Development of a certified reference material for visual inspection of unsound maize kernels

Maize unsound kernel content is one of the limited items in maize trade, and generally determined by sensory detection. A certified reference material(CRM) for visual inspection of maize unsound kernels was developed according to a national standard of China, GB 1353-2018, and five items, such as insect-damaged kernels, spotted kernels, broken kernels, sprouted kernels and moldy kernels, were included. Unsound maize kernels were collected from fields or prepared in a laboratory, then screened, and ten kernels demonstrating varying levels of unsoundness or damage for each item were embedded in epoxy resin. The CRM showed excellent homogeneity and stability, which was stable for 14 days at the temperature from-20℃ to 45℃ and for at least 18 months at room temperature out of direct sunlight and strong light. Co-laboratory confirmation showed the CRM conformed to the morphological characteristics described in GB 1353-2018. The research filled the gap of unsound maize kernel CRM in maize detection.

Multi-omics analysis reveals the pivotal role of phytohormone homeostasis in regulating maize grain water content

Grain water content(GWC)is a key determinant for mechanical harvesting of maize(Zea mays).In our previous research,we identified a quantitative trait locus,qGWC1,associated with GWC in maize.Here,we examined near-isogenic lines(NILs)NILL and NILH that differed at the qGWC1 locus.Lower GWC in NILL was primarily attributed to reduced grain water weight(GWW)and smaller fresh grain size,rather than the accumulation of dry matter.The difference in GWC between the NILs became more pronounced approximately 35 d after pollination(DAP),arising from a faster dehydration rate in NILL.Through an integrated analysis of the transcriptome,proteome,and metabolome,coupled with an examination of hormones and their derivatives,we detected a marked decrease in JA,along with an increase in cytokinin,storage forms of IAA(IAA-Glu,IAA-ASP),and IAA precursor IPA in immature NILL kernels.During kernel development,genes associated with sucrose synthases,starch biosynthesis,and zein production in NILL,exhibited an initial up-regulation followed by a gradual down-regulation,compared to those in NILH.This discovery highlights the crucial role of phytohormone homeostasis and genes related to kernel development in balancing GWC and dry matter accumulation in maize kernels.

Integrating microRNAs and mRNAs reveals the hormones synthesis and signal transduction of maize under different N rates

The effect of nitrogen(N)fertilizer on the development of maize kernels has yet to be fully explored.MicroRNA-mRNA analyses could help advance our understanding of how kernels respond to N.This study analyzed the morphological,physiological,and transcriptomic changes in maize kernels under different N rates(0,100,200,and 300 kg ha–1).The result showed that increasing N application significantly increased maize grains’fresh and dry weight until N reached 200 kg ha–1.Higher levels of indole-3-acetic acid,cytokinin,gibberellin,and a lower level of ethylene were associated with increased N applications.We obtained 31 differentially expressed genes(DEGs)in hormone synthesis and transduction,and 9 DEGs were regulated by 14 differentially expressed microRNAs(DEMIs)in 26 pairs.The candidate DEGs and DEMIs provide valuable insight for manipulating grain filling under different N rates.

Effect of Drought Stress on Proteome of Maize Grain during Grain Filling

Based on isobaric tags for relative and absolute quantification(iTRAQ)technology,the proteome of grains of a maize cultivar Huangzao 4 under drought stress at grain filling stage was analyzed.The results show that under drought stress,438 proteins were differentially expressed in the maize grains during grain filling.Among them,200 were up-regulated and 238 were down-regulated.The gene ontology(GO)analysis shows that the biological processes in which differential proteins are more involved are cellular processes,metabolic processes and single biological processes;proteins in the cell component category are mainly distributed in cells,cell parts and organelles;and the proteins the molecular function category mainly possess catalytic activity and binding function.Differentially expressed proteins classified by COG are mainly involved in protein post-translational modification and transport,molecular chaperones,general functional genes,translation,ribosomal structure,biosynthesis,energy production and transformation,carbohydrate transport and metabolism,amino acid transport and metabolism,etc.The subcellular structure of the differentially expressed proteins is mainly located in the cell chloroplast and cytosol.The proportions are 35.01%and 30.21%respectively.KEGG metabolic pathway enrichment analysis shows that the differentially expressed proteins are mostly involved in antibiotic biosynthesis,microbial metabolism in different environments,and endoplasmic reticulum protein processing;the metabolic pathways with higher enrichment are the carbon fixation pathway and estrogen signaling pathway of prokaryotes;and the higher enrichment and greater significance are in the tricarboxylic acid cycle,carbon fixation of photosynthetic organisms and proteasome.The results of this study preliminarily reveal the adaptive mechanism of maize grains in response to drought stress during grain filling,providing a theoretical reference for maize drought-resistant molecular breeding.

