1 revealed significant differences between different haplotypes, with inbred lines carrying hap6 having more EN. Overall, this is the first report about using GWAS and BSA to dissect the genetic architecture of EN in maize, which can be valuable for breeding specialty maize varieties and improving maize yield.

Physicochemical Characteristics and the Scale Inhibition Effect of Air Nanobubbles (A-NBs) in a Circulating Cooling Water System.

Air nanobubbles (A-NBs) in a circulating cooling water system have not been investigated, although their role is significant. In this paper, the influences of the contents of main salts and other parameters on the physicochemical characteristics and scale inhibition performance of A-NBs in circulating cooling water were investigated and the scale inhibition mechanism of A-NBs in a simulated circulating cooling water system was explored. A-NBs realized a higher scale inhibition rate of 90%, which was higher than that of 1-hydroxyethane-1,1-diphosphonic acid (40%), and A-NBs stably existed for more than 5 days in the complex water environment. Four interface functions were proposed to interpret the scale inhibition effect of A-NBs in circulating cooling water as follows. First, the negatively charged surface of A-NBs adsorbed cations (Ca2+) reduced the concentration of scaling ions. Second, the negatively charged surface of A-NBs could also adsorb microcrystals, and their crystal-like seed action was conducive to the formation of large-size crystals, broke the rules of crystal growth, and reduced the adhesion of scales to the pipe wall. Third, A-NBs could also form a bubble layer after they were adsorbed on the inner surface of pipes, thereby preventing the deposition of scales on the surface. Fourth, A-NB burst caused local turbulence, increased the shear force onto the pipe surface, and reduced the scales adhering to the pipe surface. The interface effect of A-NBs in metal pipes is important in many industrial applications. This study laid the basis for the development of a new green A-NB scale-inhibiting technology.

Identification of novel loci associated with starch content in maize kernels by a genome-wide association study using an enlarged SNP panel.

Starch is a major component of cereals, comprising over 70% of dry weight. It serves as a primary carbon source for humans and animals. In addition, starch is an indispensable industrial raw material. While maize (Zea mays) is a key crop and the primary source of starch, the genetic basis for starch content in maize kernels remains poorly understood. In this study, using an enlarged panel, we conducted a genome-wide association study (GWAS) based on best linear unbiased prediction (BLUP) value for starch content of 261 inbred lines across three environments. Compared with previous study, we identified 14 additional significant quantitative trait loci (QTL), encompassed a total of 42 genes, and indicated that increased marker density contributes to improved statistical power. By integrating gene expression profiling, Gene Ontology (GO) enrichment and haplotype analysis, several potential target genes that may play a role in regulating starch content in maize kernels have been identified. Notably, we found that ZmAPC4, associated with the significant SNP chr4.S_175584318, which encodes a WD40 repeat-like superfamily protein and is highly expressed in maize endosperm, might be a crucial regulator of maize kernel starch synthesis. Out of the 261 inbred lines analyzed, they were categorized into four haplotypes. Remarkably, it was observed that the inbred lines harboring hap4 demonstrated the highest starch content compared to the other haplotypes. Additionally, as a significant achievement, we have developed molecular markers that effectively differentiate maize inbred lines based on their starch content. Overall, our study provides valuable insights into the genetic basis of starch content and the molecular markers can be useful in breeding programs aimed at developing maize varieties with high starch content, thereby improving breeding efficiency. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01437-6.

Dissection of the genetic architecture of peduncle vascular bundle-related traits in maize by a genome-wide association study.

