Physiological and transcriptome analyses reveal the response of Ammopiptanthus mongolicus to extreme seasonal temperatures in a cold plateau desert ecosystem
DEGs under low-temperature stressThe results from the field experiments indicated that the daily mean values of A, Fvʹ/Fmʹ, ETR and Fv/Fm decreased in the LT group, the PSII function was impaired, and the photosynthetic capacity was weakened. Through the specific analysis of the “Photosynthesis” pathway (pathway ID ko00195) in the LT group, it was found that PSII, the cytochrome b6f. complex (Cyt b6f.), PSI and ATPase exhibited differential gene expressions. Figure 9 shows the structural pattern diagram for photosynthesis. The parts marked by white boxes indicate that the structure has DEGs. The gene expressions of CP43, CP47, D1 protein and Cytb559 of PSII changed. The inner peripheral antenna pigment proteins, CP43 and CP47, of PSII bind to chlorophyll. They accept the excitation energy transferred from the surrounding antenna complex and transfer this energy to the reaction centre complex. Changes in CP43 and CP47 affect the absorption and transmission of light energy. In the PSII reaction centre, light energy is converted into chemical energy. P680 absorbs light and is excited to become P680*, and then transfers electrons to pheophytin (Pheo). At the same time, the PSII oxygen-evolving complex obtains electrons from water molecules, the water molecules are split and releases oxygen and protons. As one of the two core proteins that compose the reaction centre complex, the D1 protein combines with various cofactors that are related to the original charge separation and electron transfer. The D1 protein plays an important role in the process of photosynthetic electron transfer. Studies have found that low temperatures can induce allosteric inactivation of the D1 protein, which results in changes in the structure of thylakoid membranes and hinders electron transfer8. As part of the reaction centre, Cytb559 can adjust the photoinhibition sensitivity of PSII through redox changes so that the PSII reaction centre is protected from damage9. The light energy absorption, energy conversion and electron transfer functions of PSII are impaired, which result in significant decreases in Fv/Fm to levels far below the normal value. The results of Xiangchun Song are similar to those presented in this paper: the PS II reaction centre of A. mongolicus seedlings is irreversibly inactivated or the thylakoid membrane is damaged under subzero low temperature stress, which may produce serious photoinhibition. However, Song believes that the peripheral antenna component of the optical system is more affected than the core complex at low temperatures, which was not observed in the corresponding results in this study10.Figure 9Photosynthesis of A. mongolicus under low-temperature stress. The areas outlined by white boxes indicate the differentially expressed genes in these structures.Full size imageThe gene expressions of Cyt b6, PrtD and Cyt f in Cyt b6f. changed. Cyt b6f. changes not only affect the electron transport function of photosynthesis but also affect ATP synthesis. Pheo transfers the received electrons to plastid quinone (PQ). PQ receives electrons and protons to form plastid hydroquinone (PQH2). Then, the electrons of PQH2 are transferred to plastid cyanin (PC) on PSI through Cyt b6f., and hydrogen protons are released into the cavity of the thylakoid to form a transmembrane proton gradient. The transmembrane proton gradient is the driving force for ATP synthesis.The function of PSI is to transfer electrons from PC to ferredoxin for the reduction of NADP+. Recent studies have found that PSI is more sensitive to light and more prone to selective photoinhibition than PS II under low temperature and weak light conditions11,12. The KEGG analysis results indicated that the LHCI complex, PsaF and PsaE subunits of PSI showed differential gene expressions. The main function of the LHCI light-harvesting pigment protein complex is to capture light energy. PsaF is a low-molecular-weight protein that is distributed in the membrane. Some studies have suggested that the N-terminal amino acid sequence of eukaryotic PsaF is involved in the binding of PSI and PC13. PsaE, PsaD and PsaC together form the docking site of ferredoxin on the PSI receptor side14,15. Ferredoxin and ferredoxin-NADP+ reductase in the photosynthetic electron transport chain are also affected, which results in hindrance of NADPH synthesis. The F-type H+/Na+ transport ATPase subunits also show differential gene expressions, which