The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG), phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins), and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1α), tubulin beta (β-TUB), cyclophilin (CYP), and eukaryotic initiation factor 4A (EIF4A) were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein), GLU1(beta-glucosidase), and UBQ9 (ubiquitin 9) were the least stable and most unsuitable genes. In addition, the suitability of EF1α, β-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types.

Magnesium (Mg(2+)) is an essential macronutrient for plant growth and development, and the CorA/MRS2/MGT-type Mg(2+) transporters play important roles in maintaining Mg(2+) homeostasis in plants. Although the MRS2/MGT genes have been identified in two model plant species, Arabidopsis and rice, a comprehensive analysis of the MRS2/MGT gene family in other plants is lacking. In this work, 12 putative MRS2/MGT genes (ZmMGT1- ZmMGT12) were identified in maize and all of them were classified into five distinct subfamilies by phylogenetic analysis. A complementation assay in the Salmonella typhimurium MM281 strain showed that five representatives of the 12 members possess Mg(2+) transport abilities. Inhibition of ZmMGT protein activity using the hexaamminecobalt (III) (Co-Hex) inhibitor indicated that the ZmMGT protein mediated both low-affinity and high-affinity Mg(2+) transport in maize. A semi-quantitative reverse transcription-PCR (RT-PCR) analysis revealed that eight genes were constitutively expressed in all of the detected tissues, with one being specifically expressed in roots and three having no detectable expression signals. A quantitative RT-PCR analysis showed that some ZmMGT members displayed differential responses to Mg(2+) deficiency and aluminum (Al) stress. Furthermore, root growth inhibition and Mg(2+) accumulation analyses in two maize inbred lines, which conferred different levels of Al tolerance, revealed that ZmMGT proteins contributed to the Al resistance of the Al tolerance genotype. We hypothesize that ZmMGT family members function as Mg(2+) transporters and may play a role in linking Mg(2+) deficiency and Al stress responses. Our results will be valuable in a further analysis of the important biological functions of ZmMGT members in maize.

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SUMMARYInorganic phosphorus (Pi) is an essential element in numerous metabolic reactions and signaling pathways, but the molecular details of these pathways remain largely unknown. In this study, metabolite profiles of maize (Zea mays L.) leaves and roots were compared between six low‐Pi‐sensitive lines and six low‐Pi‐tolerant lines under Pi‐sufficient and Pi‐deficient conditions to identify pathways and genes associated with the low‐Pi stress response. Results showed that under Pi deprivation the concentrations of nucleic acids, organic acids and sugars were increased, but that the concentrations of phosphorylated metabolites, certain amino acids, lipid metabolites and nitrogenous compounds were decreased. The levels of secondary metabolites involved in plant immune reactions, including benzoxazinoids and flavonoids, were significantly different in plants grown under Pi‐deficient conditions. Among them, the 11 most stable metabolites showed significant differences under low‐ and normal‐Pi conditions based on the coefficient of variation (CV). Isoleucine and alanine were the most stable metabolites for the identification of Pi‐sensitive and Pi‐resistant maize inbred lines. With the significant correlation between morphological traits and metabolites, five low‐Pi‐responding consensus genes associated with morphological traits and simultaneously involved in metabolic pathways were mined by combining metabolites profiles and genome‐wide association study (GWAS). The consensus genes induced by Pi deficiency in maize seedlings were also validated by reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR). Moreover, these genes were further validated in a recombinant inbred line (RIL) population, in which the glucose‐6‐phosphate‐1‐epimerase encoding gene mediated yield and correlated traits to phosphorus availability. Together, our results provide a framework for understanding the metabolic processes underlying Pi‐deficient responses and give multiple insights into improving the efficiency of Pi use in maize.

Iron is an essential element for almost all living organisms, actively involved in a variety of cellular activities. To acquire iron from soil, strategy I plants such as Arabidopsis (Arabidopsis thaliana) must first reduce ferric to ferrous iron by Fe(III)-chelate reductases (FROs). FRO genes display distinctive expression patterns in several plant species. However, regulation of FRO genes is not well understood. Here, we report a systematic characterization of the AtFRO6 expression during plant growth and development. AtFRO6, encoding a putative FRO, is specifically expressed in green-aerial tissues in a light-dependent manner. Analysis of mutant promoter-b-glucuronidase reporter genes in transgenic Arabidopsis plants revealed the presence of multiple light-responsive elements in the AtFRO6 promoter. These light-responsive elements may act synergistically to confer light responsiveness to the AtFRO6 promoter. Moreover, no AtFRO6 expression was detected in dedifferentiated green calli of the korrigan1-2 (kor1-2) mutant or undifferentiated calli derived from wild-type explants. Conversely, AtFRO6 is expressed in redifferentiated kor1-2 shoot-like structures and differentiating calli of wild-type explants. In addition, AtFRO7, but not AtFRO5 and AtFRO8, also shows a reduced expression level in kor1-2 green calli. These results suggest that whereas photosynthesis is necessary but not sufficient, both light and cell differentiation are necessary for AtFRO6 expression. We propose that AtFRO6 expression is light regulated in a tissue-or cell differentiation-specific manner to facilitate the acquisition of iron in response to distinctive developmental cues.