Comparative transcriptomic and metabolomic study reveal that exogenous 24‐epiandrosterone mitigate alkaline stress in broomcorn millet (Panicum miliaceum L.) via regulating photosynthesis and antioxidant capacity

Globally, land alkalinization affecting agricultural development. Considering the increasingly serious effects of alkaline stress on agriculture and environment, phytoremediation may be an efficient way to addressed land alkalinization. Broomcorn millet (Panicum miliaceum L.) is a saline‐alkaline‐tolerant crop and bioenergy crop. However, the molecular mechanism of alkaline response on broomcorn millet remains large gap. To explore the alkaline stress on broomcorn millet and the mitigation of 24‐epicastasterone (BR), the effects of CK (nutrient solution only), CB (nutrient solution + 0.5 mg L−1 BR), AS (alkaline stress), and AB (alkaline stress + 0.5 mg L−1 BR) on TA289 (alkaline‐tolerant) and SA223 (alkaline‐sensitive) were investigated. Alkaline stress enhanced reactive oxygen species and membrane lipid peroxidation. BR boosted antioxidant enzyme activities to reduce oxidative stress. Simultaneously, BR attenuated Na+ toxicity and maintained ion homeostasis. Additionally, BR significantly maintained the physiological structure and photosynthetic properties. Transcriptomic and metabolomic analyses were applied to further evaluate the effect of BR on photosynthetic and antioxidant defense. The results showed that BR significantly reduced the transcriptional responses of photosynthesis and antioxidant defense and promoted the accumulation of effective metabolites such as biliverdin, l‐glutamate, and phosphoric acid. Taken together, BR application can significantly alleviate the damage of alkaline stress to broomcorn millet by altering transcriptional expression and metabolite accumulation and is a simple and effective strategy to alleviate alkaline stress. This study reveals the molecular mechanism of BR to enhance photosynthetic capacity and antioxidant defense of broomcorn millet under alkaline stress, which provides theoretical support for the cultivation of bioenergy crops on alkaline lands and the breeding of alkaline‐tolerant bioenergy varieties.


| INTRODUCTION
The development of global bioenergy crops has faced a series of major environmental challenges, including extensive exploration of resources, extreme changes in climate, and pollution of arable land. Soil salt-alkalinization has restricted the crop productivity in arid and semi-arid regions worldwide . Consequently, intensified the growth of food and bioenergy crops in the world. Low crop productivity results in a considerable amount of waste of saline-alkaline land resources, which reduced the cultivation of bioenergy crops globally. Therefore, the development of alkaline-tolerant bioenergy crops is increasingly important to alleviate the challenges of environmental stress, promote sustainable development of world agriculture and ensure global food security and energy supply.
Broomcorn millet (Panicum miliaceum L.) is one of the earliest domesticated cereal/bioenergy crops in the world Yuan et al., 2022;Zou et al., 2019). In the northern China, broomcorn millet is an indispensable food crop and forage grass. Additionally, broomcorn millet is a good fermentation substrate with excellent fermentation yield, which advances its contribution to the industrial production of fuel ethanol (Rose & Santra, 2013). Importantly, Broomcorn millet has strong adaptability to harsh environments and is a pioneer crop of resistance to stress (Yuan, Li, et al., 2021). Although the broomcorn millet genome has been published (Shi et al., 2019;Zou et al., 2019), the breeding of broomcorn millet is still relatively delayed (Dwivedi et al., 2012). Currently, most of the existing studies have focused on broomcorn millet under low nitrogen, drought, and neutral salt stress (Liu et al., 2020;Yuan, Li, et al., 2021; rather than alkaline stress. The research on alkaline resistance of broomcorn millet is still at the stage of alkaline-tolerant resource screening  and physiological and biochemical responses in germination (Ma et al., 2022). Additionally, breeding is a long-term and challenging project. Therefore, while advancing breeding, mitigation measurements under alkaline stress are necessary to ensure sustainable development of energy and food crops.
Brassinolide induced a series of physiological and biochemical mechanisms related to stress tolerance, including the upregulation of stress-responsive genes (Bajguz & Hayat, 2009), translation protection mechanisms (Dhaubhadel et al., 2002), and the improvement of photosynthetic efficiency under abiotic stress (Xia et al., 2009). One of the most active forms of brassinolide is 24-Epicastasterone (BR), which has can alleviate environmental stress in plants (Yao et al., 2017). BR promotes the maintenance of a relatively normal nitrogen metabolism and antioxidant system under cadmium and/or NaCl stress (Wani et al., 2017). In addition, BR reduced the toxic effects of lead on rice by promoting the activity of antioxidant enzymes and inhibiting the oxidative stress on the photosynthetic mechanism (Guedes et al., 2021). However, there is a gap in research on BR for mitigation of alkaline stress in bioenergy crops.
To date, there is no information on the mitigation effects of brassinolide on bioenergy crops to alkaline stress. The objective of this study was to investigate the mitigation effect of BR on the damage induced by alkaline stress in broomcorn millet seedlings. We measured the anatomical structure of the two broomcorn millet varieties (one alkaline-tolerant and one alkaline-sensitive) and evaluated the osmotic adjustment ability, antioxidant defense capacity, photosynthetic pigments, gas exchange, and chlorophyll fluorescence of broomcorn millet under alkaline stress. This study is the first to reveal the ability of BR to reduce the damage caused by alkaline stress in broomcorn millet. This study contributes to the understanding of BR mitigation of alkaline stress in bioenergy crops and provide valuable information for the development of sustainable agriculture on alkalized lands, food security, and biofuel production in the world.

