The objective of this work was to develop a model for simulating the leaf color dynamics of winter wheat in relation to crop growth stages and leaf positions under different nitrogen (N) rates.  RGB (red, green and blue) data of each main stem leaf were collected throughout two crop growing seasons for two winter wheat cultivars under different N rates.  A color model for simulating the leaf color dynamics of winter wheat was developed using the collected RGB values.  The results indicated that leaf color changes went through three distinct stages, including early development stage (ES), early maturity stage (MS) and early senescence stage (SS), with respective color characteristics of light green, dark green and yellow for the three stages.  In the ES stage, the R and G colors gradually decreased from their initial values to steady values, but the B value generally remained unchanged.  RGB values remained steady in the MS, but all three gradually increased to steady values in the SS.  Different linear functions were used to simulate the dynamics of RGB values in time and space.  A cultivar parameter of leaf color matrix (M_RGB) and a nitrogen impact factor (FN) were added to the color model to quantify their respective effects.  The model was validated with an independent experimental dataset.  RMSEs (root mean square errors) between the observed and simulated RGB values ranged between 7.0 and 10.0, and relative RMSEs (RRMSEs) ranged between 7 and 9%.  In addition, the model was used to render wheat leaves in three-dimensional space (3D).  The 3D visualizations of leaves were in good agreement with the observed leaf color dynamics in winter wheat.  The developed color model could provide a solid foundation for simulating dynamic crop growth and development in space and time. 

Aerated irrigation has been proven to increase crop production and quality, but studies on its environmental impacts are sparse.  The effects of aeration and irrigation regimes on soil CO₂ and N₂O emissions in two consecutive greenhouse tomato rotation cycles in Northwest China were studied via the static closed chamber and gas chromatography technique.  Four treatments, aerated deficit irrigation (AI1), non-aerated deficit irrigation (CK1), aerated full irrigation (AI2) and non-aerated full irrigation (CK2), were performed.  The results showed that the tomato yield under aeration of each irrigation regime increased by 18.8% on average compared to non-aeration, and the difference was significant under full irrigation (P<0.05).  Full irrigation significantly increased the tomato yield by 23.9% on average in comparison to deficit irrigation.  Moreover, aeration increased the cumulative CO₂ emissions compared to non-aeration, and treatment effects were significant in the autumn-winter season (P<0.05).  A slight increase of CO₂ emissions in the two seasons was observed under full irrigation (P>0.05).  There was no significant difference between aeration and non-aeration in soil N₂O emissions in the spring-summer season, whereas aeration enhanced N₂O emissions significantly in the autumn-winter season.  Furthermore, full irrigation over the two seasons greatly increased soil N₂O emissions compared to the deficit irrigation treatment (P<0.05).  Correlation analysis indicated that soil temperature was the primary factor influencing CO₂ fluxes.  Soil temperature, soil moisture and NO₃^– were the primary factors influencing N₂O fluxes.  Irrigation coupled with particular soil aeration practices may allow for a balance between crop production yield and greenhouse gas mitigation in greenhouse vegetable fields.

Visualization of simulated crop growth and development is of significant interest to crop research and production.  This study aims to address the phenomenon of organs cross-drawing by developing a method of collision detection for improving vivid 3D visualizations of virtual wheat crops.  First, the triangular data of leaves are generated with the tessellation of non-uniform rational B-splines surfaces.  Second, the bounding volumes (BVs) and bounding volume hierarchies (BVHs) of leaves are constructed based on the leaf morphological characteristics and the collision detection of two leaves are performed using the Separating Axis Theorem.  Third, the detecting effect of the above method is compared with the methods of traditional BVHs, Axis-Aligned Bounding Box (AABB) tree, and Oriented Bounding Box (OBB) tree.  Finally, the BVs of other organs (ear, stem, and leaf sheath) in virtual wheat plant are constructed based on their geometric morphology, and the collision detections are conducted at the organ, individual and population scales.  The results indicate that the collision detection method developed in this study can accurately detect collisions between organs, especially at the plant canopy level with high collision frequency.  This collision detection-based virtual crop visualization method could reduce the phenomenon of organs cross-drawing effectively and enhance the reality of visualizations.

