When plants are subjected to drought stress, total Chl (e.g., Chla, Chl b, and total Chl) and Car levels may decrease²⁴. Drought stress inhibits the synthesis of Chl a/b²⁴, affects light energy absorption, and transmission and conversion of photosystem I (PSI) and photosystem II (PSII) as well as decreases the photosynthetic rate²⁵. In our study, Chl content decreased under drought stress in industrial hemp (Fig. 1a–d). Our finding that P_N was reduced in the leaves of industrial hemp seedlings under drought stress (Fig. 1e) was similar to that documented by Jhou et al.²⁶. Reportedly, uniconazole application markedly improves contents of both Chl and Car and protects plants against drought²⁷. Similar results were observed in this study (Fig. 1a–d). Many genes involved in porphyrin biosynthesis, such as those for glutamyl-tRNA reductase, protoporphyrinogen oxidase, and the Mg chelatase subunit, are regulated to provide better drought tolerance in plants²⁸. Glutamyl-tRNA reductase, a key enzyme in plant porphyrin biosynthesis, affects Mg chelatase and enzymes involved in Chl synthesis²⁹. Mg chelatase catalyses the first step of Chl synthesis³⁰. Mg protoporphyrin IX monomethyl ester (oxidative) cyclase is a major enzyme involved in Chl biosynthesis, and induction of the corresponding genes improves Chl content and restores chloroplast structure³¹. Chlorophyll(ide) b reductase is a necessary enzyme for Chl b degradation, and inhibition of corresponding genes reduces Chl and Car degradation and promotes thylakoid grana retention³². In the present study, the expression of genes corresponding to glutamyl-tRNA reductase, Mg chelatase subunit, Mg protoporphyrin IX monomethyl ester (oxidative) cyclase, protochlorophyllide reductase, and Chlorophyll(ide) b reductase were regulated by uniconazole under drought stress (Fig. 6a, Supplementary Table S2). The differential expression of porphyrin and Chl metabolism pathway genes suggested that contents of Chl and Car were affected by uniconazole under drought stress (Fig. 1a–d). Some reports showed that under drought stress, plants treated with uniconazole had a higher P_N compared to that in untreated plants¹⁵ same to our study (Fig. 1e). Uniconazole application under drought stress can increase the levels of compounds involved in osmoregulation, lessen lipid peroxidation¹⁶, and protect cell integrity. These positive effects can improve absorption of light energy and thereby enhance photosynthesis capacity. Light harvesting Chl a/b-binding proteins (LHCs), as the most abundant membrane proteins, are involved in ABA signal transduction by regulating ROS (reactive oxygen species) homeostasis to improve tolerance to drought stress and grain yield³³. In the present study, photosynthesis-antenna proteins pathway genes encoding LHCs were up-regulated by uniconazole under drought stress (Fig. 6b, Supplementary Table S3). These results suggest that uniconazole may modulate the drought resistance mechanisms of plants by changes in LHC gene expression. Photosystem II is a unique photosynthesis pigment-protein complex and a site for photosynthetic water oxidation³⁴. The three photosystem II oxygen-evolving enhancer proteins and two photosystem II reaction centre subunits encoded by psbQ, psbP, psb28, and psbW are required for oxidation of water to O₂ and thus are essential enzymes (Fig. 6c, Supplementary Table S4). Cytochrome c6 and plastocyanin are soluble electron carriers that transfer electrons to PSI³⁵. In our study, one cytochrome c6 gene and one PC gene were up-regulated by uniconazole under drought stress. Moreover, uniconazole treatment under drought stress regulated seven PSI reaction centre subunit genes coding for proteins that catalysed the transfer of electrons from PC to FD through a series of electron transporters (Fig. 6c, Supplementary Table S4). FD receives electrons from PSI and transfers electrons to FD-NADP reductase (FNR), FNR induces the transfer of electrons from reduced FD to NADP⁺, and generates reduced NADPH for CO₂ fixation and other chloroplast metabolic processes in the Calvin cycle³⁶. Our study showed that four FD genes and two FD-NADP reductase genes were induced by uniconazole in plants grown under drought stress (Fig. 6c, Supplementary Table S4). The differential expression of photosynthetic pathway genes indicated that photosynthesis plays a significant role in the response to drought stress by uniconazole.

