Identification of the RRM1 gene family in rice (Oryza sativa) and its response to rice blast.

To better understand RNA-binding proteins in rice, a comprehensive investigation was conducted on the RRM1 gene family of rice. It encompassed genome-wide identification and exploration of its role in rice blast resistance. The physicochemical properties of the rice OsRRM1 gene family were analyzed. There genes were also analyzed for their conserved domains, motifs, location information, gene structure, phylogenetic trees, collinearity, and cis-acting elements. Furthermore, alterations in the expression patterns of selected OsRRM1 genes were assessed using quantitative real-time PCR (qRT-PCR). A total of 212 members of the OsRRM1 gene family were identified, which were dispersed across 12 chromosomes. These genes all exhibit multiple exons and introns, all of which encompass the conserved RRM1 domain and share analogous motifs. This observation suggests a high degree of conservation within the encoded sequence domain of these genes. Phylogenetic analysis revealed the existence of five subfamilies within the OsRRM1 gene family. Furthermore, investigation of the promoter region identified cis-regulatory elements that are involved in nucleic acid binding and interaction with multiple transcription factors. By employing GO and KEGG analyses, four RRM1 genes were tentatively identified as crucial contributors to plant immunity, while the RRM1 gene family was also found to have a significant involvement in the complex of alternative splicing. The qRT-PCR results revealed distinct temporal changes in the expression patterns of OsRRM1 genes following rice blast infection. Additionally, gene expression analysis indicates that the majority of OsRRM1 genes exhibited constitutive expressions. These findings enrich our understanding of the OsRRM1 gene family. They also provide a foundation for further research on immune mechanisms rice and the management of rice blast.

Non-targeted GC-MS metabolomics-based differences in Indica rice seeds of different varieties.

Rice seeds of different varieties exhibited distinct metabolic profiles in our study. We analyzed the metabolites in seeds of six rice varieties (CH, HM, NX, YX, HY, and MX) using non-targeted GC-MS. Our findings revealed that amino acids, sugars, and organic acids were predominant in all varieties, with significant differences observed in CH compared to the others. Specifically phenylalanine and glycine content differed notably in NX and YX, respectively. Additionally, 1,5-anhydroglucitol content in NX, and glutamate, aspartate, and lactulose in NX, YX, HM, HY, and MX were up-regulated. Due to the biological functions of these amino acids and sugars, these indicated that compared to CH, rice of NX were more conducive to metabolism of carbohydrate and fat, and healthy growth maintenance in the human body, but mightThese variations suggest that NX rice may be more beneficial for carbohydrate and fat metabolism and overall health maintenance compared to CH. However, it may not be suitable for diabetic patients. YX rice may not be an ideal glycine supplement, rice ofwhile HM, HY, and MX rice could serve as potential lactulose sources. Furthermore, NX and YX rice exhibited higher levels of main storage proteins compared to CH. This study offers valuable insights into the metabolic differences among various rice varieties.

Alternative polyadenylation profiles of susceptible and resistant rice (Oryza sativa L.) in response to bacterial leaf blight using RNA-seq.

BACKGROUND: Alternative polyadenylation (APA) is an important pattern of post-transcriptional regulation of genes widely existing in eukaryotes, involving plant physiological and pathological processes. However, there is a dearth of studies investigating the role of APA profile in rice leaf blight. RESULTS: In this study, we compared the APA profile of leaf blight-susceptible varieties (CT 9737-613P-M) and resistant varieties (NSIC RC154) following bacterial blight infection. Through gene enrichment analysis, we found that the genes of two varieties typically exhibited distal poly(A) (PA) sites that play different roles in two kinds of rice, indicating differential APA regulatory mechanisms. In this process, many disease-resistance genes displayed multiple transcripts via APA. Moreover, we also found five polyadenylation factors of similar expression patterns of rice, highlighting the critical roles of these five factors in rice response to leaf blight about PA locus diversity. CONCLUSION: Notably, the present study provides the first dynamic changes of APA in rice in early response to biotic stresses and proposes a possible functional conjecture of APA in plant immune response, which lays the theoretical foundation for in-depth determination of the role of APA events in plant stress response and other life processes.

Analysis of anthocyanins and total flavonoids content in functional rice and its recombination inbred lines.