A study was conducted in attempting to identify the cold-resistant apple rootstocks and to establish a comprehensive evaluation system.  In this study, 10 elite apple dwarfing rootstocks (GM256, JM7, M26, M7, SC1, SH1, SH38, SH6, M9, and T337) were employed for the experiment and the following parameters were investigated under different low temperature stress conditions (0, –15, –20, –25, –30, and –35°C): the changes of the relative electrical conductivity (REC), anthocyanin content, protein content, soluble sugar content, soluble starch content, proline content, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and peroxidase (POD) activity of the dormant branches.  The inflection temperature that could represent the plant tissue semi-lethal temperature (LT₅₀) was obtained by the measurements of REC.  The LT₅₀ was used to evaluate eight other indices.  The results showed that there was no significant correlation between LT₅₀ and POD activity as well as between the soluble sugar, protein and proline contents at 0 and –15°C.  Soluble starch content at 0 and –15°C and anthocyanin content at –15–(–30)°C were significantly but negatively correlated to the LT₅₀ and the MDA content at 0–(–20)°C was significantly positively correlated to the LT₅₀.  Statistical analysis based on principal component analysis and LT₅₀ showed that cold resistant apple rootstocks in the decreasing order from high to low as GM256, SH6, SH38, SH1, SC1, M26, M7, JM7, T337, and M9.

Leaf area index (LAI) is used for crop growth monitoring in agronomic research, and is promising to diagnose the nitrogen (N) status of crops.  This study was conducted to develop appropriate LAI-based N diagnostic models in irrigated lowland rice.  Four field experiments were carried out in Jiangsu Province of East China from 2009 to 2014.  Different N application rates and plant densities were used to generate contrasting conditions of N availability or population densities in rice.  LAI was determined by LI-3000, and estimated indirectly by LAI-2000 during vegetative growth period.  Group and individual plant characters (e.g., tiller number (TN) and plant height (H)) were investigated simultaneously.  Two N indicators of plant N accumulation (NA) and N nutrition index (NNI) were measured as well.  A calibration equation (LAI=1.7787LAI₂₀₀₀–0.8816, R²=0.870**) was developed for LAI-2000.  The linear regression analysis showed a significant relationship between NA and actual LAI (R²=0.863**).  For the NNI, the relative LAI (R2=0.808**) was a relatively unbiased variable in the regression than the LAI (R²=0.33**).  The results were used to formulate two LAI-based N diagnostic models for irrigated lowland rice (NA=29.778LAI–5.9397; NNI=0.7705RLAI+0.2764).  Finally, a simple LAI deterministic model was developed to estimate the actual LAI using the characters of TN and H (LAI=–0.3375(TH×H×0.01)²+3.665(TH×H×0.01)–1.8249, R²=0.875**).  With these models, the N status of rice can be diagnosed conveniently in the field.

The objective of this work was to develop a dynamic model for describing leaf curves and a detailed spatial geometry model of the rice leaf (including sub-models for unexpanded leaf blades, expanded leaf blades, and leaf sheaths), and to realize three-dimensional (3D) dynamic visualization of rice leaves by combining relevant models.  Based on the experimental data of different cultivars and nitrogen (N) rates, the time-course spatial data of leaf curves on the main stem were collected during the rice development stage, then a dynamic model of the rice leaf curve was developed using quantitative modeling technology.  Further, a detailed 3D geometric model of rice leaves was built based on the spatial geometry technique and the non-uniform rational B-spline (NURBS) method.  Validating the rice leaf curve model with independent field experiment data showed that the average distances between observed and predicted curves were less than 0.89 and 1.20 cm at the tilling and jointing stages, respectively.  The proposed leaf curve model and leaf spatial geometry model together with the relevant previous models were used to simulate the spatial morphology and the color dynamics of a single leaf and of leaves on the rice plant after different growing days by 3D visualization technology.  The validation of the leaf curve model and the results of leaf 3D visualization indicated that our leaf curve model and leaf spatial geometry model could efficiently predict the dynamics of rice leaf spatial morphology during leaf development stages.  These results provide a technical support for related research on virtual rice.