| Experimental treatments
The experiments were conducted in a plant growth greenhouse at Northwest A&F University in Yangling, Shaanxi, China. Broomcorn millet seedlings were cultivated using hydroponics (1/2 Hoagland nutrient solution) under controlled conditions (30 ± 1°C day/18 ± 1°C night, 24,000 lx illumination intensity, 14 h light/10 h dark cycle, and 55%-60% relative humidity). The plants were planted in black rectangular pots with a size of 37.5 cm × 27.5 cm. When the seedlings grow to the three-leaf one-heart stage, they are subjected to alkaline stress. According to our previous research , two varieties of broomcorn millet were selected: alkaline-tolerant variety TA289 and the alkaline-sensitive variety SA223 (provided by the Science K E Y W O R D S alkaline stress, antioxidant, metabolome, mitigation, photosynthetic parameters, transcriptome and Technology Innovation Team of Minor Grain Crops of Northwest A&F University). An alkaline concentration of 40 mmol L −1 (molar ratio NaHCO 3 :Na 2 CO 3 = 9:1) was determined to be an appropriate stress condition . Four treatments were used in the experiment: CK (nutrient solution only), CE (nutrient solution + 0.5 mg L −1 BR), AS (alkaline stress), and AB (alkaline stress + 0.5 mg L −1 BR). Each treatment included three replicates. The position of the planting pots was randomly oriented every day to avoid the impact of uneven lighting. The BR solution was sprayed onto plants at 09:00 and 21:00 every day at a concentration of 0.5 mg L −1 . The nutrient solution was renewed every 2 days. Samples were taken after 5 days of alkaline stress.

| Growth parameters
Five days after the application of alkaline, the plants were harvested. Fresh weight (FW) of the plants was measured. More than half of the leaves that remain green are considered green. The green leaf area is calculated by multiplying the leaf length by the leaf width by the coefficient 0.75.

| Leaf relative water content
The 2-3 leaves from the top of the plant were immediately weighed to obtain the FW and then cut into small segments and immediately placed in deionized water to obtain turgid weight (TW). Finally, the leaves were dried at 80°C for 48 h before the dry weight (DW) of each leaf sample was recorded (Patel & Parida, 2021 Estimation of electrolyte leakage (EL) in the leaves was performed according to the method described by Ma et al. (2021) with slight modifications. Briefly, fresh leaves were cut into small sections and the EL EC1 was measured using a conductivity meter (DDS-307A) after incubating (2 h at 25°C) with double distilled water (10 mL). The samples were incubated in boiling water for 10 min and cooled to 25°C before the EC2 was determined.
To quantify the malondialdehyde (MDA) content, fresh leaf samples were homogenized in 5% trichloroacetic acid (w/v) and centrifuged. The homogenate was mixed with 0.5% thiobarbituric acid solution prepared in 20% trichloroacetic acid (V/V = 1:4). The mixture was incubated in boiling water for 30 min and immediately cooled on ice. MDA was quantified using an extinction coefficient of 155 mM −1 cm −1 after obtaining the absorbance at 532 and 600 nm.