Anthocyanin is one of the flavonoids, which has strong antioxidant properties. Functional rice rich in anthocyanins can not only improve immunity, but also anti-radiation, beauty, anti-aging effect, very popular in the market. In this study, we used Zibaoxiangnuo 1 (ZBXN 1), a functional rice variety which is rich in total flavonoids and anthocyanins, as the experimental material to construct Recombination Inbred Lines (RILs) with Minghui63 (MH63), a variety without anthocyanins. The contents of anthocyanins and total flavonoids of RILs and two parents were determined for three consecutive generations. The average anthocyanin content of parent ZBXN 1 was 319.31 mg/kg, and the anthocyanin inheritance of RIL population was relatively stable, with 10 samples higher than ZBXN 1. In addition, there was no significant difference in the total flavonoids content between the two parents, the total flavonoids content of Z25 in RIL population was 0.33%. Based on these studies, we believe that ZBXN 1 has abundant and stable anthocyanins, which can be used as an intermediate breeding material for breeding high-quality varieties with high anthocyanins, and lay a foundation for breeding more anthocyanin-rich rice varieties.

A base substitution in OsphyC disturbs its Interaction with OsphyB and affects flowering time and chlorophyll synthesis in rice.

BACKGROUND: Phytochromes are important photoreceptors in plants, and play essential roles in photomorphogenesis. The functions of PhyA and PhyB in plants have been fully analyzed, while those of PhyC in plant are not well understood. RESULTS: A rice mutant, late heading date 3 (lhd3), was characterized, and the gene LHD3 was identified with a map-based cloning strategy. LHD3 encodes phytochrome C in rice. Animo acid substitution in OsphyC disrupted its interaction with OsphyB or itself, restraining functional forms of homodimer or heterodimer formation. Compared with wild-type plants, the lhd3 mutant exhibited delayed flowering under both LD (long-day) and SD (short-day) conditions, and delayed flowering time was positively associated with the day length via the Ehd1 pathway. In addition, lhd3 showed a pale-green-leaf phenotype and a slower chlorophyll synthesis rate during the greening process. The transcription patterns of many key genes involved in photoperiod-mediated flowering and chlorophyll synthesis were altered in lhd3. CONCLUSION: The dimerization of OsPhyC is important for its functions in the regulation of chlorophyll synthesis and heading. Our findings will facilitate efforts to further elucidate the function and mechanism of OsphyC and during light signal transduction in rice.

Research on Discrete Dynamic Modeling of Learner Behavior Analysis in English Teaching.

The current English teaching mode focuses on the traditional offline teaching and online teaching. In order to solve the problems that some students are inefficient and cannot teach students according to their aptitude in the teaching process, this paper uses the big data analysis strategy based on a neural network algorithm. This paper studies the discrete dynamic modeling method of learner behavior analysis in English teaching. Firstly, it summarizes the current situation of English teaching and the research status of the hybrid application of discrete dynamic modeling technology. Secondly, combined with English teaching content and teaching objectives, through the analysis of various data of students' learning behavior, this paper evaluates students' English teaching quality from five aspects that affect the students' English teaching quality and puts forward a personalized English teaching quality evaluation model based on discrete dynamic modeling technology and learners' behavior analysis. Finally, through the practical teaching application in a university, the feasibility of the discrete dynamic English teaching model is verified. The results show that compared with the current innovative English teaching methods based on a dynamic iterative decision algorithm, the personalized discrete dynamic English teaching model based on learner behavior analysis significantly improves the quality of English teaching and students' academic performance.

Fine Mapping of a Novel Major Quantitative Trait Locus, qPAA7, That Controls Panicle Apical Abortion in Rice.

The panicle apical abortion (PAA) causes severe yield losses in rice production, but details about its development and molecular basis remain elusive. Here, we detected PAA quantitative trait loci (QTLs) in three environments using a set of chromosome segment substitution lines (CSSLs) that was constructed with indica Changhui121 as the recurrent parent and japonica Koshihikari as the donor parent. First, we identified a novel major effector quantitative trait locus, qPAA7, and selected a severe PAA line, CSSL176, which had the highest PAA rate among CSSLs having Koshihikari segments at this locus. Next, an F2 population was constructed from a cross between CSS176 and CH121. Using F2 to make recombinantion analysis, qPAA7 was mapped to an 73.8-kb interval in chromosome 7. Among nine candidate genes within this interval, there isn't any known genes affecting PAA. According to the gene annotation, gene expression profile and alignment of genomic DNA, LOC_Os07g41220 and LOC_Os07g41280 were predicted as putative candidate genes of qPAA7. Our study provides a foundation for cloning and functional characterization of the target gene from this locus.

