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Plant seedling morphogenesis is considerably related to photosynthesis, pigment synthesis, and circadian periodicity during seedling development. We identified and cloned a maize zebra or crossbanding leaves mutant wk3735 , which produces pale white kernels and was identified and plays a role in the equilibrium of the Redox state the in/out of ETC by active oxygen scavenging. Interestingly, it produces the zebra leaves during the production of the first seven leaves, which is apparently different from the mutation of homologs AtPTOX in Arabidopsis . It is intriguing to investigate how and why yellow crossbands (zebra leaf phenotype) emerge on leaves. As expected, chlorophyll concentration and photosynthetic efficiency both significantly declined in the yellow sector of wk3735 leaves. Meanwhile, we observed the circadian expression pattern of ZmPTOX1 , which was further validated by protein interaction assays of the circadian clock protein TIM1 and ZmPTOX1. The transcriptome data of yellow (muW) and green (muG) sectors of knock-out lines and normal leaves of overexpression lines (OE) at the 5th-leaf seedling stage were analyzed. Zebra leaf etiolated sections exhibit a marked defect in the expression of genes involved in the circadian rhythm and rhythmic stress (light and cold stress) responses than green sections. According to the analysis of co-DEGs of muW vs. OE and muG vs. OE, terms linked to cell repair function were upregulated while those linked to environmental adaptability and stress response were downregulated due to the mutation of ZmPTOX1 . Further gene expression level analyses of reactive oxygen species (ROS) scavenging enzymes and detection of ROS deposition indicated that ZmPTOX1 played an essential role in plant stress resistance and ROS homeostasis. The pleiotropic roles of ZmPTOX1 in plant ROS homeostasis maintenance, stress response, and circadian rhythm character may collectively explain the phenotype of zebra leaves during wk3735 seedling development.

Anthocyanin, a kind of flavonoid, plays a crucial role in plant resistance to abiotic stress. Salt stress is a kind of abiotic stress that can damage the growth and development of plant seedlings. However, limited research has been conducted on the involvement of maize seedlings in salt stress resistance via anthocyanin accumulation, and its potential molecular mechanism is still unclear. Therefore, it is of great significance for the normal growth and development of maize seedlings to explore the potential molecular mechanism of anthocyanin improving salt tolerance of seedlings via transcriptome analysis. In this study, we identified two W22 inbred lines (tolerant line pur-W22 and sensitive line bro-W22) exhibiting differential tolerance to salt stress during seedling growth and development but showing no significant differences in seedling characteristics under non-treatment conditions. In order to identify the specific genes involved in seedlings' salt stress response, we generated two recombinant inbred lines (RIL pur-W22 and RIL bro-W22 ) by crossing pur-W22 and bro-W22, and then performed transcriptome analysis on seedlings grown under both non-treatment and salt treatment conditions. A total of 6100 and 5710 differentially expressed genes (DEGs) were identified in RIL pur-W22 and RIL bro-W22 seedlings, respectively, under salt-stressed conditions when compared to the non-treated groups. Among these DEGs, 3160 were identified as being present in both RIL pur-W22 and RIL bro-W22 , and these served as commonly stressed EDGs that were mainly enriched in the redox process, the monomer metabolic process, catalytic activity, the plasma membrane, and metabolic process regulation. Furthermore, we detected 1728 specific DEGs in the salt-tolerant RIL pur-W22 line that were not detected in the salt-sensitive RIL bro-W22 line, of which 887 were upregulated and 841 were downregulated. These DEGs are primarily associated with redox processes, biological regulation, and the plasma membrane. Notably, the anthocyanin synthesis related genes in RIL pur-W22 were strongly induced under salt treatment conditions, which was consistented with the salt tolerance phenotype of its seedlings. In summary, the results of the transcriptome analysis not only expanded our understanding of the complex molecular mechanism of anthocyanin in improving the salt tolerance of maize seedlings, but also, the DEGs specifically expressed in the salt-tolerant line (RIL pur-W22 ) provided candidate genes for further genetic analysis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Typically, sweet corn, particularly sh2 sweet corn, has low seed vigor owing to its high sugar and low starch content, which is a major problem in sweet corn production, particularly at low temperatures. There is considerable variation in the germination rates among sweet corn varieties under low-temperature conditions, and the underlying mechanisms behind this phenomenon remain unclear. In this study, we screened two inbred sweet corn lines (tolerant line L282 and sensitive line L693) differing in their low-temperature germination rates; while no difference was observed in their germination rates at normal temperatures. To identify the specifically induced genes influencing the germination capacity of sweet corn at low temperatures, a transcriptome analysis of the two lines was conducted at both normal and low temperatures. Compared to the lines at a normal temperature, 3926 and 1404 differently expressed genes (DEGs) were identified from L282 and L693, respectively, under low-temperature conditions. Of them, 830 DEGs were common DEGs (cDEGs) that were identified from both L282 and L693, which were majorly enriched in terms of microtubule-based processes, histone H3-K9 modification, single-organism cellular processes, and carbohydrate metabolic processes. In addition, 3096 special DEGs (sDEGs), with 2199 upregulated and 897 downregulated, were detected in the tolerant line L282, but not in the sensitive line L693. These sDEGs were primarily related to plasma membranes and oxygen-containing compounds. Furthermore, electric conductivity measurements demonstrated that the membrane of L282 experienced less damage, which is consistent with its strong tolerance at low temperatures. These results expand our understanding of the complex mechanisms involved in the cold germination of sweet corn and provide a set of candidate genes for further genetic analysis.

