Reproductive-stage heat stress (RSHS) poses a major constraint to cereal crop production by damaging main plant reproductive structures and hampering reproductive processes, including pollen and stigma viability, pollination, fertilization, grain setting and grain filling. Despite this well-recognized fact, research on crop heat stress (HS) is relatively recent compared to other abiotic stresses, such as drought and salinity, and in particular, RSHS studies in cereals are considerably few in comparison with seedling-stage and vegetative-stage-centered studies. Meanwhile, climate change-exacerbated HS, independently or synergistically with drought, will have huge implications on crop performance and future global food security. Fortunately, due to their sedentary nature, crop plants have evolved complex and diverse transient and long-term mechanisms to perceive, transduce, respond and adapt to HS at the molecular, cell, physiological and whole plant levels. Therefore, uncovering the molecular and physiological mechanisms governing plant response and tolerance to RSHS facilitates the designing of effective strategies to improve HS tolerance in cereal crops. In this review, we update our understanding of several aspects of RSHS in cereals, particularly impacts on physiological processes and yield; HS signal perception and transduction; and transcriptional regulation by heat shock factors and heat stress-responsive genes. We also discuss the epigenetic, post-translational modification and HS memory mechanisms modulating plant HS tolerance. Moreover, we offer a critical set of strategies (encompassing genomics and plant breeding, transgenesis, omics and agronomy) that could accelerate the development of RSHS-resilient cereal crop cultivars. We underline that a judicious combination of all of these strategies offers the best foot forward in RSHS tolerance improvement in cereals. Further, we highlight critical shortcomings to RSHS tolerance investigations in cereals and propositions for their circumvention, as well as some knowledge gaps, which should guide future research priorities. Overall, our review furthers our understanding of HS tolerance in plants and supports the rational designing of RSHS-tolerant cereal crop cultivars for the warming climate.

Drought is the major abiotic stress threatening maize (Zea mays L.) production globally. Despite recent scientific headway in deciphering maize drought stress responses, the overall picture of key genes, pathways, and co-expression networks regulating maize drought tolerance is still fragmented. Therefore, deciphering the molecular basis of maize drought tolerance remains pertinent. Here, through a comprehensive comparative leaf transcriptome analysis of drought-tolerant hybrid ND476 plants subjected to water-sufficient and water-deficit treatment conditions at flared (V12), tasseling (VT), the prophase of grain filling (R2), and the anaphase of grain filling (R4) crop growth stages, we report growth-stage-specific molecular mechanisms regulating maize drought stress responses. Based on the transcriptome analysis, a total of 3,451 differentially expressed genes (DEGs) were identified from the four experimental comparisons, with 2,403, 650, 397, and 313 DEGs observed at the V12, VT, R1, and R4 stages, respectively. Subsequently, 3,451 DEGs were divided into 12 modules by weighted gene co-expression network analysis (WGCNA), comprising 277 hub genes. Interestingly, the co-expressed genes that clustered into similar modules exhibited diverse expression tendencies and got annotated to different GO terms at different stages. MapMan analysis revealed that DEGs related to stress signal transduction, detoxification, transcription factor regulation, hormone signaling, and secondary metabolites biosynthesis were universal across the four growth stages. However, DEGs associated with photosynthesis and amino acid metabolism; protein degradation; transport; and RNA transcriptional regulation were uniquely enriched at the V12, VT, R2, and R4 stages, respectively. Our results affirmed that maize drought stress adaptation is a growth-stage-specific response process, and aid in clarifying the fundamental growth-stage-specific mechanisms regulating drought stress responses in maize. Moreover, genes and metabolic pathways identified here can serve as valuable genetic resources or selection targets for further functional validation experiments.

Nitrogen is one of the essential nutrients for plant growth and development. However, large amounts of nitrogen fertilizer not only increase the production costs, but also lead to serious environmental problems. Therefore, it is particularly important to reduce the application of nitrogen fertilizer and develop maize varieties with low nitrogen tolerance. The aim of this study was to determine the phenotypic and proteomic alterations of maize affected by nitrogen deficiency and to elucidate the molecular and physiological mechanisms underpinning maize tolerance to low nitrogen. Two maize hybrids with contrasting low nitrogen tolerance were used as the experimental materials. Maize plants were grown under different nitrogen application levels (N0 and N240) and proteomic analysis performed to analyze leaf differentially abundant proteins (DAPs) under different nitrogen conditions. The results showed that under the nitrogen deficiency condition, the nitrogen content, leaf dry weight, leaf area, and leaf area index of XY335 decreased by 15.58%, 8.83%, 3.44%, and 3.44%, respectively. However, in the variety HN138, the same parameters decreased by 56.94%, 11.97%, 8.79%, and 8.79%, respectively. Through proteomic analysis, we found that the low nitrogen tolerance variety responded to low nitrogen stress through lignin biosynthesis, ubiquitin-mediated proteolysis, and stress defense proteins. Transmembrane transporters were differentially expressed in both hybrids after low nitrogen treatment, suggesting that this was a common response to low nitrogen stress. Using bioinformatics analysis, we selected the key candidate gene (ZmTGA) that was assumed to respond to low nitrogen stress, and its function was characterized by maize mutants. The results showed that when compared with normal nitrogen treatment, the root length of the mutants under low nitrogen treatment increased by 10.1%, while that of the wild-type increased by 14.8%; the root surface area of the wild type under low nitrogen treatment increased by 9.6%, while that of the mutants decreased by 5.2%; the root surface area of the wild type was higher than that of the mutant at both nitrogen levels; and the activities of glutathione and guaiacol peroxidase enzymes in the mutant were lower than those in the wild-type under low nitrogen treatment. In summary, the mutant was less adaptable to a low nitrogen environment than the wild type. Our results provide maize genetic resources and a new direction for a further understanding of maize response to low nitrogen stress.

