Hatching and development of maize cyst nematode Heterodera zeae infecting different plant hosts

The occurrence, distribution, and rapid molecular detection technology of Heterodera zeae Koshy et al. 1971, have been reported in China. We explored the biological characteristics of H. zeae sampled in Henan Province, China to understand its interaction with plants. Cysts and second-stage juveniles(J2s) were identified under an optical and scanning electron microscope, internal transcribed spacer(ITS) phylogenetic tree, and sequence characterized amplified region(SCAR)-PCR analyses. The optimum hatching temperatures of H. zeae were 30°C and 28°C, with cumulative hatching rates of 16.5 and 16.1%, respectively, at 30 days post-hatching(dph). The hatching rate of H. zeae eggs was improved by 20-and 50-time maize soil leachate and root juice, and 10-time root exudates. The hatching rate in 10-time root exudates was the highest(25.9%). The 10-time root exudates of maize and millet produced the highest hatching rate at 30 dph(25.9 and 22.9%, respectively), followed by wheat(19.9%), barley(18.3%), and rice(17.6%). Heterodera zeae developed faster in maize than in other crops. Fourth-stage juveniles(J4s) were detected in maize roots 8 days post-inoculation(dpi) at 28°C but not in other crops. Combined with hatching tests, the Huang–Huai–Hai summer maize region and the south and central-southwest mountainous maize areas are highly suitable for H. zeae in China. This is the first systematically study of the hatching and infection characteristics on different plant hosts of corn cyst nematode H. zeae in temperate regions. This study laid a theoretical foundation for the rapid spread and high environmental adaptability of corn cyst nematode.

ZmCYP90D1 regulates maize internode development by modulating brassinosteroid-mediated cell division and growth

Plant height(PH)is associated with lodging resistance and planting density,which is regulated by a complicated gene network.In this study,we identified a spontaneous dwarfing mutation in maize,m30,with decreased internode number and length but increased internode diameter.A candidate gene,ZmCYP90D1,which encodes a member of the cytochrome P450 family,was isolated by map-based cloning.ZmCYP90D1 was constitutively expressed and showed highest expression in basal internodes,and its protein was targeted to the nucleus.A G-to-A substitution was identified to be the causal mutation,which resulted in a truncated protein in m30.Loss of function of ZmCYP90D1 changed expression of hormoneresponsive genes,in particular brassinosteroid(BR)-responsive genes which is mainly involved in cell cycle regulation and cell wall extension and modification in plants.The concentration of typhasterol(TY),a downstream intermediate of ZmCYP90D1 in the BR pathway,was reduced.A haplotype conferring dwarfing without reducing yield was identified.ZmCYP90D1 was inferred to influence plant height and stalk diameter via hormone-mediated cell division and cell growth via the BR pathway.

A peptide chain release factor 2a gene regulates maize kernel development by modulating mitochondrial function

Mitochondrial protein translation that is essential for aerobic energy production includes four essential steps of the mitochondrial ribosome cycle,namely,initiation,elongation,termination of the polypeptide,and ribosome recycling.Translation termination initiates when a stop codon enters the A site of the mitochondrial ribosome where it is recognized by a dedicated peptide release factor(RF).However,RFs and mechanisms involved in translation in plant mitochondria,especially in monocotyledons,remain largely unknown.Here,we identified a crumpled kernel(crk5 allele)mutant,with significantly decreased kernel size,100-kernel weight,and an embryo-lethal phenotype.The Crk5 allele was isolated using map-based cloning and found to encode a mitochondrial localization RF2a.As it is an ortholog of Arabidopsis mitochondrial RF2a,we named the gene ZmmtRF2a.ZmmtRF2a is missing the 5th–7th exons in the crk5 resulting in deletion of domains containing motifs GGQ and SPF that are essential for release activity of RF,mitochondrial ribosome binding,and stop codon recognition.Western blot and qRT-PCR analyses indicate that the crk5 mutation results in abnormal mitochondrion structure and function.Intriguingly,we observed a feedback loop in the crk5 with up-regulated transcript levels detected for several mitochondrial ribosome and mitochondrial-related components,in particular mitochondrial complexes CI,CIV,and a ribosome assembly related PPR.Together,our data support a crucial role for ZmmtRF2a in regulation of mitochondrial structure and function in maize.