Although bulked segregant analysis (BSA) has been used extensively in genetic mapping, user-friendly tools which can integrate current algorithms for researchers with no background in bioinformatics are scarce. To address this issue, we developed an R package, PNGSeqR, which takes single-nucleotide polymorphism (SNP) markers from next-generation sequencing (NGS) data in variant call format (VCF) as the input file, provides four BSA algorithms to indicate the magnitude of genome-wide signals, and rapidly defines the candidate region through the permutation test and fractile quantile. Users can choose the analysis methods according to their data and experimental design. In addition, it also supports differential expression gene analysis (DEG) and gene ontology analysis (GO) to prioritize the target gene. Once the analysis is completed, the plots can conveniently be exported.

Maize originated from tropical regions and is extremely sensitive to low temperature during germination. Our previous work identified a major QTL, qp1ER1-1 , for low temperature germination ability (LTGA) of maize. Here, we introgressed qp1ER1-1 from the tolerant line L220 into the sensitive line PH4CV to generate two near isogenic lines NIL 220-3 and NIL 220-25 . When germinated under cold temperature for 25 days (Cold-25), the NILs showed similar seedling root length and shoot length to L220, but significantly higher than PH4CV. However, when germinated under cold temperature for 15 days (Cold-15) or under normal temperature (25 °C) for 3 days (CK-3), all lines showed similar seedling performance, indicating that introgression of qp1ER1-1 from L220 into PH4CV could improve LTGA of NIL 220-3 and NIL 220-25 . The whole seedlings, including root and shoot, of Cold-15 and CK-3 were harvested for transcriptome analysis, when both stayed at a similar developmental stage. Dry seed embryo was sequenced as a non-germination control (CK-0). Compared with PH4CV, the tolerant line (L220, NIL 220-3 , and NIL 220-25 ) specifically expressed genes (different expressed genes, DEGs) were identified for CK-0, Cold-15, and CK-3. Then, DEGs identified from Cold-15, but not from CK-0 or CK-3, were defined as tolerant line specifically expressed LTGA genes. Finally, 1786, 174, and 133 DEGs were identified as L220, NIL 220-3 , and NIL 220-25 specifically expressed LTGA genes, respectively. Of them, 27 were common LTGA genes that could be identified from all three tolerant lines, with two ( Zm00001d031209 and Zm00001d031292 ) locating in the confidence interval of qp1ER1-1 . In addition, GO analysis revealed that L220 specifically expressed LTGA genes were majorly enriched in the cell division process and plasma membrane related categories. Taken together, these results provided new insight into the molecular mechanism of maize seed LTGA and facilitated the cloning of the qp1ER1-1 gene.