Despite the improvement in cultivar characters and management practices, large gaps between the attainable and potential yields still exist in winter wheat of China.  Quantifying the crop potential yield is essential for estimating the food production capacity and improving agricultural policies to ensure food security.  Gradually descending models and geographic information system (GIS) technology were employed to characterize the spatial variability of potential yields and yield gaps in winter wheat across the main production region of China.  The results showed that during 2000–2010, the average potential yield limited by thermal resource (YG_T) was 23.2 Mg ha^–1, with larger value in the northern area relative to the southern area.  The potential yield limited by the water supply (YG_W) generally decreased from north to south, with an average value of 1.9 Mg ha^–1 across the entire study region.  The highest YG_W in the north sub-region (NS) implied that the irrigation and drainage conditions in this sub-region must be improved.  The averaged yield loss of winter wheat from nutrient deficiency (YG_N) varied between 2.1 and 3.1 Mg ha^–1 in the study area, which was greater than the yield loss caused by water limitation.  The potential decrease in yield from photo-thermal-water-nutrient-limited production to actual yield (YG_O) was over 6.0 Mg ha^–1, ranging from 4.9 to 8.3 Mg ha^–1 across the entire study region, and it was more obvious in the southern area than in the northern area.  These findings suggest that across the main winter wheat production region, the highest yield gap was induced by thermal resources, followed by other factors, such as the level of farming technology, social policy and economic feasibility.  Furthermore, there are opportunities to narrow the yield gaps by making full use of climatic resources and developing a reasonable production plan for winter wheat crops.  Thus, meeting the challenges of food security and sustainability in the coming decades is possible but will require considerable changes in water and nutrient management and socio-economic policies.

This paper was to develop a model for simulating the leaf color changes in rice (Oryza sativa L.) based on RGB (red, green, and blue) values. Based on rice experiment data with different cultivars and nitrogen (N) rates, the time-course RGB values of each leaf on main stem were collected during the growth period in rice, and a model for simulating the dynamics of leaf color in rice was then developed using quantitative modeling technology. The results showed that the RGB values of leaf color gradually decreased from the initial values (light green) to the steady values (green) during the first stage, remained the steady values (green) during the second stage, then gradually increased to the final values (from green to yellow) during the third stage. The decreasing linear functions, constant functions and increasing linear functions were used to simulate the changes in RGB values of leaf color at the first, second and third stages with growing degree days (GDD), respectively; two cultivar parameters, MatRGB (leaf color matrix) and AR (a vector composed of the ratio of the cumulative GDD of each stage during color change process of leaf n to that during leaf n drawn under adequate N status), were introduced to quantify the genetic characters in RGB values of leaf color and in durations of different stages during leaf color change, respectively; FN (N impact factor) was used to quantify the effects of N levels on RGB values of leaf color and on durations of different stages during leaf color change; linear functions were applied to simulate the changes in leaf color along the leaf midvein direction during leaf development process. Validation of the models with the independent experiment dataset exhibited that the root mean square errors (RMSE) between the observed and simulated RGB values were among 8 to 13, the relative RMSE (RRMSE) were among 8 to 10%, the mean absolute differences (da) were among 3.85 to 6.90, and the ratio of da to the mean observation values (dap) were among 3.04 to 4.90%. In addition, the leaf color model was used to render the leaf color change over growth progress using the technology of visualization, with a good performance on predicting dynamic changes in rice leaf color. These results would provide a technical support for further developing virtual plant during rice growth and development.