Carbon and nitrogen metabolisms, as the two most basic metabolic processes in plant physiology, affect the formation, transformation, and transportation of photosynthetic products³⁷. Plants grown under drought stress are characterized by reduced Chl contents and decreased photosynthesis, which inhibits starch synthesis. In addition, starch was converted into soluble sugar to maintain cell osmotic pressure³⁸. Some studies showed that the content of soluble sugar and sucrose increases but of starch content decreases³⁹, which may be related to the increase of carbohydrate metabolism activity under drought stress. Analogous consequences were observed in our study: soluble sugar and sucrose content improved, whereas starch content decreased with prolonged drought stress (Fig. 2a–c). SPS, SS, S-AI, and NI are key enzymes involved in starch biosynthesis. Zahoor et al.³⁹ reported an increase in SPS and SS activities but a decrease in S-AI activity in response to drought stress. In other reports, SS activity was lowered⁴⁰ and SPS activity was enhanced⁴¹ by stress. These results were replicated in our studies, indicating that S-AI, NI, and SS activities were significantly reduced but SPS activity was increased in drought-treated plants (Fig. 2d–g). SS can catalyse both the synthesis and decomposition of sucrose. Considering our results, SS might play a role in sucrose decomposition. Improved SPS activity changed the distribution of carbon assimilates, resulting in increased contents of soluble sugar and sucrose, which are important for regulating cell osmotic pressure to resist drought stress⁴². Uniconazole markedly raised the soluble sugar content during waterlogging⁴³, a similar finding was observed here (Fig. 2a). It was reported that exogenous spraying with uniconazole increased starch accumulation, starch granule content, and key enzyme activities involved in starch synthesis ²², same effects were noted in our study (Fig. 2b). An increase in sucrose content and a decrease in starch content were induced by drought stress, indicating that drought promoted the distribution of photosynthetic products to sucrose. Exogenous uniconazole application improved starch content, whereas it reduced the sucrose content of industrial hemp leaves under drought stress (Fig. 2b, c). A reasonable explanation is that uniconazole could enhance the photosynthetic capacity of drought-stressed industrial hemp leaves, which possibly adjusted the synthesis of sucrose and starch influenced by drought stress. Our results revealed that in plants under drought stress, uniconazole supplementation increased S-AI, NI, and SS activities compared with no supplementation (Fig. 2d–f), suggesting that uniconazole promotes sucrose decomposition and regulates photo-assimilation and translocation. SPS affects the distribution of photosynthetic products between starch and sucrose, and its activity is negatively correlated with starch accumulation and positively with sucrose accumulation⁴⁴. We observed similar results: uniconazole decreased SPS activity, increased starch content, and reduced sucrose content under drought stress (Fig. 2b–g). Drought stress has been reported to influence SPS transcripts levels, indicating that SPS enzymes play central roles in starch and sucrose metabolism pathways⁴⁵. In the present study, five SPS genes were regulated by uniconazole under drought stress (Supplementary Table S5). Previous studies have shown that sucrose synthase genes are regulated by drought stress, improving hyperosmotic stress tolerance of plants in relation to the accumulation of starch and sucrose metabolism proteins⁴⁶. The present study described that two SuSy genes were differentially expressed in drought stress induced by uniconazole (Fig. 7a, Supplementary Table S6). In addition, INV (1), scrK (4), bglX (8), GN4 (3), glgC (2), PYG (3), GBE1 (2), TPS (7), TREH (3), and beta-amylase (1) genes were differentially expressed (Fig. 7a, Supplementary Table S6). The differential expression of these genes suggested that the starch and sucrose metabolic pathways were involved in the responses of industrial hemp leaves to drought stress under uniconazole treatment.