lead to impaired ATP synthesis. Low temperatures affect the ability to absorb light energy, transfer electrons, convert light energy into electric energy, and synthesize NADPH as well as ATP, which ultimately lead to declines in Fv’/Fm’ and ETR and impair the photosynthesis capacity of A. mongolicus.Compared with the light reaction, low temperatures have a greater impact on the dark reaction. Because the dark reaction process is composed of many complex enzymatic reactions, the enzyme activity is very susceptible to temperature. The KEGG results show that 13 related enzymes were differentially expressed in the “carbon sequestration of photosynthesis” (ko00710). The Rubisco enzyme is a key enzyme that determines the direction and efficiency of photosynthetic carbon metabolism in C3 plants and is sensitive to temperature16. The results also show that the expression levels of 10 differentially expressed genes of Rubisco enzymes all declined. In the Calvin cycle, the gene expressions of only transketolase and glyceraldehyde-3-phosphate dehydrogenase are not sensitive to temperature. In addition, the reduction phase of the dark reaction requires the use of NADPH and ATP that are produced by the light reaction. The inhibition of NADPH and ATP synthesis will inevitably affect the normal progression of the Calvin cycle.Chloroplast respiration is an O2-dependent electron transport pathway in chloroplasts. Chloroplast respiration includes the nonphotochemical reduction of PQ by NAD(P) H and the reoxidation of PQ by terminal oxidase, which can consume excess electrons to protect plants from damage due to photooxidation.Figure 10 shows the partial KEGG enrichment metabolic pathway in the LT group. There were three significant enrichment pathways related to carbohydrate metabolism: fructose and mannose metabolism (ko00051), butanoate metabolism (ko00650) and C5-branched dibasic acid metabolism (ko00660). The metabolism of fructose and mannose includes the ascorbic acid biosynthetic pathway. Ascorbic acid (ASA), also known as vitamin C, can be used as a cofactor of violaxanthin de-epoxidase to participate in the lutein cycle and consume excess light energy and protect plants from harm.Figure 10The regulatory mechanism of A. mongolicus under low-temperature stress. The white ovals represent the enriched metabolic pathways. The blue rectangles represent significantly enriched KEGG metabolic pathways. The pathways are followed by the physiological structures and substances or physiological processes in which the expressions of related genes change.Full size imageLow temperatures damage cell membranes first. Increasing the mass fraction of unsaturated fatty acids in the membrane is beneficial to improve the stability and fluidity of the membrane. Some studies have shown that the degree of unsaturation of fatty acids in adult leaves of A. mongolicus that grow naturally in the field is lower in summer and higher in autumn and winter17. The significantly enriched pathways related to unsaturated fatty acid metabolism were alpha-linolenic acid metabolism (ko00592), linoleic acid metabolism (ko00591) and arachidonic acid metabolism (ko00590). Various proteins, such as linoleate 13S-lipoxygenase and cytochrome P450 family 2 subfamily J (CYP2J), which are involved in the metabolism of linoleic acid, showed differences in their gene expressions. Linoleate 13S-lipoxygenase is a common lipoxygenase in plants that can catalyse the production of precursors of several important compounds, including jasmonic acid. CYP2J is a group of P450 haem thiolate proteins, which are mainly distributed on the endoplasmic reticulum and inner mitochondrial membrane and are involved in the synthesis of sterol hormones, including brassinosteroids. Because light systems are distributed on the thylakoid membrane, damage to this membrane will affect the progress of plant photosynthesis.Plant hormone signal transduction (ko04075) plays an important role in plant resistance to stress. Studies have shown that JAs have physiological functions, such as inducing stomatal closure, inhibiting photosynthesis, promoting respiration and promoting leaf senescence18,19. Treating plants with exogenous methyl jasmonate can induce the transcription of the heat shock protein family, increase the synthesis of antioxidants, reduce lipoxygenase activity and enhance the ability of plants to resist cold damage20.Figure 11 shows the regulatory mechanism of A. mongolicus in the HL group. The MapMan analysis results show that the DEGs of the LHCII complex and those for the assembly and