OsPDCD5 negatively regulates plant architecture and grain yield in rice.

Plant architecture is an important agronomic trait that affects crop yield. Here, we report that a gene involved in programmed cell death, OsPDCD5, negatively regulates plant architecture and grain yield in rice. We used the CRISPR/Cas9 system to introduce loss-of-function mutations into OsPDCD5 in 11 rice cultivars. Targeted mutagenesis of OsPDCD5 enhanced grain yield and improved plant architecture by increasing plant height and optimizing panicle type and grain shape. Transcriptome analysis showed that OsPDCD5 knockout affected auxin biosynthesis, as well as the gibberellin and cytokinin biosynthesis and signaling pathways. OsPDCD5 interacted directly with OsAGAP, and OsAGAP positively regulated plant architecture and grain yield in rice. Collectively, these findings demonstrate that OsPDCD5 is a promising candidate gene for breeding super rice cultivars with increased yield potential and superior quality.

Genome-wide characterization of MATE gene family and expression profiles in response to abiotic stresses in rice (Oryza sativa).

Multidrug and toxic compound extrusion (MATE) proteins are involved in many physiological functions of plant growth and development. Although an increasing number of MATE proteins have been identified, the understanding of MATE proteins is still very limited in rice. In this study, 46 MATE proteins were identified from the rice (Oryza sativa) genome by homology searches and domain prediction. The rice MATE family was divided into four subfamilies based on the phylogenetic tree. Tandem repeats and fragment replication contribute to the expansion of the rice MATE gene family. Gene structure and cis-regulatory elements reveal the potential functions of MATE genes. Analysis of gene expression showed that most of MATE genes were constitutively expressed and the expression patterns of genes in different tissues were analyzed using RNA-seq. Furthermore, qRT-PCR-based analysis showed differential expression patterns in response to salt and drought stress. The analysis results of this study provide comprehensive information on the MATE gene family in rice and will aid in understanding the functional divergence of MATE genes.

qTGW12a, a naturally varying QTL, regulates grain weight in rice.

KEY MESSAGE: A stable QTL associated with rice grain type with a large effect value was found in multiple environments, and its candidate genes were verified by genetic transformation. Rice (Oryza sativa L.) grain size is critical to both yield and appearance quality. Therefore, the discovery and identification of rice grain size genes can provide pathways for the cultivation of high-yielding varieties. In the present work, 45,607 SNP markers were used to construct a high-density genetic map of rice recombinant inbred lines, and hence a total of 14 quantitative trait loci (QTLs) were detected based on the phenotypic data of grain weight, grain length and grain width under four different environments. qTGW12a and qGL12 are newly detected QTLs related to grain weight, and are located between 22.43 Mb and 22.45 Mb on chromosome 12. Gene annotation shows that the QTL region contains the LOC_Os12g36660 annotated gene, which encodes the multidrug and toxic compound extrusion (MATE) transporter. Mutations in exons and the splice site were responsible for the changes in grain type and weight. Gene knockout experiments were used to verify these results. Hence, these results provide a basis for the cloning of qTGW12a. This discovery provides new insights for studying the genetic mechanism of rice grain morphology, and reveals a promising gene to ultimately increase rice yield.

Combined proteomics, metabolomics and physiological analyses of rice growth and grain yield with heavy nitrogen application before and after drought.