Maize (Zea mays L.) is one of the major staple crops providing human food, animal feed, and raw material support for biofuel production. For its growth and development, maize requires essential macronutrients. In particular, nitrogen (N) plays an important role in determining the final yield and quality of a maize crop. However, the excessive application of N fertilizer is causing serious pollution of land area and water bodies. Therefore, cultivating high-yield and low-N-tolerant maize varieties is crucial for minimizing the nitrate pollution of land and water bodies. Here, based on the analysis of the maize leaf transcriptome and proteome at the grain filling stage, we identified 3957 differentially expressed genes (DEGs) and 329 differentially abundant proteins (DAPs) from the two maize hybrids contrasting in N stress tolerance (low-N-tolerant XY335 and low-N-sensitive HN138) and screened four sets of low-N-responsive genes and proteins through Venn diagram analysis. We identified 761 DEGs (253 up- and 508 down-regulated) specific to XY335, whereas 259 DEGs (198 up- and 61 down-regulated) were specific to HN138, and 59 DEGs (41 up- and 18 down-regulated) were shared between the two cultivars under low-N-stress conditions. Meanwhile, among the low-N-responsive DAPs, thirty were unique to XY335, thirty were specific to HN138, and three DAPs were shared between the two cultivars under low-N treatment. Key among those genes/proteins were leucine-rich repeat protein, DEAD-box ATP-dependent RNA helicase family proteins, copper transport protein, and photosynthesis-related proteins. These genes/proteins were involved in the MAPK signaling pathway, regulating membrane lipid peroxidation, and photosynthesis. Our results may suggest that XY335 better tolerates low-N stress than HN138, possibly through robust low-N-stress sensing and signaling, amplified protein phosphorylation and stress response, and increased photosynthesis efficiency, as well as the down-regulation of ‘lavish’ or redundant proteins to minimize N demand. Additionally, we screened glutathione transferase 42 (ZmGST42) and performed physiological and biochemical characterizations of the wild-type (B73) and gst42 mutant at the seedling stage. Resultantly, the wild-type exhibited stronger tolerance to low N than the mutant line. Our findings provide a better understanding of the molecular mechanisms underlying low-N tolerance during the maize grain filling stage and reveal key candidate genes for low-N-tolerance breeding in maize.

Clarifying the genetic basis of trace element accumulation is of great significance to breed new maize varieties with high quality. In this study, an integrated variant map with 1.25 million (M) SNPs and 489 inbred lines was used for a genome-wide association study on the accumulation of iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), cadmium (Cd) and Arsenic (As) in maize kernels. Seventeen SNPs linked with six genes were overlapped by different trace elements. After further analysis, 65 SNPs located in 28 genes with a p-value lower than 10⁻¹⁰ were associated with Cd content by genome-wide association studies (GWAS). There was a 3.1-fold difference in Cd content between different groups, which was divided by SNP haplotype in chr2.S_160782359, chr2.S_161045498 and chr2.S_161273716. The amino acid sequences of GRMZM2G150608 and GRMZM2G051367 only shared 68.85% and 88.16% similarity between B73 and Mo17, and the Cd content of Mo17 was 2.2-fold that of B73. In addition, 19 lines with higher contents of Fe, Zn, Cu, Mn and fewer contents of As and Cd were screened from GWAS associated populations. This study will lay a foundation for revealing the molecular mechanism of trace element accumulation in maize kernels and provide candidate genes for breeding new maize varieties with high nutritional quality.

Drought stress is seriously affecting the growth and production of crops, especially when agricultural irrigation still remains quantitatively restricted in some arid and semi-arid areas. The identification of drought-tolerant genes is important for improving the adaptability of maize under stress. Here, we found that a new member of the actin-depolymerizing factor (ADF) family; the ZmADF5 gene was tightly linked with a consensus drought-tolerant quantitative trait locus, and the significantly associated signals were detected through genome wide association analysis. ZmADF5 expression could be induced by osmotic stress and the application of exogenous abscisic acid. Its overexpression in Arabidopsis and maize helped plants to keep a higher survival rate after water-deficit stress, which reduced the stomatal aperture and the water-loss rate, as well as improved clearance of reactive oxygen species. Moreover, seventeen differentially expressed genes were identified as regulated by both drought stress and ZmADF5, four of which were involved in the ABA-dependent drought stress response. ZmADF5-overexpressing plants were also identified as sensitive to ABA during the seed germination and seedling stages. These results suggested that ZmADF5 played an important role in the response to drought stress.