maintenance of PSII are significantly changed. LHCII contains chlorophyll and carotenoids, which can capture and transmit light energy. Chlorophyll is an important photosynthetic pigment that captures light energy and drives electrons to the reaction centre. The chlorophyll molecule in the reaction centre is related to photochemical quenching. The entire chlorophyll biosynthesis process (e.g., L-glutamyl-tRNA → chlorophyll a → chlorophyll b) involves 15 enzymes. The analysis found that 4/5 of the enzymes’ expression genes were changed. Carotenoids include carotene and lutein, and their synthesis is affected by high temperatures. Lutein participates in the lutein cycle, which can dissipate excess light energy and prevent membrane lipids from being peroxidized and thus maintain the stability of the thylakoid membrane structure and protect A. mongolicus. from high temperature stress and strong light stress.Figure 11The regulatory mechanism of A. mongolicus. under high-temperature stress. The white ovals represent enriched metabolic pathways. The red rectangles represent significantly enriched KEGG metabolic pathways. The pathways are followed by the physiological structures and substances or physiological processes in which the expressions of related genes change.Full size imageThe D1 protein in the PSII reaction centre is rapidly degraded under strong light conditions. To maintain the normal physiological needs of plants, the degraded D1 protein will be replaced by the new D1 protein that is produced by the repair mechanism. The reversible inactivation of the PSII reaction centre can protect the photosynthetic system and avoid destruction. This may be the reason for the significant changes in the DEGs that are involved in the assembly and maintenance of PSII.Rubisco is the main site for high-temperature inhibition of the Calvin cycle16. The KEGG analysis found that there were 7 (4↑, 3↓) DEGs of Rubisco. SBPase catalyses the conversion of sedum heptulose-1,7-diphosphate (SBP) into sedum heptulose-7-phosphate (S7P) in the renewal phase. Under low-temperature stress, only transketolase and glyceraldehyde-3-phosphate dehydrogenase remained unchanged in the Calvin cycle. In addition, NDH-mediated cyclic electron transfer may decreased the photooxidation damage that is caused by high-temperature stress by shunting the excess electrons that were generated by the inhibition of CO2 assimilation to the chloroplast respiratory pathway21.In the HT group, the net photosynthetic rates of the leaves showed two peaks on the diurnal change curves, and there was an obvious phenomenon of midday photosynthesis depression. The daily average A values were greater than those of the CK group. These results show that A. mongolicus has a complete photosynthetic structure protection mechanism and can adapt to high-temperature environments. The pathway of significant enrichment related to carbohydrate metabolism in the HT group was the same as that in the LT group. The enrichment degrees of the fructose and mannose metabolic pathways were higher only in the HT group, and C5-branched dibasic acid metabolism and butanoate metabolism were higher in the LT group.Under high temperature and strong light conditions, the balance between production and removal of reactive oxygen species (ROS) in plant cells was broken, and large amounts of reactive oxygen species accumulated in the cells. Active oxygen can cause lipid peroxidation of the biomembrane, enlarge membrane pores, increase the permeability, and affect the spatial structures of enzymes on the membrane, which thus leads to chloroplast destruction. In severe cases, ROS will cause serious injury or even death to plants22. The gene expressions of FabH and acetyl-CoA carboxylase (ACCase) changed during the synthesis of unsaturated fatty acids in the HT group.There are two types of active oxygen scavenging mechanisms in plants. (1) The enzymatic detoxification system: superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT). (2) Nonenzymatic antioxidants: ASA, carotenoids, glutathione, mannitol, and flavonoids23.Secondary metabolites result from long-term adaptation of plants to their environments. They can improve the ability of plants to protect themselves, compete for survival, and coordinate the relationship between plants and the environment. The significant enrichment pathways related to the biosynthesis of secondary metabolites in the HT group consisted of phenylpropane biosynthesis (ko00940), flavonoid biosynthesis (ko00941) and isoflavone biosynthesis (ko00943). The phenylpropanoid biosynthesis pathway