BACKGROUND: Nitrogen application can effectively mitigate the damage to crop growth and yield caused by drought. However, the efficiency of heavy nitrogen application before drought (NBD) and heavy nitrogen application after drought (NAD) to regulate rice response to drought stress remains controversial. In this study, we profiled physiology, proteomics and metabolomics in rice variety Wufengyou 286 of two nitrogen management modes (NBD and NAD) to investigate their yield formation and the mechanism of nitrogen regulation for drought resistance. RESULTS: Results revealed that the yield of NBD and NAD decreased significantly when it was subjected to drought stress at the stage of young panicle differentiation, while the yield of NBD was 33.85 and 36.33% higher than that of NAD in 2017 and 2018, reaching significant levels. Under drought conditions, NBD increased chlorophyll content and net photosynthetic rate in leaves, significantly improved the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase and catalase, and decreased malondialdehyde (MDA) content compared with NAD. NBD promoted nitrogen assimilation in leaves, which was characterized by increased activities of nitrate reductase (NR) and glutamine synthetase (GS). In addition, NBD significantly increased the contents of osmotic regulatory substances such as soluble sugar, soluble protein and free proline. Gene ontology and KEGG enrichment analysis of 234 differentially expressed proteins and 518 differential metabolites showed that different nitrogen management induced strong changes in photosynthesis pathway, energy metabolism pathway, nitrogen metabolism and oxidation-reduction pathways. CONCLUSION: Different nitrogen management methods have significant differences in drought resistance of rice. These results suggest that heavy nitrogen application before drought may be an important pathway to improve the yield and stress resistance of rice, and provide a new ecological perspective on nitrogen regulation in rice.

Ribosome profiling reveals the effects of nitrogen application translational regulation of yield recovery after abrupt drought-flood alternation in rice.

Abrupt drought-flood alternation is a frequent meteorological disaster during the summer in Southern China. The study of physiological and translation mechanisms of rice yield recovery after abrupt drought-flood alternation has great potential benefits in field production. Our results showed that yield recovery upon nitrogen (N) application after abrupt drought-flood alternation was due to the increase in effective panicle numbers per plant. The N application resulted in the regulation of physiological and biochemical as well as growth development processes, which led to a rapid growth recovery effect after abrupt drought-flood alternation stress in rice. Using ribosome profiling combined with RNA sequencing (RNA-seq) technology, the interactions between transcription and translation for N application after abrupt drought-flood alternation were analyzed. It was found that a small proportion of response genes were shared at the transcriptional and translational levels, that is, 14% of the expressed genes were upregulated and 6.6% downregulated. Further analysis revealed that the translation efficiency (TE) of the genes was influenced by their sequence characteristics, including their GC content, coding sequence length and normalized minimal free energy. Compared with the number of untranslated upstream open reading frames (uORFs), the increased number of translated uORFs promoted the improvement of TE. The TE of the uORFs for N application was lower than the control without N application after abrupt drought-flood alternation. This study characterizes the translational regulatory pattern in response to N application after abrupt drought-flood alternation stress.

OsPLS4 Is Involved in Cuticular Wax Biosynthesis and Affects Leaf Senescence in Rice.

Leaf senescence is one of the most common factors that affects the growth and yield of rice. Although numerous genes affecting leaf senescence have been identified, few involved in cuticular wax synthesis have been described for rice premature leaf senescence. Here, we cloned and characterized Premature Leaf Senescence 4 (PLS4) in rice (Oryza sativa), which encodes a putative 3-oxoacyl-reductase in the fatty acid biosynthetic pathway. Subcellular localization of OsPLS4 was observed in the chloroplast. A single nucleotide substitution in OsPLS4 reduced leaf cuticular wax, and the expression levels of most wax biosynthesis-associated genes were downregulated. TEM showed chloroplast development were defective in the pls4 mutant. Further investigation revealed that the chlorophyll (Chl) content was reduced in the pls4 mutant compared with the WT and that the photosynthesis rate was lower, which caused ROS dramatic accumulation at the heading stage. These results confirmed premature leaf senescence in pls4 plants. Cold treatment indicated that the mutant was more sensitive than the WT was to cold stress. Together, all the above results indicate that the OsPLS4 mutation affects cuticular wax biosynthesis and chloroplast development in rice, causing reduced cuticular wax and premature leaf senescence.

Comprehensive metabolomic, proteomic and physiological analyses of grain yield reduction in rice under abrupt drought-flood alternation stress.

Abrupt drought-flood alternation (T1) is a meteorological disaster that frequently occurs during summer in southern China and the Yangtze river basin, often causing a significant loss of rice production. In this study, the response mechanism of yield decline under abrupt drought-flood alternation stress at the panicle differentiation stage was analyzed by looking at the metabolome, proteome as well as yield and physiological and biochemical indexes. The results showed that drought and flood stress caused a decrease in the yield of rice at the panicle differentiation stage, and abrupt drought-flood alternation stress created a synergistic effect for the reduction of yield. The main reason for the decrease of yield per plant under abrupt drought-flood alternation was the decrease of seed setting rate. Compared with CK0 (no drought and no flood), the net photosynthetic rate and soluble sugar content of T1 decreased significantly and its hydrogen peroxidase, superoxide dismutase, peroxidase activity increased significantly. The identified differential metabolites and differentially expressed proteins indicated that photosynthesis metabolism, energy metabolism pathway and reactive oxygen species response have changed strongly under abrupt drought-flood alteration stress, which are factors that leads to the rice grain yield reduction.