This study was aimed to evaluate the potential effects of rest grazing on organic carbon storage in Stipa grandis steppe of Inner Mongolia, China. Using potassium dichromate heating method, we analyzed the organic carbon storage of plant and soil in Stipa grandis steppe after rest grazing for 3, 6, and 9 yr. The results indicated that as the rest grazing ages prolonged, the biomass of aboveground parts, litter and belowground plant parts (roots) of the plant communities all increased, meanwhile the C content of the biomass increased with the rest grazing ages prolonging. For RG0, RG3a, RG6a, and RG9a, C storage in aboveground vegetation were 60.7, 76.9, 82.8 and 122.2 g C m-2, respectively; C storage of litter were 5.1, 5.8, 20.4 and 25.5 g C m-2, respectively; C storage of belowground roots (0-100 cm) were 475.2, 663.0, 1 115.0 and 1 867.3 g C m-2, respectively; C storage in 0-100 cm soil were 13.97, 15.76, 18.60 and 32.41 kg C m-2, respectively. As the rest grazing ages prolonged, the organic C storage in plant communities and soil increased. The C storage of belowground roots and soil organic C was mainly concentrated in 0-40 cm soil body. The increased soil organic C for RG3a accounted for 89.8% of the increased carbon in vegetation-soil system, 87.2% for RG6a, and 92.6% for RG9a. From the perspective of C sequestration cost, total cost for RG3a, RG6a, and RG9a were 2 903.4, 5 806.8 and 8 710.2 CNY ha-1, respectively. The cost reduced with the extension of rest grazing ages, 0.15 CNY kg-1 C for RG3a, 0.11 CNY kg-1 C for RG6a and 0.04 CNY kg-1 C for RG9a. From the growth characteristics of grassland plants, the spring was one of the two avoided grazing periods, timely rest grazing could effectively restore and update grassland vegetation, and was beneficial to the sustainable use of grassland. Organic C storage for RG9a was the highest, while the cost of C sequestration was the lowest. Therefore, spring rest grazing should be encouraged because it was proved to be a very efficient grassland use pattern.

The accurate assessment of the spatiotemporal changes in soil nutrients influenced by agricultural production provides the basis for development of management strategies to maintain soil fertility and balance soil nutrients. In this paper, we combined spatial measurements from 2 157 soil samples and geostatistical analysis to assess the spatiotemporal changes in soil organic carbon (SOC), total nitrogen (TN), available phosphorus (AP) and available potassium content (AK) from the first soil survey (in the 1980s) to the second soil survey (in the 2000s) in the Taihu region of Jiangsu Province in China. The results showed that average soil nutrients in three soil types all exhibited the increased levels in the 2000s (except for AK in the yellow brown soil). The standard deviation of soil nutrient contents increased (except for TN in the paddy soil). Agricultural production in the 20 years led to increases in SOC, TN, AP and AK by 74, 82, 89 and 65%, respectively, of the Taihu areas analyzed. From the 1980s to 2000s all the nugget/sill ratios of soil nutrients indices were between 25 and 75% (except for AK in the yellow brown soil in the 2000s), indicating moderate spatial dependence. The ratio of AP in the yellow brown soil in the 2000s was 88.74%, showing weak spatial dependence. The spatial correlation range values for SOC, TN, AP and AK in the 2000s all decreased. The main areas showing declines in SOC, TN and AP were in the northwest. For AK, the main region with declining levels was in the east and middle of western areas. Apparently, the increase in soil nutrients in the Taihu region can be mainly attributed to the large increase in fertilizer inputs, change in crop systems and enhanced residues management since the 1980s. Future emphasis should be placed on avoiding excess fertilizer inputs and balancing the effects of the fertilizers in soils.

Plant nitrogen (N) uptake is a good indicator of crop N status. In this study, a new method was designed to determine the central wavelength, optimal bandwidth and vegetation indices for predicting plant N uptake (g N m-2) in winter wheat (Triticum aestivum L.). The data were collected from the ground-based hyperspectral reflectance measurements in eight field experiments on winter wheat of different years, eco-sites, varieties, N rates, sowing dates, and densities. The plant N uptake index (PNUI) based on NDVI of 807 nm combined with 736 nm was selected as the optimal vegetation index, and a linear model was developed with R2 of 0.870 and RMSE of 1.546 g N m-2 for calibration, and R2 of 0.834, RMSE of 1.316 g N m-2, slope of 0.934, and intercept of 0.001 for validation. Then, the effect of the bandwidth of central wavelengths on model performance was determined based on the interaction between central wavelength and bandwidth expansion. The results indicated that the optimal bandwidth varies with the changes of the central wavelength and with the interaction between the two bands in one vegetation index. These findings are important for prediction and diagnosis of plant N uptake more precise and accurate in crop management.