Nitrogen metabolism has great significances in plant growth, ecosystem structure and function⁴⁷. Plants absorb nitrate nitrogen (NO₃⁻) and ammonium nitrogen (NH₄⁺) from soil, which are then converted into amino acids, proteins, and nucleotides by the key enzymes of NR, nitrite reductase, GS, GOGAT, and GDH⁴⁷ that are crucial to nitrogen assimilation. Reportedly, the activities of NR, GS, and GOGAT in the functional leaves of plants were significantly reduced by drought stress, which affected the assimilation of NO₃⁻ and NH₄⁺, then inhibited nitrogen metabolism in the leaves of plants⁴⁰. Our results were similar to these: NR, GS, and GOGAT activities of industrial hemp leaves were lower in drought stressed seedlings than in controls (Fig. 3a–c). Hessini et al.⁴⁸ reported that GS levels decreased but GDH activity increased and proline levels improved, suggesting that GDH might be implicated in the synthesis of compatible solute under drought stress. In our results, the activity of GDH increased in drought stressed plants (Fig. 3d). Previous studies with uniconazole revealed that it has important roles in regulating levels of metabolites and promoting an increase in NR activity⁴⁹. These results are similar to those seen in our study, where uniconazole showed increased NR activity under drought stress (Fig. 3a). Overexpression of the NR gene delayed the decrease in NR activity under drought stress, and the plants rapidly recovered N assimilation after rehydration⁵⁰. In our study, one nitrate/nitrite transporter gene was up-regulated significantly by uniconazole under drought stress (Fig. 7b, Supplementary Table S7). Previous studies indicated that the regulation of a nitrate transporter gene implicated in N utilization decreased NO₃⁻ accumulation of guard cells and enhanced drought tolerance of plants⁵¹. In plants under drought stress, exogenous uniconazole application enhanced GS activity compared with no application (Fig. 3b), which is consistent with the results of a previous study⁵². The GS/GOGAT pathway is the major system for utilizing NH₄⁺ in plants, in our study, exogenous uniconazole application increased the activities of GS and GOGAT to relieve the toxic effect of NH₄⁺ over-accumulation on plant seedlings by converting NH₄⁺ to glutamate⁵³. Previous studies showed that the transcription of glutamine synthetase genes and glutamate synthase genes in different tissues was up-regulated or down-regulated with changes in nitrogen levels⁵⁴. Overexpression of glutamine synthetase OsGS1;1 and OsGS2 genes could strengthen the tolerance of plants to adverse abiotic stresses at the seedling stage in rice⁵⁵. Lu et al.⁵⁶ revealed that the suppression of glutamate synthase genes affected nitrogen assimilation in plants and had significant roles in carbon and nitrogen metabolism. In our studies, one glutamine synthetase gene (glnA) and two glutamate synthase genes (GLT1) were regulated by uniconazole under drought stress (Fig. 7b, Supplementary Table S7). These results revealed that uniconazole promoted the GS/GOGAT cycle, which is important in nitrogen metabolism. Reportedly, GS activity was reduced and GDH activity was increased under conditions of adversity stress, suggesting that glutamate and proline were produced by GDH^48,57. Our study showed that DS plants exhibited lower GDH activity than D plants (Fig. 3d). Lightfoot et al.⁵⁸ found that gdhA transgenic plants had improved seed germination rate, growth level, and grain biomass production and showed enhanced plant resistance by promoting nitrogen uptake⁵⁹. In our results, four glutamate dehydrogenase genes were induced by uniconazole under drought stress (Fig. 7b, Supplementary Table S7). In addition, four CA genes (cynT) were differentially expressed under drought stress treated with uniconazole (Fig. 7b, Supplementary Table S7), which may catalyse the conversion between CO₂ and HCO₃⁻, and play important functions in photosynthesizing cells of plants under environmental stress. The differential expression of nitrogen metabolism pathway genes revealed that nitrogen metabolism had an effect on plants under drought stress treated with uniconazole.