| Scanning electron microscopy of leaves
To observe the structure of leaves, approximately one third of the second leaves were taken, washed with deionized water, dehydrated with an ethanol concentration gradient, and then dried. The samples were sputtered with 60:40 gold: palladium ratio and observed under a scanning electron microscope (S4800; Hitachi) .

| Photosynthetic performance and chlorophyll fluorescence
Photosynthetic pigments contents were determined according to the description of Yuan, Li, et al. (2021). Fresh leaf samples (0.5-1 g) after 5 days of treated were homogenized in 10 mL of 80% acetone and then centrifuged at 10,000 g for 10 min. A spectrophotometer was used to measure the absorbance of the supernatant at 645, 663, and 450 nm.
Photosynthetic parameters and chlorophyll fluorescence were determined after 5 days of alkaline stress. Photosynthetic parameters and chlorophyll fluorescence measurements were based on previous research (Liu et al., 2020). Photosynthetic parameters, including net photosynthetic rate (P n ), transpiration rate (T r ), intercellular CO 2 concentration (C i ), and stomatal conductance (g s ) were measured using a CIRAS-3 portable photosynthesis system (PP Systems). Chlorophyll fluorescence was analyzed using a MINI-PAM-II fluorometer (Imaging PAM; Walz). The maximal quantum yield of PSII photochemistry (F v /F m ), photochemical quenching coefficient (q P ), and non-photochemical quenching coefficient (NPQ) were measured after the plants were dark-adapted for 30 min. The actual PSII efficiency (Φ PSII ) was calculated as

| Transcriptome and metabolomics analysis
Samples of the two cultivars under control and alkaline stress treatments were collected, with three biological replicates for each treatment. Total RNA was extracted from the root samples using the RNAprep Pure Plant Kit (Tiangen) for transcriptome analysis. Metabolomic analyses were performed by Allwegene Biotechnology Co., Ltd., and each treatment had six biological replicates. Ultrahigh pressure liquid chromatography separation was performed using an EXIONLC System (Sciex). See details in Appendix of Method (Appendix S1).

| Data analysis
The data were analyzed by one-way analysis of variance using IBM SPSS 23.0. Means of different treatments were compared using Duncan's test (p < 0.05). All figures were drawn using the Origin Pro 2020 (OriginLab).

| BR reduced the growth inhibition of broomcorn millet under alkaline stress
BR significantly improved the growth of broomcorn millet under alkaline stress conditions. There was no significant difference in the phenotypes between CB and CK groups of SA223 and TA289 varieties ( Figure 1a). Under alkaline stress conditions, the alkaline sensitive SA223 variety showed extreme growth inhibition, indicated by most of its leaves withering or even dying, while the TA289 variety showed an excellent growth. Compared with the AS group, the FW and green leaf area of SA223 were significantly increased in AB groups (Figure 1b,c). Alkaline stress significantly reduced the relative water content (RWC). Compared with the AS group, the RWC of SA223 and TA289 were increased by 13.36% and 6.07%, respectively ( Figure 1c). BR alleviated the wilting of leaf surface cells under alkaline stress ( Figure 1e). Moreover, the stomata of TA289 were tightly closed in alkaline treatment; however, the stomata of SA223 were deformed or damaged in the AS group. BR reduced the injury of stomatal and promoted the maintenance of stomatal structure, thus reducing alkaline toxicity. These results indicated that BR significantly reduced the growth inhibition of alkaline stress.