Comparative QTL analysis of maize seed artificial aging between an immortalized F_2 population and its corresponding RILs

Seed aging decreases the quality and vigor of crop seeds,thereby causing substantial agricultural and economic losses in crops.To identify genetic differences in seed aging between homozygotes and heterozygotes in maize,the seeds of a set of recombinant inbred lines(RILs) and an immortalized F_2(IF_2) population were subjected to artificial aging treatments for 0,2,3,and 4 days under 45℃ and 85%relative humidity and seed vigor was then evaluated in a field experiment.Seed vigor of all entries tested decreased sharply with longer aging treatment and seed vigor decreased more slowly in heterozygotes than in homozygotes.Forty-nine QTL were detected for four measured seed vigor traits in the RIL(28QTL) and IF_2(21 QTL) populations.Only one QTL,qGP5,was detected in both populations,indicating that the genes involved in anti-aging mechanisms differed between inbred lines and hybrids.Several QTL were identified to be responsible for multiple seed vigor traits simultaneously in the RIL and IF_2 populations under artificial aging conditions.These QTL may include major genes for seed vigor or seed aging.QTL qVI4 b and qGE3 a detected in the RIL population coincided with genes ZmLOX1 and ZmPLD1 in the same respective chromosomal regions.These QTL would be useful for screening for anti-aging genes in maize breeding.

Identification and isolation of Mu-flanking fragments from maize

Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics. Isolation of genomic segments flanking the insertion Mutator （Mu） is a key step in insertion mutagenesis studies. Herein, we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize. The method consists of the following steps： 1） double-digestion of genomic DNA with Bgl II/Msp I and ligation of digested fragments to the Bgl II- and Msp I-adaptors; 2） enrichment of a subset of Bgl II/Msp I fragments followed by selective amplification of the Mu-flanking fragments; 3） simultaneous display of AFLP bands derived from the flanking regions for both insert and native Mu transposons; 4） identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5） confirmation of flanking fragments related to these Mu insertions. Using this approach, we have isolated flanking fragment（s） resulting from Mu insertion for every Mu-induced mutant, and one such fragment, M196-FF, is found to contain a partial sequence of the DNA topoisomerase I gene Topl. Moreover, the modified AFLP method including all restriction enzymes, adaptors and primers has been optimized in this study. The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.

Systematic dissection of disease resistance to southern corn rust by bulked-segregant and transcriptome analysis

Southern corn rust(SCR) is a destructive maize disease caused by Puccinia polysora Underw. To investigate the mechanism of SCR resistance in maize, a highly resistant inbred line, L119 A, and a highly susceptible line, Lx9801, were subjected to gene mapping and transcriptome analysis. Bulked-segregant analysis coupled with whole-genome sequencing revealed several quantitative trait loci(QTL) on chromosomes 1, 6, 8, and 10. A set of 25 genes, including two coiled-coil nucleotide-binding site leucine-rich repeat(CC-NBS-LRR) genes, were identified as candidate genes for a major-effect QTL on chromosome 10. To investigate the mechanism of SCR resistance in L119 A, RNA-seq of P. polysorainoculated and non-inoculated plants of L119 A and Lx9801 was performed. Unexpectedly, the number of differentially expressed genes in inoculated versus non-inoculated L119 A plants was about 10 times that of Lx9801, with only 29 common genes identified in both lines, suggesting extensive gene expression changes in the highly resistant but not in the susceptible line. Based on the transcriptome analysis, one of the CC-NBS-LRR candidate genes was confirmed to be upregulated in L119 A relative to Lx9801 independently of P. polysora inoculation. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that transcription factors, as well as genes involved in defense responses and metabolic processes, were dominantly enriched, with the phenylpropanoid biosynthesis pathway most specifically activated. Consistently, accumulation of phenylpropanoid-derived lignin, especially S lignin, was drastically increased in L119 A after P. polysora inoculation, but remained unchanged in Lx9801, suggesting a critical role of lignin in SCR resistance. A regulatory network of defense activation and metabolic change in SCR-resistant maize upon P. polysora infection is described.

Single-cell RNA sequencing of meiocytes and microspores reveals the involvement of the Rf4 gene in redox homeostasis of CMS-C maize

Normal microsporogenesis is determined by both nuclear and mitochondrial genes. In maize C-type cytoplasmic male sterility, it is unclear how the development of meiocytes and microspores is affected by the mitochondrial sterility gene and the nuclear restorer gene. In this study, we sequenced the transcriptomes of single meiocytes(tetrad stage) and early mononucleate microspores from sterile and restorer lines. The numbers of expressed genes varied in individual cells and fewer than half of the expressed genes were common to the same cell types. Four comparisons revealed 3379 differentially expressed genes(DEGs), with 277 putatively associated with mitochondria, 226 encoding transcription factors,and 467 possibly targeted by RF4. KEGG analysis indicated that the DEGs in the two lines at the tetrad stage were involved predominantly in carbon metabolism and in amino acid biosynthesis and metabolism, whereas the DEGs during the transition from the tetrad stage to the early mononucleate stage were associated mostly with regulation of protein metabolism, fatty acid metabolism, and anatomical structure morphogenesis. Thus, meiocyte and microspore development was affected by the surrounding cells and the restorer gene, and the restorer gene helped restore the redox homeostasis of microspores and the normal cellular reconstruction during the transition.