A series of changes in plant endogenous hormones under drought stress reflects the adaptability of plants to stress. Moreover, drought resistance in plants is achieved by the complex coordination of various hormones, rather than by a single hormone. ABA and ZT, as critical endogenous hormones, take part in regulating the abiotic stress response. Some studies showed that drought stress substantially enhanced endogenous hormone ABA level but reduced ZT content, indicating that ABA and ZT jointly controlled plant photosynthetic performance in response to drought stress⁶⁰. Our results similarly showed that ABA content was increased but ZT level was decreased under drought stress (Fig. 4a, d). Some reports revealed that ABA and ZT levels significantly increased in plants grown under abiotic stress and treated with uniconazole¹⁴, promoting the accumulation of proline and abiotic stress-related amino acids¹³. We noticed that uniconazole application notably increased ABA and ZT contents under drought stress for 4 days (Fig. 4a, d). Previous studies showed that histidine kinases (AHKs) positively affected drought stress responses through the ABA signalling pathway⁶¹. Two-component response regulator genes are also employed by plants for stress adaptation⁶². A previous study revealed that ABA signalling key kinases interacted with the CTK signalling regulator ARR in response to drought stress⁶³. Liu et al.^21,22 reported that uniconazole regulated AHKs and type-B ARRs to affect Chl degradation. In our studies, histidine kinase genes (AHK2_3_4) and two-component response regulator gene (ARR-A) were induced by uniconazole under drought stress (Fig. 7c, Supplementary Table S8). Protein phosphatase genes have been isolated and cloned from plants and may play key roles in signal transduction in drought stress⁶⁴. Serine/threonine-protein kinases are the key regulatory factors of ABA signalling pathways, which are involved in abiotic stress adaptation in plants⁶⁵. In our studies, protein phosphatase (PP2C) and serine/threonine-protein kinase (SnRK2) were down-regulated by uniconazole as an adaptation to drought stress (Fig. 7c, Supplementary Table S8). Drought stress blocks IAA signal transduction, decreases IAA biosynthesis, and markedly reduces IAA content⁶⁶. In our study, IAA content was decreased with prolonged drought stress (Fig. 4b). Exogenous uniconazole treatment increased IAA content compared with no treatment in seedlings under drought stress (Fig. 4b). A logical explanation is that uniconazole inhibited IAA oxidase activity under drought stress, thus increasing IAA concentration. Similar conditions have been identified wherein uniconazole-treated plants had increased IAA content under water deficit stress¹⁵. It has been reported that auxin transporter genes (AUX/LAX)⁶⁷, auxin-responsive protein (IAA and SAUR)⁶⁸, and indole-3-acetic acid-amido synthetase gene (GH3)⁶⁹ are involved in the auxin signalling pathway and play important roles in drought tolerance. In the present study, auxin-related genes (AUX1, IAA, SAUR, and GH3) were induced by uniconazole in response to drought stress (Fig. 7c, Supplementary Table S8). Gibberellin plays a key role in different stages of plant growth, such as seed germination, internode elongation, and flower and fruit formation and is involved in regulating abiotic stress processes⁷⁰. The GA₃ level decreased under drought stress, restricting the growth of plants¹⁵. In our studies, the content of endogenous GA₃ reduced during the final d of drought stress (Fig. 4c). Uniconazole is an inhibitor of gibberellin biosynthesis, reports also found that endogenous GA₃ content decreased in uniconazole-treated plants^21,22 under stress¹⁵. These results were similar to those obtained in our study, where supplemental uniconazole under drought stress decreased the GA₃ content (Fig. 4c). DELLA proteins, members of the plant-specific GRAS family, negatively regulate plant growth⁷¹. In previous studies, abiotic stress promoted the accumulation of DELLA proteins and then reduced endogenous hormone GA levels to inhibit plant growth and enhance plant tolerance to stress⁷⁰. However, a study had shown that the overexpression of DELLA proteins (S-della) increased stomatal sensitivity to ABA and reduced water loss⁷². Uniconazole affected not only the GA biosynthesis pathway but also related genes in this pathway²³. A previous study showed that uniconazole regulated the expression of DELLA proteins to affect plant growth²². In our study, one DELLA protein was up-regulated by uniconazole under drought stress (Fig. 7c, Supplementary Table S8). In addition, BSK (1) and CYCD3 (1) genes involved in brassinosteroid biosynthesis; JAZ (2) and MYC2 (1) genes involved in jasmonic acid metabolism; and two transcription factor TGA genes involved in salicylic acid biosynthesis were differentially expressed (induced) by uniconazole (Fig. 7c, Supplementary Table S8). Numerous genes were differentially expressed, showing that plant hormone signal transduction pathways are involved in the response of industrial hemp leaves in plants grown under drought stress and treated with uniconazole.

Based on the above discussion, a hypothetical model of the effects of uniconazole on physiology and genome-wide gene expression in industrial hemp seedlings grown under drought stress is presented in Fig. 8.