The peduncle vascular system of maize is critical for the transport of photosynthetic products, nutrients, and water from the roots and leaves to the ear. Accordingly, it positively affects the grain yield. However, the genetic basis of peduncle vascular bundle (PVB)-related traits in maize remains unknown. Thus, 15 PVB-related traits of 386 maize inbred lines were investigated at three locations (Yongcheng, 17YC; Kaifeng, 20KF; and Yuanyang, 20YY). The repeatability for the 15 traits ranged from 35.53% to 92.13%. A genome-wide association study was performed and 69 non-redundant quantitative trait loci (QTL) were detected, including 9, 41, and 27 QTL identified at 17YC, 20KF, and 20YY, respectively. These QTL jointly explained 4.72% (SLL) to 37.30% (NSVB) of the phenotypic variation. Eight QTL were associated with the same trait at two locations. Furthermore, four pleiotropic QTL were identified. Moreover, one QTL (qPVB44), associated with NSVB_20KF, was co-localized with a previously reported locus related to kernel width, implying qPVB44 may affect the kernel width by modulating the number of small vascular bundles. Examinations of the 69 QTL identified 348 candidate genes that were classified in five groups. Additionally, 26 known VB-related homologous genes (e.g. VLN2, KNOX1, and UGT72B3) were detected in 20 of the 69 QTL. A comparison of the NSVB between a Zmvln2 EMS mutant and its wild type elucidated the function of the candidate gene ZmVLN2. These results are important for clarifying the genetic basis of PVB-related traits and may be useful for breeding new high-yielding maize cultivars.

Integrative Dissection of Lignin Composition in Tartary Buckwheat Seed Hulls for Enhanced Dehulling Efficiency.

The rigid hull encasing Tartary buckwheat seeds necessitates a laborious dehulling process before flour milling, resulting in considerable nutrient loss. Investigation of lignin composition is pivotal in understanding the structural properties of tartary buckwheat seeds hulls, as lignin is key determinant of rigidity in plant cell walls, thus directly impacting the dehulling process. Here, the lignin composition of seed hulls from 274 Tartary buckwheat accessions is analyzed, unveiling a unique lignin chemotype primarily consisting of G lignin, a common feature in gymnosperms. Furthermore, the hardness of the seed hull showed a strong negative correlation with the S lignin content. Genome-wide detection of selective sweeps uncovered that genes governing the biosynthesis of S lignin, specifically two caffeic acid O-methyltransferases (COMTs) and one ferulate 5-hydroxylases, are selected during domestication. This likely contributed to the increased S lignin content and decreased hardness of seed hulls from more domesticated varieties. Genome-wide association studies identified robust associations between FtCOMT1 and the accumulation of S lignin in seed hull. Transgenic Arabidopsis comt1 plants expressing FtCOMT1 successfully reinstated S lignin content, confirming its conserved function across plant species. These findings provide valuable metabolic and genetic insights for the potential redesign of Tartary buckwheat seed hulls.

Identification of Novel QTL for Mercury Accumulation in Maize Using an Enlarged SNP Panel.

Mercury (Hg) pollution not only poses a threat to the environment but also adversely affects the growth and development of plants, with potential repercussions for animals and humans through bioaccumulation in the food chain. Maize, a crucial source of food, industrial materials, and livestock feed, requires special attention in understanding the genetic factors influencing mercury accumulation. Developing maize varieties with low mercury accumulation is vital for both maize production and human health. In this study, a comprehensive genome-wide association study (GWAS) was conducted using an enlarged SNP panel comprising 1.25 million single nucleotide polymorphisms (SNPs) in 230 maize inbred lines across three environments. The analysis identified 111 significant SNPs within 78 quantitative trait loci (QTL), involving 169 candidate genes under the Q model. Compared to the previous study, the increased marker density and optimized statistical model led to the discovery of 74 additional QTL, demonstrating improved statistical power. Gene ontology (GO) enrichment analysis revealed that most genes participate in arsenate reduction and stress responses. Notably, GRMZM2G440968, which has been reported in previous studies, is associated with the significant SNP chr6.S_155668107 in axis tissue. It encodes a cysteine proteinase inhibitor, implying its potential role in mitigating mercury toxicity by inhibiting cysteine. Haplotype analyses provided further insights, indicating that lines carrying hap3 exhibited the lowest mercury content compared to other haplotypes. In summary, our study significantly enhances the statistical power of GWAS, identifying additional genes related to mercury accumulation and metabolism. These findings offer valuable insights into unraveling the genetic basis of mercury content in maize and contribute to the development of maize varieties with low mercury accumulation.

VAMP726 from maize and Arabidopsis confers pollen resistance to heat and UV radiation by influencing lignin content of sporopollenin.