The vast area and marked variation of China make it difficult to predict the impact of climate changes on rice productivity in different regions. Therefore, analyzing the spatial and temporal characteristics of rice potential productivity and predicting the possible yield increment in main rice production regions of China is important for guiding rice production and ensuring food security. Using meteorological data of main rice production regions from 1961 to 1970 (the 1960s) and from 1996 to 2005 (the 2000s) provided by 333 stations, the potential photosynthetic, photo-thermal and climatic productivities in rice crop of the 1960s and 2000s in main rice production regions of China were predicted, and differences in the spatial and temporal distribution characteristics between two decades were analyzed. Additionally, the potential yield increment based on the high yield target and actual yield of rice in the 2000s were predicted. Compared with the 1960s, the potential photosynthetic productivity of the 2000s was seen to have decreased by 5.40%, with rates in northeastern and southwestern China found to be lower than those in central and southern China. The potential photo-thermal productivity was generally seen to decrease (2.56%) throughout main rice production regions, decreasing most in central and southern China. However, an increase was seen in northeastern and southwestern China. The potential climatic productivity was observed to be lower (7.44%) in the 2000s compared to the 1960s, but increased in parts of central and southern China. The potential yield increment from the actual yield to high yield target in the 2000s were no more than 6×103 kg ha-1 and ranged from 6×103 to 12×103 kg ha-1 in most of the single- and double-cropping rice growing regions, respectively. The yield increasing potential from the high yield target to the potential photo-thermal productivity in 2000s were less than 10×103 kg ha-1 and ranged from 10×103 to 30×103 kg ha-1 in most of the single- and double-cropping rice growing regions, respectively. The potential yield increment contributed by irrigation was between 5×103 and 20×103 kg ha-1, and between 20×103 and 40×103 kg ha-1 in most of the single- and double-cropping rice growing regions, respectively. These findings suggested that the high yield could be optimized by making full use of climatic resources and through a reasonable management plan in rice crop.

1 Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R.China 2 Graduate University of Chinese Academy of Sciences, Beijing 100049, P.R.China 3 Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R.China Carotenoid biosynthetic pathway produces not only pigments that protect photosynthetic system against photo-oxidative damage, but also precursors of abscisic acid, the major hormone regulates stress responses. To understand the response of carotenoid biosynthetic pathway to salt stress, the expression of the genes involved in carotenoid and ABA biosynthesis were compared in cultivated tomato Solanum lycopersicon cv. Moneymaker and its relative wild genotype S. pimpinellifolium (PI365967) together with the contents of carotenoids and ABA. The results showed that 11 of the 15 genes investigated were up-regulated and four unaltered in Moneymaker after 5 h of salt stress; whereas only four genes were up-regulated, four unaltered, and seven down-regulated in PI365967 after stress. Further comparison revealed that 11 salinity-induced genes were expressed significantly lower in Moneymaker than in PI365967 under normal condition, and 8 of them were induced to similar levels after salt stress. In consistence, ABA level was doubled in Moneymaker but kept consistent in PI365967 after salt stress, though the contents of neoxanthin, violaxanthin, β-carotene, lutein, and total carotenoids were kept unchanged in both species. Since it is known that PI365967 is more tolerant to salt stress than Moneymaker, we proposed that the constitutive high level of carotenoid and ABA biosynthetic pathway under normal growth condition could be benefit to PI365967 for establishing the early response to salt stress. In addition, CrtR-b1 and CrtR-b2 that encode β-carotenoid hydroxylases were the only genes in carotenoid biosynthetic pathway that were up-regulated by salt stress in both species. The CrtR-b2 gene was cloned from both species and no essential difference was found in the encoded amino acid sequences. Transformation of CrtR-b2 to tobacco improved the seed germination under salt stress condition, indicating that the hydrolysis of β-carotenoid is the target of transcriptional regulation of the carotenoid biosynthesis in both tomato cultivar and wild relative.