Genome-wide analysis of long non-coding RNAs affecting roots development at an early stage in the rice response to cadmium stress.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been found to play a vital role in several gene regulatory networks involved in the various biological processes in plants related to stress response. However, systematic analyses of lncRNAs expressed in rice Cadmium (Cd) stress are seldom studied. Thus, we presented the characterization and expression of lncRNAs in rice root development at an early stage in response to Cd stress. RESULTS: The lncRNA deep sequencing revealed differentially expressed lncRNAs among Cd stress and normal condition. In the Cd stress group, 69 lncRNAs were up-regulated and 75 lncRNAs were down-regulated. Furthermore, 386 matched lncRNA-mRNA pairs were detected for 120 differentially expressed lncRNAs and 362 differentially expressed genes in cis, and target gene-related pathway analyses exhibited significant variations in cysteine and methionine metabolism pathway-related genes. For the genes in trans, overall, 28,276 interaction relationships for 144 lncRNAs and differentially expressed protein-coding genes were detected. The pathway analyses found that secondary metabolites, such as phenylpropanoids and phenylalanine, and photosynthesis pathway-related genes were significantly altered by Cd stress. All of these results indicate that lncRNAs may regulate genes of cysteine-rich peptide metabolism in cis, as well as secondary metabolites and photosynthesis in trans, to activate various physiological and biochemical reactions to respond to excessive Cd. CONCLUSION: The present study could provide a valuable resource for lncRNA studies in response to Cd treatment in rice. It also expands our knowledge about lncRNA biological function and contributes to the annotation of the rice genome.

Genetic analysis for the grain number heterosis of a super-hybrid rice WFYT025 combination using RNA-Seq.

BACKGROUND: Despite the great contributions of utilizing heterosis to crop productivity worldwide, the molecular mechanism of heterosis remains largely unexplored. Thus, the present research is focused on the grain number heterosis of a widely used late-cropping indica super hybrid rice combination in China using a high-throughput next-generation RNA-seq strategy. RESULTS: Here, we obtained 872 million clean reads, and at least one read could maps 27,917 transcripts out of 35,679 annotations. Transcript differential expression analysis revealed a total of 5910 differentially expressed genes (DGHP) between super-hybrid rice Wufengyou T025 (WFYT025) and its parents were identified in the young panicles. Out of the 5910 DGHP, 63.1% had a genetic action mode of over-dominance, 17.3% had a complete-dominance action, 15.6% had a partial-dominance action and 4.0% had an additive action. DGHP were significantly enriched in carotenoid biosynthesis, diterpenoid biosynthesis and plant hormone signal transduction pathways, with the key genes involved in the three pathways being up-regulated in the hybrid. By comparing the DGHP enriched in the KEGG pathway with QTLs associated with grain number, several DGHP were located on the same chromosomal segment with some of these grain number QTLs. CONCLUSION: Through young panicle development transcriptome analysis, we conclude that the over-dominant effect is probably the major contributor to the grain number heterosis of WFYT025. The DGHP sharing the same location with grain number QTLs could be considered a candidate gene and provide valuable targets for the cloning and functional analysis of these grain number QTLs.

Mapping QTL for Seed Germinability under Low Temperature Using a New High-Density Genetic Map of Rice.

Mapping major quantitative trait loci (QTL) responsible for rice seed germinability under low temperature (GULT) can provide valuable genetic source for improving cold tolerance in rice breeding. In this study, 124 rice backcross recombinant inbred lines (BRILs) derived from a cross indica cv. Changhui 891 and japonica cv. 02428 were genotyped through re-sequencing technology. A bin map was generated which includes 3057 bins covering distance of 1266.5 cM with an average of 0.41 cM between markers. On the basis of newly constructed high-density genetic map, six QTL were detected ranging from 40 to 140 kb on Nipponbare genome. Among these, two QTL qCGR8 and qGRR11 alleles shared by 02428 could increase GULT and seed germination recovery rate after cold stress, respectively. However, qNGR1 and qNGR4 may be two major QTL affecting indica Changhui 891germination under normal condition. QTL qGRR1 and qGRR8 affected the seed germination recovery rate after cold stress and the alleles with increasing effects were shared by the Changhui 891 could improve seed germination rate after cold stress dramatically. These QTL could be a highly valuable genetic factors for cold tolerance improvement in rice lines. Moreover, the BRILs developed in this study will serve as an appropriate choice for mapping and studying genetic basis of rice complex traits.