| BR maintained ionic homeostasis of broomcorn millet under alkaline stress
Alkaline stress increased the Na + content of leaves; however, BR significantly reduced the Na + content under alkaline stress (Figure 2). Compare with CK groups, the K + content in leaves of AS groups were reduced by 40.66% in SA223 and reduced by 18.15% in TA289, respectively. Alkaline stress inhibited the accumulation of Ca 2+ and Mg 2+ in leaves. BR attenuated the inhibitions of the K + , Ca 2+ , and Mg 2+ by alkaline stress. The responses of Fe 2+ , Cu 2+ , and Mn 2+ were like Mg 2+ in SA223 and TA289 ( Figure 2).

| BR reduced the oxidative damage to broomcorn millet by alkaline stress
Under alkaline stress, the activities of antioxidant enzymes (SOD, POD, APX, and GPX) were activated to resist oxidative stress. BR enhanced the activity of antioxidant enzymes under alkaline stress, especially SOD, POD, and GPX of SA223 (Figure 3a)

| BR improved photosynthetic capacity and gas exchange to broomcorn millet under alkaline stress
Alkaline stress significantly reduced photosynthetic pigment content, especially in SA223 in the AS group ( Figure 4a). Under alkaline stress, total chlorophyll and carotenoids were increased by BR by 49.65% and 30.48% in SA223 and by 19.90% and 7.37% in TA289, respectively. Compared with CK groups, the net photosynthetic rate (P n ) of SA223 and TA289 leaves decreased by 84.22% and 46.05% in AS groups, respectively. However, BR increased the P n under alkaline stress by 353.45% (of SA223) and 41.12% (of TA289). Under alkaline stress, BR improved the T r and g s by 219.70% and 214.56% in SA223 and 19.35% and 6.88% in TA289, respectively. Alkaline stress promoted the intercellular CO 2 concentration (C i ), but BR reduced Ci by 18.95% in SA223 and 4.17% in TA289 under alkaline stress (Figure 4b). Furthermore, alkaline stress greatly reduced the maximal photochemical efficiency of PSII (F v /F m ), photochemical quenching coefficient (q P ), NPQ coefficient, and the actual photosynthetic efficiency of PSII (Φ PSII ) (Figure 4c). Similarly, relative to AS groups, F v /F m , NPQ, q P , and Φ PSII were promoted in AB groups by 13.61%, 38.26%, 84.47%, and 111.66%, respectively (Figure 4c).

| BR suppressed photosynthesisrelated transcriptional and metabolic changes under alkaline stress
Photosynthesis significantly affected the expression of genes encoding key enzymes in photosynthesis pathway, involving photosystem II, photosystem I, cytochrome b6/f complex, photosynthetic electron transport, and F-type ATPase ( Figure 5). Detailed transcriptome quality control was shown in Figure S1a stress. The genes encoding maeB (malate dehydrogenase (oxaloacetate-decarboxylating) (NADP + )), PPC (phosphoenolpyruvate carboxylase), and GGAT (glutamate-glyoxylate aminotransferase) in carbon fixation in photosynthetic organisms were upregulated by alkaline stress. Most of the genes in the porphyrin and chlorophyll metabolism were downregulated by alkaline stress, and BR compensated for the expression levels of these genes. Additionally, five differential accumulation metabolites (DAMs)-l-alanine, laspartic acid, biliverdin, l-glutamic acid (l-glutamate), and phosphoric acid-were detected in photosynthesis-related pathways. Alkaline stress inhibited the accumulation of photosynthesis-related metabolites; however, BR mitigated the inhibition of metabolites by alkaline stress.