Real-time monitoring of nitrogen status in rice and wheat plant is of significant importance for nitrogen diagnosis, fertilization recommendation, and productivity prediction. With 11 field experiments involving different cultivars, nitrogen rates, and water regimes, time-course measurements were taken of canopy hyperspectral reflectance between 350-2 500 nm and leaf nitrogen accumulation (LNA) in rice and wheat. A new spectral analysis method through the consideration of characteristics of canopy components and plant growth status varied with phenological growth stages was designed to explore the common central bands in rice and wheat. Comprehensive analyses were made on the quantitative relationships of LNA to soil adjusted vegetation index (SAVI) and ratio vegetation index (RVI) composed of any two bands between 350-2 500 nm in rice and wheat. The results showed that the ranges of indicative spectral reflectance were largely located in 770-913 and 729-742 nm in both rice and wheat. The optimum spectral vegetation index for estimating LNA was SAVI (R822,R738) during the early-mid period (from jointing to booting), and it was RVI (R822,R738) during the mid-late period (from heading to filling) with the common central bands of 822 and 738 nm in rice and wheat. Comparison of the present spectral vegetation indices with previously reported vegetation indices gave a satisfactory performance in estimating LNA. It is concluded that the spectral bands of 822 and 738 nm can be used as common reflectance indicators for monitoring leaf nitrogen accumulation in rice and wheat.

Due to their sessile nature, plants require strong adaptability to complex environments, with stress tolerance often associated with trade-offs in growth and development (Major et al. 2020).  This antagonistic relationship between defense and growth has been interpreted as a competition for limited resources that are allocated to defense at the expense of growth, or vice versa. Recent studies have demonstrated that hormone-based signaling networks trigger transcriptional changes in key genes, leading to trade-offs between growth rates and stress defense (Huot et al. 2014).  Several genes involved in biotic and abiotic stress response have been identified.  These genes contain nonsynonymous variants that show convergent changes in allele frequency across different breeding eras in both China and the United States (Wang et al. 2020), which may reflect the selection of biotic and abiotic stress response genes during modern maize breeding.

Transcription factors (TFs) play vital roles in regulation of gene expression in plant cells, with specific key TFs exhibiting multifunctionality by coordinating various regulatory pathways to promote plant growth (Hufford et al. 2021).  Jasmonates (JAs) are identified among phytohormones for their significant roles in regulating various plant processes, particularly in defense mechanisms against pests. MYC2 is a central transcription factor that orchestrates the JA signaling pathway and defense responses in plants by regulating the expression of numerous genes (Du et al. 2022).  Although MYC2 has been extensively characterized in Arabidopsis, studies in crops have revealed the complexity of MYC2’s function, with reports addressing different aspects, such as growth in wheat (Li et al. 2023) or stress defense in maize (Ma et al. 2023).  However, lack of systematic understanding of the complex regulatory network of MYC2 in crops, particularly in maize constrain the further utilization of MYC2 and its downstream genes in maize genetic modification for breeding elite varieties.  Here, we reported that ZmMYC2 had undergone selection during domestication and modern breeding; it acts as a key regulator of the trade-off between development and defense gene expression in maize, elucidating its regulatory network, which holds significant importance in balancing yield and resistance.