Sporopollenin in the pollen cell wall protects male gametophytes from stresses. Phenylpropanoid derivatives, including guaiacyl (G) lignin units, are known to be structural components of sporopollenin, but the exact composition of sporopollenin remains to be fully resolved. We analyzed the phenylpropanoid derivatives in sporopollenin from maize and Arabidopsis by thioacidolysis coupled with nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The NMR and GC-MS results confirmed the presence of p-hydroxyphenyl (H), G, and syringyl (S) lignin units in sporopollenin from maize and Arabidopsis. Strikingly, H units account for the majority of lignin monomers in sporopollenin from these species. We next performed a genome-wide association study to explore the genetic basis of maize sporopollenin composition and identified a vesicle-associated membrane protein (ZmVAMP726) that is strongly associated with lignin monomer composition of maize sporopollenin. Genetic manipulation of VAMP726 affected not only lignin monomer composition in sporopollenin but also pollen resistance to heat and UV radiation in maize and Arabidopsis, indicating that VAMP726 is functionally conserved in monocot and dicot plants. Our work provides new insight into the lignin monomers that serve as structural components of sporopollenin and characterizes VAMP726, which affects sporopollenin composition and stress resistance in pollen.

Candidate loci for leaf angle in maize revealed by a combination of genome-wide association study and meta-analysis.

Leaf angle (LA) is a key component of maize plant architecture that can simultaneously govern planting density and improve final yield. However, the genetic mechanisms underlying LA have not been fully addressed. To broaden our understanding of its genetic basis, we scored three LA-related traits on upper, middle, and low leaves of 492 maize inbred lines in five environments. Phenotypic data revealed that the three LA-related traits were normally distributed, and significant variation was observed among environments and genotypes. A genome-wide association study (GWAS) was then performed to dissect the genetic factors that control natural variation in maize LA. In total, 85 significant SNPs (involving 32 non-redundant QTLs) were detected (p ≤ 2.04 × 10-6), and individual QTL explained 4.80%-24.09% of the phenotypic variation. Five co-located QTL were detected in at least two environments, and two QTLs were co-located with multiple LA-related traits. Forty-seven meta-QTLs were identified based on meta-analysis combing 294 LA-related QTLs extracted from 18 previously published studies, 816 genes were identified within these meta-QTLs, and seven co-located QTLs were jointly identified by both GWAS and meta-analysis. ZmULA1 was located in one of the co-located QTLs, qLA7, and its haplotypes, hap1 and hap2, differed significantly in LA-related traits. Interestingly, the temperate materials with hap2 had smallest LA. Finally, we also performed haplotype analysis using the reported genes that regulate LA, and identified a lot of maize germplasms that aggregated favorable haplotypes. These results will be helpful for elucidating the genetic basis of LA and breeding new maize varieties with ideal plant architecture.

Multi-Omics Analyses Reveal Systemic Insights into Maize Vivipary.

Maize vivipary, precocious seed germination on the ear, affects yield and seed quality. The application of multi-omics approaches, such as transcriptomics or metabolomics, to classic vivipary mutants can potentially reveal the underlying mechanism. Seven maize vivipary mutants were selected for transcriptomic and metabolomic analyses. A suite of transporters and transcription factors were found to be upregulated in all mutants, indicating that their functions are required during seed germination. Moreover, vivipary mutants exhibited a uniform expression pattern of genes related to abscisic acid (ABA) biosynthesis, gibberellin (GA) biosynthesis, and ABA core signaling. NCED4 (Zm00001d007876), which is involved in ABA biosynthesis, was markedly downregulated and GA3ox (Zm00001d039634) was upregulated in all vivipary mutants, indicating antagonism between these two phytohormones. The ABA core signaling components (PYL-ABI1-SnRK2-ABI3) were affected in most of the mutants, but the expression of these genes was not significantly different between the vp8 mutant and wild-type seeds. Metabolomics analysis integrated with co-expression network analysis identified unique metabolites, their corresponding pathways, and the gene networks affected by each individual mutation. Collectively, our multi-omics analyses characterized the transcriptional and metabolic landscape during vivipary, providing a valuable resource for improving seed quality.