The growth of rapeseed was stunted under drought stress and the loss in biomass was more seriously under potassium deficiency. Youyan57 kept much higher potassium level in tissue and larger biomass both under NK and LK, showing higher drought tolerance than Chuanyou36. Large difference was observed in transcriptom profile between the two contrast cultivars in response to drought stress, being 1689 and 1050 DEGs identified between Youayn57 and Chuanyou36 under NK and LK, respectively. Furthermore, there were 461 commonly expressed DEGs, and 1228 and 589 unique DEGs were detected in NK and LK, respectively, indicating that potassium deficiency hindered ability of transcription regulation and induced diverse transcriptome in rapeseed encountering drought stress.
Glutathione (GSH) is an important antioxidant to detoxify reactive oxygen species in plant under stress, and improved expression of glutathione synthetase (GS) enhances the glutathione pool which results in greater tolerance to environmental stresses (Park et al., 2017). Glutathione S-transferases (GST) play major roles in oxidative stress metabolism, which catalyze the conjugation of reduced GSH to electrophilic substrates (Cummins et al., 2011). Drought stress could induce the expression of GST and improved tolerance was observed in GST overexpression plants (Srivastava et al., 2019). Different molecular regulation mechanism was observed in Youyan57 and Chuanyou36 under NK and LK. GS and 6-phosphogluconate dehydrogenase gene which promoted glutathione biosynthesis were both up-regulated under NK and LK, indicating that Youyan57 was capable of biosynthesis of GSH under drought stress. A total of 11 GST were down-regulated under NK, and the expression pattern of GST was affected largely under LK with 3 GST down-regulated. The results indicated that Youyan57 could improve GSH pool in tissues to eliminate the excess reactive oxygen radicals, while Chuanyou36 adapted to drought stress through consuming of GSH by enhanced expression of GST which was sensitive to potassium deficiency. Glucosinolate is a kind of secondary metabolites found in Brassicaceae that protect plants from herbivory and pathogen attack, and also plays important roles in drought tolerance (Mohammad et al., 2019). Drought-induced accumulation of glucosinolate in leaves directly or indirectly controls stomatal closure to prevent water loss in rapeseed (Seung Hee et al., 2018). Cytochrome P450 plays important role in yield of glucosinolate in plants (Kai et al., 2011). Methylthioalkylmalate synthase catalyzes the committed step in the side chain elongation of methionine, yielding important precursors for glucosinolate biosynthesis in Brassicaceae species (Kraker and Gershenzon, 2011). In this study, 3 cytochrome P450 and 5 methylthioalkylmalate synthase were down-regulated under NK. Meanwhile, 2 cytochrome P450 and 3 methylthioalkylmalate synthase were down-regulated under LK. Coincidently, we found that potassium deficiency led to massively accumulation of glucosinolate in seeds, and Youyan57 kept much less glucosinolate in seeds than that of Chuanyou36 under drought stress with different potassium levels in our later study (Zhu et al., 2021). The findings indicated that biosynthesis of glucosinolate was an important mechanism for Chuanyou36 in response to drought stress.
The photosynthesis decreased dramatically under LK compared to NK. Large difference in photosynthesis was observed with two genotypes under NK and LK, and Pn of Youyan57 was 1.73-timefolds to that of Chuanyou36 under LK. Changed molecular regulation mechanism of photosynthesis was observed in response to drought stress under different potassium status. There were 6 DEGs up-regulated related to photosynthesis under NK, including 5 Chlorophyll a-b binding protein which involved in light harvesting chlorophyll protein complex (Horn et al., 2007). A total of 5 DEGs up-regulated related to photosynthesis under LK, including 1 chlorophyll a-b binding protein, 2 oxygen-evolving enhancer protein involved in photosystem II, 1 ferredoxin and 1 ferredoxin-NADP reductase which involved in photosynthetic electron transportation (Kimata-Ariga et al., 2019). The results indicated that photosynthetic electron transportation was affected seriously in Chuanyou36 under LK as potassium depletion in tissue. Mitochondrial oxidative phosphorylation (OXPHOS) provides ATP for driving cellular functions, and is composed of 5 protein complex, including NADH dehydrogenase complex (complex I), succinate dehydrogenase complex (complex II), cytochrome c reductase complex (complex III), cytochrome c oxidase complex (complex IV) and ATP synthase complex (complex V). At daytime, OXPHOS takes place in the context of photosynthesis in plants, and NADH produced by photorespiration is the main substrate of respiratory electron transfer chain rather than NADH produced by the TCA cycle (Braun, 2020). Besides, plants possesses type II NADH dehydrogenase enzymes bypass the complex I allowing turnover of NADH without translocating protons into OXPHOS, which can avoid electron transfer chain over-reduction and help prevent cellular damage under environmental stress (Rasmusson et al., 2020; Sweetman et al., 2019). In this study, four genes encoding NADH dehydrogenase were down-regulated under NK. By contrast, five up-regulated DEGs were identified related to ATP synthesis under NK, including 2 soluble inoranic pyrophosphatase and 3 ATP synthase. We deduced that enhanced expression of NADH dehydrogenase genes in Chuanyou36 could alleviate photoinhibition under drought stress by sustaining non-phosphorylating pathway of electron transportation. Four out of 6 DEGs associated with oxidative phosphorylation were up-regulated under LK, including NADH dehydrogenase, cytochrome c reductase, soluble inorganic pyrophosphatase, ATP synthase. The results indicated that the drought tolerant cultivar Youyan57 kept higher level of oxidative phosphorylation to biosynthesis of ATP than that of drought-sensitive cultivar both under LK and NK.