is one of the three main secondary metabolic pathways in plants. It starts from phenylalanine and generates different phenylpropane metabolites through multistep reactions, such as flavonoids, isoflavones, anthocyanins and lignin24,25. Anthocyanins can protect plants from light damage by quenching free oxygen radicals and reducing the absorption of light energy. Hughes studied 10 species of evergreen broad-leaved trees and found that red leaves containing anthocyanins always maintained higher Fv/Fm levels than green leaves. Fv’/Fm’ is related to nonphotochemical quenching. This means that trees with red leaves rely more on the light-damage defence function of anthocyanins than on the light-damage defence mediated by lutein26.Riboflavin metabolism (ko00740) and biotin metabolism (ko00780) are two significantly enriched cofactors and vitamin metabolic pathways. Riboflavin is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). As a prosthetic group of flavinases, FAD participates in multiple biochemical processes, such as mitochondrial electron transport, photosynthesis, fatty acid oxidation and folate metabolism, in plants27. Riboflavin can induce antioxidant accumulations in plant cells and can also promote plant growth by affecting the ethylene signalling pathway28. Biotin (e.g., VH or VB7), as an essential cofactor for biotin-dependent carboxylase, plays an important role in the life activities of plants. Common biotin-dependent carboxylase enzymes are pyruvate carboxylase (PC) and ACCase. PC is present in the mitochondria and participates in the replenishment mechanism of the tricarboxylic acid cycle. ACCase plays a pivotal role in the feedback regulation of fatty acid synthesis and is the site of action for the feedback regulation of fatty acid synthesis29.The four pathways related to amino acid metabolism showed differences in the HT group. The enrichment degrees of each pathway were as follows: valine, leucine and isoleucine biosynthesis (ko00290) > biosynthesis of amino acids (ko01230) > lysine biosynthesis (ko00300) > glycine, serine and threonine metabolism (ko00260). The branched chain amino acids, valine, leucine and isoleucine and their derivatives, are beneficial to plant growth and plant responses to stress30. As an essential amino acid, lysine metabolism affects many physiological reactions, such as the tricarboxylic acid cycle, abiotic and biotic stress responses, and starch metabolism31. The glycine, serine and threonine metabolic pathways combined with the GO enrichment results showed that the genes related to glycine catabolism and glycine dehydrogenation/decarboxylase activity changed greatly. It is known that when the activity of mitochondrial glycine decarboxylase increases, both photorespiration and photosynthesis will increase32.In terms of hormones, salicylic acid, cytokinin, and abscisic acid (ABA) can improve plant active oxygen scavenging ability. Salicylic acid can decrease the damage to seedlings due to high temperatures by improving the ability of plants to resist oxidative stress and increasing the contents of osmotic adjustment substances in cells33. Salicylic acid also has the function of delaying the degradation of D1 protein and speeding up the recovery of D1 protein when high temperatures are no longer present34. ABA can improve the heat tolerance of plants by regulating the expressions of heat stress-induced genes at the transcriptional level35.In conclusion, A. mongolicus has weak resistance to low temperatures and good adaptation to high temperatures. At the physiological level, under low-temperature stress, the proportion of Y (NO) increased, the function of PSII was damaged, and photosynthesis was inhibited. A. mongolica maintains normal physiological activities by regulating the circadian rhythm, increasing the synthesis of unsaturated fatty acids and changing the effects of plant hormones. Under high-temperature stress, A. mongolicus maintains normal photosynthesis by adjusting gsw as well as water utilization and by increasing the proportion of Y (NPQ). At the same time, A. mongolicus uses LHCII to consume excess energy, continuously assembles and maintains the normal function of PSII, and changes the types of antioxidants, such as by synthesizing anthocyanins, flavonoids, and isoflavones, to protect itself from injury. In addition, the porphyrin and chlorophyll metabolisms, carotenoid metabolism, plant hormones, amino acid metabolism, unsaturated fatty acid synthesis and other metabolic pathways that are related to the differentially expressed genes changed greatly. More