基于定量机电阻抗方法的混凝土固化过程模量和内耗测量监测

测定新鲜混凝土凝固状态对于控制建筑质量和安全至关重要.实际上,混凝土通常需要约28天时间进行养护,期间混凝土凝固并加强,直至达到可使用标准.传统的混凝土养护过程通常使用超声波等方法测量杨氏模量.在这项工作中,我们提出了一种基于定量机电阻抗方法(Q-EMI)精确测量模量和内耗的实时混凝土养护监测方法.结果表明,模量和内耗均可用于监测混凝土养护过程,且模量测量更准确,因其受支撑条件影响较小.对于特定的混凝土,杨氏模量在4天后趋于稳定,剪切模量在7天后才能稳定,因此剪切模量测量更适合用于监测混凝土养护过程.此外,还发现养护时间对砂/水泥比非常敏感.这项工作为使用小样本监测混凝土养护过程提供了一种便捷方法.

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.

Regulation of maize growth and immunity by ZmSKI3-mediated RNA decay and post-transcriptional gene silencing

Disease resistance is often associated with compromised plant growth and yield due to defense-growth tradeoffs.However,key components and mechanisms underlying the defensegrowth tradeoffs are rarely explored in maize.In this study,we find that ZmSKI3,a putative subunit of the SUPERKILLER(SKI)complex that mediates the 3′-5′degradation of RNA,regulates both plant development and disease resistance in maize.The Zmski3 mutants showed retarded plant growth and constitutively activated defense responses,while the ZmSKI3 overexpression lines are more susceptible to Curvularia lunata and Bipolaris maydis.Consistently,the expression of defense-related genes was generally up-regulated,while expressions of growth-related genes were mostly down-regulated in leaves of the Zmski3-1 mutant compared to that of wild type.In addition,223 differentially expressed genes that are up-regulated in Zmski3-1 mutant but down-regulated in the ZmSKI3 overexpression line are identified as potential target genes of ZmSKI3.Moreover,small interfering RNAs targeting the transcripts of the defense-and growth-related genes are differentially accumulated,likely to combat the increase of defense-related transcripts but decrease of growthrelated transcripts in Zmski3-1 mutant.Taken together,our study indicates that plant growth and immunity could be regulated by both ZmSKI3-mediated RNA decay and post-transcriptional gene silencing in maize.

The RppC-AvrRppC NLR-effector interaction mediates the resistance to southern corn rust in maize

Southern corn rust(SCR),caused by the fungal pathogen Puccinia polysora,is a major threat to maize pro-duction worldwide.Efficient breeding and deployment of resistant hybrids are key to achieving durable control of SCR.Here,we report the molecular cloning and characterization of RppC,which encodes an NLR-type immune receptor and is responsible for a major SCR resistance quantitative trait locus.Further-more,we identified the corresponding avirulence effector,AvrRppC,which is secreted by P.polysora and triggers RppC-mediated resistance.Allelic variation of AvrRppC directly determines the effectiveness of RppC-mediated resistance,indicating that monitoring of AvrRppC variants in the field can guide the rational deployment of RppC-containing hybrids in maize production.Currently,RppC is the most frequently deployed SCR resistance gene in China,and a better understanding of its mode of action is crit-ical for extending its durability.

The chimeric gene atp6c confers cytoplasmic male sterility in maize by impairing the assembly of the mitochondrial ATP synthase complex

Cytoplasmic male sterility(CMS)is a powerful tool for the exploitation of hybrid heterosis and the study of signaling and interactions between the nucleus and the cytoplasm.C-type CMS(CMS-C)in maize has long been used in hybrid seed production,but the underlying sterility factor and its mechanism of action remain unclear.In this study,we demonstrate that the mitochondrial gene atp6c confers male sterility in CMS-C maize.The ATP6C protein shows stronger interactions with ATP8 and ATP9 than ATP6 during the assembly of F1F0-ATP synthase(F-type ATP synthase,ATPase),thereby reducing the quantity and activity of assem-bled F_(1)F_(o)-ATP synthase.By contrast,the quantity and activity of the F1'component are increased in CMS-C lines.Reduced F1F0-ATP synthase activity causes accumulation of excess protons in the inner membrane space of the mitochondria,triggering a burst of reactive oxygen species(ROS),premature programmed cell death of the tapetai cells,and pollen abortion.Collectively,our study identifies a chimeric mitochondrial gene(ATP6C)that causes CMS in maize and documents the contribution of ATP6C to F1F0-ATP synthase assembly,thereby providing novel insights into the molecular mechanisms of male sterility in plants.