| BR reduced antioxidant-related transcriptional and metabolic changes under alkaline stress
Alkaline stress induced transcriptional changes in the peroxisome and glutathione metabolism pathways ( Figure 6). Detailed metabolome quality control was shown in Figure S1d,e. The genes encoding PEX (peroxin) 10, PEX12, and PEX14 of peroxisome biogenesis were upregulated by alkaline stress. The ROS metabolism-related gene expressions were both upregulated and downregulated. Simultaneously, most of the peroxisomal proteins genes expressions showed significant superiority under alkaline stress, including HPCL2, PHYH, and XDH and other enzymes. The GST-, PGD-, ICD-, GSR-, and ODC-related genes in the glutathione metabolism were induced to be upregulated by alkaline stress, and the gene expressions of LAP-, G6PD-, and other enzyme-related were both upregulated and downregulated. Meanwhile, four DAMs were detected in glutamine metabolism, including (5-l-glutamine)-l-amino acid (l-gamma-glutamyl-lamino acid), 5-oxoproline, Cys-Gly (l-cysteinylglycine), and l-glutamic acid (l-glutamate). Alkaline stress inhibited the production of these DAMs, but BR existed a compensatory effect on these DAMs to a certain extent.

| BR mitigates alkaline stress by regulating osmotic balance of broomcorn millet leaves
Broomcorn millet is a halophyte that can survive high NaCl stress (Yuan, Li, et al., 2021). Therefore, broomcorn millet is a suitable candidate for phytoremediation and plant stabilization of salt-alkalinized soils. BR is a brassinolide that can improve stress resistance in plants (Wani et al., 2017). Plants under stress suppress their growth to reduce energy consumption. In the present study, alkaline stress significantly inhibited the growth of SA223 as evidenced by phenotype, FW, and green leaf area. BR significantly increased the broomcorn millet growth under alkaline stress. RWC% of broomcorn millet decreased under alkaline stress, indicating that water absorption capacity was inhibited by alkaline treatment. However, the application of BR increased the RWC% under alkaline stress. Our data showed that the FW and green leaf area of broomcorn millet grown under alkaline stress were greatly reduced, and BR significantly improved these parameters under alkaline stress. These results indicated that BR could reduce the damage caused by alkaline stress in broomcorn millet. Under alkaline stress, plants maintain water balance via osmotic adjustment to ensure survival, which usually involves the absorption of ions (Parida & Jha, 2013). Na + is usually transported into the vacuole as a toxic element to reduce salt poisoning. Our results showed that the Na + concentrations in plants grown under alkaline conditions were much higher than that under non-alkaline conditions, suggested that Na + may be the main osmotic factor under alkaline stress. Additionally, the contribution of BR to K + /Na + corresponded to the phenotype implied that K + /Na + is important for maintaining osmotic balance F I G U R E 5 Transcriptional and metabolic responses of photosynthesis, photosynthesis-antenna proteins, carbon fixation in photosynthetic organisms, and porphyrin and chlorophyll metabolism pathways. The green box represents the enzymes or proteins with DEGs and the blue box represents DAMs. The heat map of DEGs/DAMs is next to the corresponding green/blue box. F16B represents fructose-1,6-bisphosphatase I; GGAT represents glutamate-glyoxylate aminotransferase; maeB represents malate dehydrogenase (oxaloacetate-decarboxylating)(NADP+); PPC represents phosphoenolpyruvate carboxylase; TPI represents triosephosphate isomerase (TIM); Lhca represents light-harvesting complex I chlorophyll a-b binding protein; Lhcb represents light-harvesting complex II chlorophyll a-b binding protein; bchG represents bacteriochlorophyll a synthase; CAO represents chlorophyllide a oxygenase; CBR represents chlorophyll(ide) b reductase; CHL represents chlorophyllase; chlG represents chlorophyll a synthase; COX15 represents heme a synthase; CPOX represents coproporphyrinogen III oxidase; DP8V represents divinyl chlorophyllide a 8-vinyl-reductase; FECH represents coproporphyrin ferrochelatase; GBR represents geranylgeranyl-bacteriochlorophyllide a reductase; GSA represents glutamate-1-semialdehyde 2,1-aminomutase; GTR represents glutamyl-tRNA reductase; HCAR represents 7-hydroxymethyl chlorophyll a reductase; HO represents heme oxygenase (biliverdin-producing, ferredoxin); MAD represents magnesium dechelatase; MCS represents magnesium chelatase subunit D; MET1 represents uroporphyrin-III C-methyltransferase; MPME represents magnesium-protoporphyrin IX monomethyl ester (oxidative) cyclase; MPOM represents magnesium-protoporphyrin O-methyltransferase; PAO represents pheophorbide a oxygenase; POR represents protochlorophyllide reductase; PPOX represents protoporphyrinogen III oxidase; RCCR represents red chlorophyll catabolite reductase; UROD represents uroporphyrinogen decarboxylase; UROS represents uroporphyrinogen-III synthase. DAM, differential accumulation metabolite; DEG, differentially expressed gene.
under alkaline stress. Yuan, Li, et al. (2021) reported similar results. In the present study, the Ca 2+ contents were significantly reduced in the AS groups but improved in the AB groups. Similar results have been reported in Tamarix (Sghaier et al., 2015). Like arsenic stress, alkaline stress severely inhibited the uptake and transport of Mg 2+ (Panda et al., 2019), and BR mitigated the lack of Mg 2+ . Cu 2+ participates in photosynthetic electron transport and mitochondrial respiration in plant cells (Patel & Parida, 2021).
normal leaf physiological structure of alkaline-sensitive broomcorn millet under alkaline stress. Overall, these results suggest that the application of BR significantly improved the alkaline resistance of the broomcorn millet.