The plant hormone IAA regulates many aspects of plant growth and development, including stem elongation, establishment of embryonic polarity, vascular development, and cell expansion. Several major classes of auxin-responsive genes involve in plants quickly sense and respond to changes of auxin levels, including Aux/IAA family, auxin response factor (ARF) family, small auxin upregulated RNA (SAUR), and the auxin-responsive Gretchen Hagen3 (GH3) family genes (Luo et al., 2018; Spartz et al., 2012). In this study, IAA content decreased under drought stress and no significant difference was observed between the two cultivars under NK. There were 4 up-regulated DEGs related to auxin signal transduction under NK, including 2 auxin-responsive protein IAA9, 1 auxin responsive GH3 family gene, and 1 auxin-responsive protein SAUR gene. Compared to NK, IAA content at LK decreased significantly, and IAA content in Youyan57 was significantly higher than that of Chuanyou36. Furthermore, 3 out of 4 DEGs were up-regulated and involved in auxin signal transduction under LK, including 2 AUX/IAA family genes and 1 GH3 auxin-responsive gene. Thus, the drought tolerant cultivar Youyan57 kept higher level of IAA in tissue and enhanced IAA signal transduction than that of Chuanyou36 under LK. The plant hormone ABA plays a crucial role in response to drought stress, which can induce stomata movement and thus reduce water loss, ROS scavenging enzymes, and proline accumulation (Chen et al., 2021). Compared to NK, ABA content increased significantly under LK, and no significant difference was observed in ABA content between the two cultivars. Instead, large difference was observed in ABA signal transduction between Youyan57 and Chuanyou36. Protein phosphatase 2C is a negative regulator of ABA signal transduction and functions as a switch at the center of the ABA signaling network (Chen et al., 2021). Protein phosphatase 2C counteracts SnRK2 kinases by physical interaction, and thereby inhibit activation of the transcription factors that mediate ABA-responsive gene expression. Under osmotic stress conditions, Protein phosphatase 2C binds to pyrabactin resistance 1 (PYR1)/ PYR1-LIKE (PYL)/ regulatory components of ABA receptors (RCAR) intracellular ABA receptors to capture ABA and releases active SnRK2s, resulting in phosphorylation of ABFs and activation of other ABA response pathways (Dupeux et al., 2011; Jung et al., 2020). In this study, 1 protein phosphatase 2C was down-regulated under NK. By contrast, 4 protein phosphatase 2C genes were down-regulated under LK. We deduced that the enhanced expression of protein phosphatase 2C disrupted ABA signal transduction under potassium deficiency in Chuanyou36, which induced defective regulation of stomatal closure, resulting in larger water loss under drought stress. Besides, 2 ABC transporters were down-regulated under LK. ABC transporters are responsible for ABA transportation, and mutants of ABC transporter is defective in ABA signaling in guard cells (Kuromori et al., 2011). The ethylene signal is perceived by ethylene receptors in plants, such as ethylene insensitive 2 (EIN2), and then induces the accumulation of transcription factor EIN3 which is a key positive switch in ethylene perception (Binder, 2020; Yanagisawa et al., 2003). EIN3 is recognized for ubiquitination through the identification of two E3 components, EIN3 Binding F-BOX1 (EBF1) and EBF2 proteins. EBF1 and EBF2 interact directly with EIN3 and subsequently promote degradation of EIN3, which is critical not only for proper ethylene signaling but also for growth in plants (Gagne et al., 2004). EBF1 and EBF2 fine-tune ethylene responses by repressing signaling in the absence of the hormone, dampening signaling at high hormone concentrations, and promoting a more rapid recovery after ethylene levels dissipate (Binder et al., 2007). In our study, the EIN2 was down-regulated under NK, while EBF2 was up-regulated both under NK and LK, indicating that Youyan57 regulated ethylene signaling properly under drought stress. CTK affects many aspects of plant development including cell division, shoot induction, and vascular development. And histidine-containing phosphotransfer proteins act as positive regulators of CTK signaling in plants (Hutchison and Kieber, 2007). Compared to NK, CTK content decreased under LK, and Youyan57 kept higher level of CTK in tissue than that of Chuanyou36 both under NK and LK. A histidine-containing phosphotransfer protein was down regulated under NK, which could compensate the decrease of CTK in Chuanyou36.