Genome-wide identification and phylogenetic analysis of the chalcone synthase gene family in rice.

The enzymes of the chalcone synthase family are also known as type III polyketide synthases (PKS), and produce a series of secondary metabolites in bacteria, fungi and plants. In a number of plants, genes encoding PKS comprise a large multigene family. Currently, detailed reports on rice (Oryza sativa) PKS (OsPKS) family genes and tissue expression profiling are limited. Here, 27 candidate OsPKS genes were identified in the rice genome,and 23 gene structures were confirmed by EST and cDNA sequencing; phylogenetic analysis has indicated that these 23 OsPKS members could be clustered into three groups (I-III). Comparative analysis has shown OsPKS08 and OsPKS26 could be classified with the CHS genes of other species. Two members OsPKS10 and OsPKS21 were grouped into anther specific chalcone synthase-like (ASCL) clade. Intron/exon structure analysis revealed that nearly all of the OsPKS members contained one phase-1 intron at a conserved Cys. Analysis of chromosomal localization and genome distribution showed that some of the members were distributed on a chromosome as a cluster. Expression data exhibited widespread distribution of the rice OsPKS gene family within plant tissues, suggesting functional diversification of the OsPKS genes. Our results will contribute to future study of the complexity of the OsPKS gene family in rice.

Using RNA-seq to Profile Gene Expression of Spikelet Development in Response to Temperature and Nitrogen during Meiosis in Rice (Oryza sativa L.).

Rice reproductive development is sensitive to high temperature and soil nitrogen supply, both of which are predicted to be increased threats to rice crop yield. Rice spikelet development is a critical process that determines yield, yet little is known about the transcriptional regulation of rice spikelet development in response to the combination of heat stress and low nitrogen availability. Here, we profiled gene expression of rice spikelet development during meiosis under heat stress and different nitrogen levels using RNA-seq. We subjected plants to four treatments: 1) NN: normal nitrogen level (165 kg ha-1) with normal temperature (30°C); 2) HH: high nitrogen level (264 kg ha-1) with high temperature (37°C); 3) NH: normal nitrogen level and high temperature; and 4) HN: high nitrogen level and normal temperature. The de novo transcriptome assembly resulted in 52,250,482 clean reads aligned with 76,103 unigenes, which were then used to compare differentially expressed genes (DEGs) in the different treatments. Comparing gene expression in samples with the same nitrogen levels but different temperatures, we identified 70 temperature-responsive DEGs in normal nitrogen levels (NN vs NH) and 135 DEGs in high nitrogen levels (HN vs HH), with 27 overlapping DEGs. We identified 17 and seven nitrogen-responsive DEGs by comparing changes in nitrogen levels in lower temperature (NN vs HN) and higher temperature (NH vs HH), with one common DEG. The temperature-responsive genes were principally associated with cytochrome, heat shock protein, peroxidase, and ubiquitin, while the nitrogen-responsive genes were mainly involved in glutamine synthetase, amino acid transporter, pollen development, and plant hormone. Rice spikelet fertility was significantly reduced under high temperature, but less reduced under high-nitrogen treatment. In the high temperature treatments, we observed downregulation of genes involved in spikelet development, such as pollen tube growth, pollen maturation, especially sporopollenin biosynthetic process, and pollen exine formation. Moreover, we observed higher expression levels of the co-expressed DEGs in HN vs HH compared to NN vs NH. These included the six downregulated genes (one pollen maturation and five pollen exine formation genes), as well as the four upregulated DEGs in response to heat. This suggests that high-nitrogen treatment may enhance the gene expression levels to mitigate aspects of heat-stress. The spikelet genes identified in this study may play important roles in response to the combined effects of high temperature and high nitrogen, and may serve as candidates for crop improvement.