Given that some resistance genes have been selected during modern breeding, we analyzed the history of ZmMYC2 over the processes of maize evolution and artificial selection.  According to maize Haplotype Map v3 (HapMap3) database consisting of 1164 modern maize accessions, 25 landraces, and 21 teosintes (Zea mays. parviglumis) (Bukowski et al. 2018), nucleotide diversity strongly decreased sharply at the promoter region (2000 bp upstream of transcription start site) of ZmMYC2 during breeding, while the coding region and 3’-downstream region of ZmMYC2 showed less dramatic changes in nucleotide diversity changes (Fig. 1-A).  Thus, we hypothesize that the genetic diversity within the promoter region of ZmMYC2 has decreased during the breeding process, with favorable variations being selected.  Moreover, the frequency of three polymorphisms underwent convergent changes during modern breeding in both the United States and China (Fig. 1-B–D).  These three polymorphisms constituted three principal haplotypes: pZmMYC2^Hap1, pZmMYC2^Hap2, and pZmMYC2^Hap3, of which the frequency of pZmMYC2^Hap1 showed an increasing trend during modern maize breeding (Fig. 1-E).  The rare haplotype pZmMYC2^Hap3 (n=4) emerged only during the breeding era of China in 2000.  LUC signal activity for pZmMYC2^Hap3 was significantly lower than that of the other two haplotypes in the promoter region (pZmMYC2^Hap1, pZmMYC2^Hap2) (Fig. 1-F–H), suggesting a differential regulatory potential among the haplotypes.  These data indicate that ZmMYC2 was under-selected during maize evolution and breeding processes of maize.  Next, we investigated the expression level of genome-wide association studies of ZmMYC2 based on 368 maize inbred lines using RNA-seq and genome resequencing data (Fu et al. 2013; Li et al. 2013).  The results showed a strong peak signal containing the genomic region of ZmMYC2 on chromosome 1 (Fig. 1-I).

To mine the genes downstream of ZmMYC2, we performed protoplast transient expression-based RNA-sequencing (PER-seq) analysis to facilitate the discovery of new downstream genes utilizing a consistent protoplast system (Zhu et al. 2023).  In total， 281.6 million clean reads were generated, among which an average of approximately 87% of reads were mapped uniquely to the reference genome (Appendices A and B).  The results demonstrated a significant increase in the expression level of ZmMYC2 in each of three replicates of the pRTL2-ZmMYC2-GFP (MYC2-GFP) construct, exceeding a 500-fold increase compared to the pRTL2-GFP-empty (GFP-empty) construct (Fig. 1-J).  Furthermore, upon analyzing differentially expressed genes (DEGs) with a false discovery rate (FDR) <0.05 as the threshold, it was found that 4480 unique DEGs of MYC2-GFP, among which 2,677 were up-regulated compared to GFP-empty (Appendix C).  These up-regulated genes are enriched in circadian rhythm, cell cycles, plant growth, and in response to stress, indicating that these genes are regulated directly or indirectly by ZmMYC2 (Appendix D-A–B).

Several potential candidate genes were selected in an unbiased manner based on their log₂(fold-change) ≥2.5 (Fig. 1-J).  Gene expression profiling analysis of ZmMYC2 and its potential targets revealed essential coincidence (Appendix E).  The interaction between MYC2 and targets observed in the PER-seq system, were further confirmed through expression quantitative trait loci (eQTL) analysis, dual-luciferase reporter assay (DLR), and electrophoretic mobility shift assay (EMSA).  Among the target genes, the members of cytochrome P450 (CYP) gene family are widely distributed in plants involving in various biological processes, such as detoxification of xenobiotics, secondary metabolites production, and terpenoid synthesis (Chakraborty et al. 2023; Sun et al. 2024).  Our results identified an unreported gene of cytochrome P450 family ZmCYP709H1 as a target of ZmMYC2.  Additionally, eQTL analysis of ZmCYP709H1 revealed a strong trans-eQTL signal in the region of chromosome 1, which contains the genomic region of ZmMYC2 (Fig. 1-K).  Subsequent validation through DLR and EMSA confirmed that ZmMYC2 interacts with the promoter region of ZmCYP709H1 and stimulates its expression (Fig. 1-L; Appendices F-A and G-A).  Moreover, the transcriptional activation effect of ZmMYC2 on the promoter of ZmCYP709H1 was suppressed by ZmJAZ8 (Fig. 1-L).  A recent report showed reduced expression of ZmCYP709H1 in three maize dwarf mutants compared to the wild-type, reflecting its potential role in regulating growth, particularly plant height.  This result supports our proposed function of the ZmMYC2-ZmCYP709H1 model (Gao et al. 2024).  Additionally, two other CYP genes, ZmBX5 and ZmBX6, were identified as potential downstream genes of ZmMYC2 that participate in benzoxazinoid synthesis, which is consistent with the findings of a previous study (Ma et al. 2023).  We further confirmed that ZmMYC2 can physically bind to the promoter region of these two genes and activate their expression (Appendix H-A–F).  Besides, the result showed that ZmMYC2 can activate ZmBRD1 expression, which is a member of the CYP gene family and responsible for the final step of brassinosteroid synthesis (Tian et al. 2019) (Fig. 4-A and B; Appendix I-A–D).