| BR relieves alkaline stress by modulating antioxidant-related transcriptional and metabolic responses of broomcorn millet leaves
Reactive oxygen species are produced under various environmental stresses including high salt, heavy metals, and drought, and cause strong oxidative damage to the synthesis of proteins, nucleic acids, carbohydrates, and lipids (Ahmad, 2010;Rangani et al., 2016) (Rangani et al., 2016). Compared with the AS conditions, the EL of leaves from plants grown under AB conditions was significantly reduced. These results indicated that the application of BR contributed to membrane stability under alkaline stress. Like the EL results, the BR also greatly reduced the production of MDA in alkaline stress conditions, further indicating that BR had a positive effect on reducing cell damage and maintaining normal metabolism by ROS regulation under alkaline stress. Similar results have been observed in salt stress (Yuan, Li, et al., 2021). Additionally, the enhancement of SOD, POD, APX, and GSH-Px/GPX activities in leaves was detected under alkaline stress conditions. Activation of antioxidant enzymes by BR alleviated ROS accumulation and membrane lipid peroxidation under alkaline stress. In the present study, alkaline stress significantly enhanced the transcriptional response in the peroxisome pathway of broomcorn millet leaves. The expression levels of ACOX-, ACAA1-, and ECH-related differentially expressed genes (DEGs) were significantly increased, indicating that alkaline stress induced strong fatty acid oxidation. Meanwhile, extreme transcriptional changes in MVK, FAR, IDH, and PIPOX demonstrate that alkaline stress activates transcriptional reprogramming of membrane proteins, sterol precursor biosynthesis, and amino acid metabolism. The decomposition and transformation of superoxide anion in cells require SOD (Dietz et al., 2016), and the combined action of SOD and CAT can reduce the formation of hydroxyl radicals (Matheson et al., 1975). Our data showed that alkaline stress significantly altered the transcriptional expression of CAT and SOD for the H 2 O 2 metabolism of broomcorn millet, suggesting that broomcorn millet initiates the response of ROS under alkaline stress, which corresponds to SOD and POD activity. Notably, in the glutathione metabolism pathway, the DEGs related to GST, DSR, PGD, and ICD were highly expressed and most DEGs related to LAP and GPX were downregulated under alkaline stress. These results suggest that glutathione metabolism is a strategy of broomcorn millet to resist alkaline stress. In addition, the results of the DAMs in the glutathione metabolism pathway demonstrated the response of broomcorn millet to alkaline stress. Chang et al. (2010) reported that lglutamic acid effectively alleviates the growth inhibition of cucumber under salt-stress. Our data show that BR effectively increased the accumulation of l-glutamate in the glutathione metabolism pathway under alkaline stress. Collectively, these results suggest that alkaline stress strongly initiates transcriptional responses of the peroxisome and glutathione metabolism pathways and regulates the metabolite accumulation. BR alleviated transcriptional changes induced by alkaline stress and promoted the accumulation of l-glutamate to mitigate alkaline stress.