The main restorer Rf3 of maize S type cytoplasmic male sterility encodes a PPR protein that functions in reduction of the transcripts of orf355

Dear Editor,Male sterile lines,especially cytoplasmic male sterile(CMS)lines,are extensively used in commercial hybrid seed production.Therefore a better understanding of the genetic basis of fertility restoration for CMS is important for the utility of heterosis in crops.In general,restorer of fertility(Rf)is controlled by one or two major genomic genes,and most of the Rf genes encode pro-teins containing P type pentatricopeptide repeat(PPR)motifs(Wang et al.,2006;Hu et al.,2012;Tang et al.,2014;Liu et al.,2016).S type CMS(CMS-S)is the main type of maize CMS with wide cytoplasmic sources,and orf355-orf77 in CMS-S was associated with male sterility(Zabala et al.,1997).The 5'stem-loop of the transcript of orf355-orf77 was thought to be associ-ated with its stability(Xiao et al.,2006).Recently,Xiao et al.(2020)confirmed that orf355 is the causal gene of CMS-S.Rf3,the main restorer of CMS-S,has been mapped to the long arm of chromosome 2 for a long time(Laughnan and Gabay,1978).Since then,great efforts have been made on genetic mapping and cloning of this gene(Xu et al.,2009).

An MCIA-like complex is required for mitochondrial complex Ⅰ assembly and seed development in maize

During adaptive radiation,mitochondria have co-evolved with their hosts,leading to gain or loss of subunits and assembly factors of respiratory complexes.Plant mitochondrial complex Ⅰ harbors40 nuclearand 9 mitochondrial-encoded subunits,and is formed by stepwise assembly during which different intermediates are integrated via various assembly factors.In mammals,the mitochondrial complex Ⅰ intermediate assembly(MCIA)complex is required for building the membrane arm module.However,plants have lost almost all of the MCIA complex components,giving rise to the hypothesis that plants follow an ancestral pathway to assemble the membrane arm subunits.Here,we characterize a maize crumpled seed mutant,crk1,and reveal by map-based cloning that CRK1 encodes an ortholog of human complex Ⅰ assembly factor 1,zNDUFAF1,the only evolutionarily conserved MCIA subunit in plants.zNDUFAF1 is localized in the mitochondria and accumulates in two intermediate complexes that contain complex Ⅰ membrane arm subunits.Disruption of zNDUFAF1 results in severe defects in complex Ⅰ assembly and activity,a cellular bioenergetic shift to aerobic glycolysis,and mitochondrial vacuolation.Moreover,we found that zNDUFAF1,the putative mitochondrial import inner membrane translocase ZmTIM17-1,and the isovaleryl-coenzyme A dehydrogenase ZmIVD1 interact each other,and could be co-precipitated from the mitochondria and co-migrate in the same assembly intermediates.Knockout of either ZmTIM17-1 or ZmIVD1 could lead to the significantly reduced complex Ⅰ stability and activity as well as defective seeds.These results suggest that zNDUFAF1,ZmTIM17-1 and ZmIVD1 probably form an MCIA-like complex that is essential for the biogenesis of mitochondrial complex Ⅰ and seed development in maize.Our findings also imply that plants and mammals recruit MCIA subunits independently for mitochondrial complex Ⅰ assembly,highlighting the importance of parallel evolution in mitochondria adaptation to their hosts.

The Application of UAV-Based Hyperspectral Imaging to Estimate Crop Traits in Maize Inbred Lines

Crop traits such as aboveground biomass(AGB),total leaf area(TLA),leaf chlorophyll content(LCC),and thousand kernel weight(TWK)are important indices in maize breeding.How to extract multiple crop traits at the same time is helpful to improve the efficiency of breeding.Compared with digital and multispectral images,the advantages of high spatial and spectral resolution of hyperspectral images derived from unmanned aerial vehicle(UAV)are expected to accurately estimate the similar traits among breeding materials.This study is aimed at exploring the feasibility of estimating AGB,TLA,SPAD value,and TWK using UAV hyperspectral images and at determining the optimal models for facilitating the process of selecting advanced varieties.The successive projection algorithm(SPA)and competitive adaptive reweighted sampling(CARS)were used to screen sensitive bands for the maize traits.Partial least squares(PLS)and random forest(RF)algorithms were used to estimate the maize traits.The results can be summarized as follows:The sensitive bands for various traits were mainly concentrated in the near-red and red-edge regions.The sensitive bands screened by CARS were more abundant than those screened by SPA.For AGB,TLA,and SPAD value,the optimal combination was the CARS-PLS method.Regarding the TWK,the optimal combination was the CARS-RF method.Compared with the model built by RF,the model built by PLS was more stable.This study provides guiding significance and practical value for main trait estimation of maize inbred lines by UAV hyperspectral images at the plot level.