The AUXIN RESPONSE FACTOR (ARF) family consists of plant-specific TFs that are key regulators of gene expression in response to the plant hormone auxin (AUX), and participated in various developmental processes such as vascular tissue differentiation, root and shoot development, and environmental stimuli responses (Hagen and Guilfoyle 2002; Salmon et al. 2008).  However, little evidence has been found to support the regulation of ARF gene expression by the core factor ZmMYC2 in the JA signal transduction pathway in maize.  Our data showed that the expression of ZmARF3 was regulated by a trans-eQTL signal involving the gene region of ZmMYC2 (Appendix F-B).  In addition, ZmMYC2 can bind to the promoter region of the ZmARF3 gene and activate its transcription (Fig. 1-M; Appendix G-B).  Besides, MYC2 can activate expressions of senescence-associated genes in rice and wheat, which could be repressed by physical interactions with TaARF15-A1 (Li et al. 2023).  These data demonstrate the key role of MYC2 in regulating the stress resistance and growth of maize through the synergistic regulation of JA and AUX hormone signaling pathways.

Tonoplast intrinsic proteins (TIPs), a subgroup of the aquaporin family, are integral membrane proteins that are crucial for transporting water and small solutes across cellular membrane to maintain water balance (Chaumont et al. 2001).  We found that ZmTIP3c was activated by ZmMYC2 (Fig. 1-N; Appendices F-C and G-C), which supports the potential role of ZmMYC2 in jointly regulating drought stress and JA signal transduction.  The CER2 gene, which is a member of the ECERIFERUM family, is critical for the synthesis of epicuticular wax (Bourdenx et al. 2011; Zhao et al. 2024).  A recent study demonstrated that wounding-induced wax accumulation was primarily regulated by the JA signaling pathway in Arabidopsis, suggesting the potential of JA signaling in wax synthesis (Huang et al. 2024).  We identified ZmCER2 as a ZmMYC2 target (Fig. 1-O; Appendices F-D and G-D).  Additionally, we confirmed the upregulation of ZmCER2 in response to drought stress in five elite inbred lines representing distinct heterotic groups of maize (Fig. 1-P), as observed by previous studies (Zhang et al. 2018, 2020; Jiang et al. 2023).  The result indicates that the drought-induced trait of ZmCER2 can be observed across different genetic backgrounds, thus supporting the potential role of ZmMYC2 in modulating JA signaling and response to drought stress in maize mediated by ZmCER2.

In summary, our findings support the selection of ZmMYC2 during domestication and breeding, highlighting its critical role in regulating genes involving plant growth and development.  Collectively, our eQTL, DLR, and EMSA data successfully validated several targets (ZmCER2, ZmARF3, ZmBRD1 ZmTIP3c, ZmCYP709H1, ZmBX5, and ZmBX6) of ZmMYC2, that encode diverse proteins and participate in various metabolic pathways (Fig. 1-Q).  Of these, ZmCER2 was confirmed to be induced by drought stress and activated by ZmMYC2, suggesting that ZmMYC2 may play a role in the drought response by regulating synthesizing epicuticular wax.  These findings underscore the diverse functions of ZmMYC2 in maintaining the balance between plant development and defense-response, primarily via the JA signaling pathway.  Our data represent a foundation for the further function and mechanism elucidation of of ZmMYC2 and its “Yin-Yang” roles in regulating plant defense and growth (Fig. 1-Q).  Given the crucial role of ZmMYC2 in balancing development and resistance, further work is needed to confirm to unlock the full potentials of ZmMYC2 in mediating yield and resistance through JA signaling pathway by exploring the function of those downstream targets, which is a significant step toward crop precision breeding.