Potassium is the most abundant inorganic cation which can make up 10% of plant’s dry weight. It is a primary cellular osmoticum and also plays important role in neutralization of negative charges (Karley, 2010). Reduced K concentration in plant tissues should lead to disturbance of metabolism, and amino acids are the most important contributor of osmoregulation in addition to potassium for plants (Morgan, 1992). In this study, the drought tolerant cultivar Youyan57 kept higher potassium level than Chuanyou36, being 14.5% and 10.9% over than that of Chuanyou36 under NK and LK, respectively. By contrast, large number of DEGs associated with biosynthesis of amino acids were down-regulated under NK. The results indicated that the drought-sensitive cultivar Chuanyou36 largely relied on biosynthesis of amino acids for osmoregulation rather than potassium under drought stress. However, the great enhancement of transcription regulation involved in amino acids biosynthesis was eliminated in Chuanyou36, with much less DEGs identified under LK. A previous study found that biosynthesis of amino acids was increased both for Youyan57 and Chuanyou36 under potassium deficiency and no significant difference was observed in total content of amino acids between the two cultivars under LK (Zhu et al., 2020). We deduced that potassium deficiency hindered the metabolism processes of Chuanyou36 more seriously and further affected amino acids biosynthesis under drought stress.
Carbohydrates are the ultimate source of carbon skeleton for biosynthesis of amino acids (Stewart et al., 1966), and carbon starvation is observed with increased accumulation of amino acids (Smith and Stitt, 2007). Sucrose phosphate synthase is the key rate-limiting enzyme in sucrose synthesis which controls sucrose content in plants (Anur et al., 2020). β-fructofuranosidase or invertase catalyzes the hydrolysis of sucrose into fructose and glucose (Pedezzi et al., 2014). β-glucosidase catalyzes the hydrolysis of cellobiose and cellooligosaccharides containing (1, 4)- β-glycosidic bonds to glucose (Huang et al., 2021). Glucose-1-phosphate uridylyltransferase catalyzes the formation of UDP-glucose from glucose-1-phosphate and UTP(JB and HM, 2007). In this study, sucrose phosphate synthase, β-fructofuranosidase, glucose-1-phosphate uridylyltransferase and three β-glucosidases were down regulated under NK, while no significant difference was observed in these genes under LK. Instead, two DEGs encoding starch synthase which related to starch synthesis were down-regulated under LK. The enhanced expression of genes related to production of glucose and sucrose in Chuanyou36 was benefited to biosynthesis of amino acids under NK. Trehalose is a non-reducing disaccharide of glucose which plays an important role in mediating biotic and abiotic stresses in plants, and improved biosynthesis of trehalose by overexpression of trehalose phosphate synthase/phosphatase gene can enhance crops tolerance to drought and saline stresses (Joshi et al., 2020; Lyu et al., 2013). Trehalose-phosphate synthase and trehalose-phosphate phosphatase were up-regulated both under NK and LK, indicating that drought tolerant cultivar Youyan57 could accumulated more trehalose in response to drought stress.
Two-thirds of the DEGs involved in pathway of glycolysis, pyruvate metabolism and citrate cycle were down regulated under NK. Fructose-1-6-bisphosphatase, glyceraldehyde 3-phosphate dehydrogenase and enolase which involved in glycolysis were down-regulated. Meanwhile, three aldehyde dehydrogenase family genes and one alcohol dehydrogenase gene were down-regulated. Aldehyde dehydrogenase is essential for scavenging redundant aldehydes by converting aldehyde into acids when plants are exposed to stress, and enhanced tolerance to drought stress was observed in aldehyde dehydrogenase overexpression plants(Chen et al., 2015). It could be inferred that large amount of pyruvate was resolved into acetaldehyde which triggered the expression of aldehyde dehydrogenase and alcohol dehydrogenase in Chuanyou36 under NK. Even though much less DEGs were identified under LK compared with those of NK, large proportion of DEGs were up-regulated in energy production and conversion under LK. 6-phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase which involved in glycolysis were up-regulated. The gene encoding dihydrolipoamide acetyltransferase which was a component of pyruvate dehydrogenase complex was up-regulated under LK. Citrate synthase and succinyl-CoA ligase were up-regulated under LK. Citrate synthase was a key rate-limiting enzyme for citrate cycle (Schmidtmann et al., 2014). The results indicated that drought tolerant cultivar Youyan57 kept higher citrate cycle level under LK.