| BR alleviates alkaline stress by maintaining photosynthesis-related transcriptional and metabolic changes in broomcorn millet leaves
Photosynthesis is a physiological biochemical energy metabolic pathway in plants that are directly affected by photosynthetic pigments. Plant growth is inhibited under alkaline stress, probably due to reduced photosynthesis in broomcorn millet. Salt stress inhibits the synthesis of chlorophyll and accelerates its degradation (Zhang et al., 2020). A decrease in photosynthetic pigment levels was also observed in apples grown under alkaline stress . Our results suggest that alkaline stress promotes leaf chlorosis, which accelerates chlorophyll degradation as evidenced by a significant decrease in chlorophyll and carotenoid. These findings suggest that the maintenance of photosynthetic pigments is essential for photosynthesis, as this promotes the conversion of light energy to chemical energy (Staehelin, 2003). In addition, chlorophyll content affects the assembly of photosynthetic reactions and chloroplast development (Hoober et al., 2007). Moreover, the maintenance of photosynthetic pigments is essential for photosynthesis because that promotes the conversion of light energy to chemical energy (Staehelin, 2003). Photosynthetic performance and chlorophyll fluorescence of broomcorn millet leaves were significantly reduced under alkaline stress. BR significantly enhanced photosynthetic properties and chlorophyll fluorescence (P n , T r , g s , F v /F m , q P , NPQ, and ΦPSII), suggesting that BR alleviates alkaline stress by maintaining higher photosynthesis. The reduction in chlorophyll level may be a consequence of the inhibition of some related enzyme activities (Bankaji et al., 2014). In the present study, transcriptome analysis showed that genes encoding proteins related to photosynthesis, including photosystem I, photosystem II, photosynthetic electron transport, and the cytochrome b6/f complex, were suppressed under alkaline stress. Additionally, DEGs of the LHC protein complex in photosynthetic antenna proteins pathway were down-regulated under alkaline stress. Simultaneously, alkaline stress contributed to the upregulated expression of DEGs associated with the carbon fixation in photosynthetic organisms pathway, and these upregulations were attenuated by BR. Meanwhile, alkaline stress caused transcriptional changes in the porphyrin and chlorophyll metabolism pathways. For example, the downregulation of UROD-, chlG-, and GBR-related DEGs directly affected the synthesis of corproporphyrin I, chlorophyll a, and chlorophyll b. These results explain the degradation of photosynthetic pigments, chlorosis of leaves, and reduction of photosynthetic capacity induced by alkaline stress. In addition, DAMs detected in photosynthesis-related pathways, especially Pi (phosphoric acid), were inhibited by alkaline stress and also verified the decrease in photosynthetic capacity of broomcorn millet. Interestingly, various genes encoding proteins related to photosynthesis, photosynthetic antenna proteins, and porphyrin and chlorophyll metabolism maintained better expressions by BR under alkaline stress. These findings indicated that BR alleviates alkaline stress by maintaining a higher photosynthetic capacity.

| CONCLUSION
The present study was conducted to investigate the mitigation of BR on alkaline stress in broomcorn millet. The application of BR effectively reduced the damage of broomcorn millet by alkaline stress, such as inhibition of plant growth and photosynthesis, and damage of structural integrity. The effect of BR on alkaline-sensitive genotypes was better than that of alkaline-tolerant genotypes when grown in alkaline stress conditions. Transcriptomic and metabolomic results revealed the positive regulation of photosynthesis and antioxidant defense-related pathways by BR at the molecular level. In conclusion, our findings show that BR protects the broomcorn millet from alkaline stress damage associated with the improvement of antioxidant properties and photosynthesis ability. Hydroponic limited the field utility of this study. Larger field studies should be conducted in the future to explore the practical application of BR in alleviating alkaline stress. The results reveal the molecular mechanism of BR to enhance the tolerance of broomcorn millet on alkaline stress, which provides theoretical support for the cultivation of bioenergy crops on alkaline lands and the breeding of alkaline-tolerant bioenergy varieties.