Development of Three Risk Assessment Models for Deoxynivalenol and Fumonisins B1 ＋ B2 Contents in Maize Kernel

The maximal deoxynivalenol （DON） and fumonisins 131 ＋ B2 （FUM） contents in cereals are dictated by the European regulation 1126/2007. The direct measurement of these mycotoxins is a tedious and expensive process. Our study is based on an alternative tool： near infrared spectroscopy. Different models were developed on 374 maize samples to predict their DON and FUM contents. Several parameters have been determined and used in a multivariate data analysis. Three models were developed： （1） a classification model based on Discriminant Factor Analysis （DFA）, （2） a linear model based on ANalysis of COVAriance （ANCOVA） and （3） a Partial Least Squares Discriminant Analysis model （PLS-DA）. Firstly, the performances of the DFA model for assessing DON and FUM risk were similar： 69 and 72% of the validation samples were respectively well classified. In the second part, the performances of the ANCOVA model for DON were higher than for FUM. The r2 was worth respectively 0.85 and 0.69. In the last part, the performances of the PLS-DA models were better for FUM than for DON. These results show that an evaluation of the mycotoxin risk is possible by analyzing selected kernel parameters measurable by secondary analytical such as near-infrared spectroscopy. Further work is needed to improve the models, adding more samples and using non linear approaches.

An ARF24-ZmArf2 module influences kernel size in different maize haplotypes

Members of the ADP-ribosylation factor family,which are GTP-binding proteins, are involved in metabolite transport, cell division, and expansion.Although there has been a significant amount of research on small GTP-binding proteins, their roles and functions in regulating maize kernel size remain elusive. Here, we identified Zm Arf2 as a maize ADPribosylation factor-like family member that is highly conserved during evolution. Maize zmarf2 mutants showed a characteristic smaller kernel size. Conversely, ZmArf2 overexpression increased maize kernel size. Furthermore, heterologous expression of Zm Arf2 dramatically elevated Arabidopsis and yeast growth by promoting cell division. Using expression quantitative trait loci(e QTL) analysis, we determined that Zm Arf2 expression levels in various lines were mainly associated with variation at the gene locus. The promoters of Zm Arf2 genes could be divided into two types, p S and p L, that were significantly associated with both Zm Arf2 expression levels and kernel size. In yeast-one-hybrid screening, maize Auxin Response Factor 24(ARF24) is directly bound to the Zm Arf2 promoter region and negatively regulated Zm Arf2 expression.Notably, the p S and p L promoter types each contained an ARF24 binding element: an auxin response element(AuxRE) in p S and an auxin response region(Aux RR) in p L, respectively. ARF24binding affinity to Aux RR was much higher compared with Aux RE. Overall, our results establish that the small G-protein Zm Arf2 positively regulates maize kernel size and reveals the mechanism of its expression regulation.

Signaling in Early Maize Kernel Development

Developing the next plant generation within the seed requires the coordination of complex programs driving pattern formation, growth, and differentiation of the three main seed compartments： the embryo （future plant）, the endosperm （storage compartment）, representing the two filial tissues, and the surround- ing maternal tissues. This review focuses on the signaling pathways and molecular players involved in early maize kernel development. In the 2 weeks following pollination, functional tissues are shaped from single cells, readying the kernel for filling with storage compounds. Although the overall picture of the signaling pathways regulating embryo and endosperm development remains fragmentary, several types of molecu- lar actors, such as hormones, sugars, or peptides, have been shown to be involved in particular aspects of these developmental processes. These molecular actors are likely to be components of signaling pathways that lead to transcriptional programming mediated by transcriptional factors. Through the integrated action of these components, multiple types of information received by cells or tissues lead to the correct differentiation and patterning of kernel compartments. In this review, recent advances regarding the four types of molecular actors （hormones, sugars, peptides/receptors, and transcription factors） involved in early maize development are presented.

Influence of feeding crimped kernel maize silage on the course of subclinical necrotic enteritis in a broiler disease model

This experiment was carried out with 375 male broilers(Ross 308) from days 1 to 28 to evaluate the influence of crimped kernel maize silage(CKMS) on the manifestation of subclinical necrotic enteritis,microbiota counts, organic acid production and relative weights of gastrointestinal segments. A necrotic enteritis disease model was applied. Birds were allocated into 3 different dietary treatments: a maizebased feed(MBF, control diet), and 2 diets supplemented with 15%(CKMS15) or 30%(CKMS30) of crimped ensiled kernel maize. The disease model involved a 10-time overdose of an attenuated live vaccine against coccidiosis given orally on day 17, followed by oral inoculation of Clostridium perfringens Type A(S48,10~8 to 10~9 bacteria/bird) twice daily on days 18,19, 20 and 21. Scoring of intestinal lesions was performed on days 22, 23, 25 and 28. Ileal and caecal digesta samples were collected for the quantification of selected bacterial groups and organic acids. The results showed that there was no effect of dietary treatments on small intestinal lesion scores(P> 0.05). Lesions scores peaked on days 23 and 25 and decreased again on day 28(P = 0.001). No effect of age on microbiota counts was observed, but feeding of CKMS30 reduced the number of coliforms in ileal contents(P = 0.01). Dietary treatments did not affect organic acid concentrations in ileum and caeca, but there was an effect of age; butyric acid was higher on days 22, 23 and 25 than on day 28(P = 0.04). Acetic acid and propionic acid concentrations in caeca were the highest on days 22 and 28 but the lowest on days 23 and 25. Relative gizzard and caeca weights were increased, and relative ileum weights were decreased when birds were fed CKMS30(P < 0.05). In conclusion, the inclusion of CKMS in broiler diets had no effects on the course of necrotic enteritis but had potential benefits in terms of inhibition of potentially harmful microorganisms.