Transcriptomic and physiological analysis of Spirodela polyrrhiza responses to sodium nitroprusside

doi:10.21203/rs.3.rs-2368117/v1

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Transcriptomic and physiological analysis of Spirodela polyrrhiza responses to sodium nitroprusside

https://doi.org/10.21203/rs.3.rs-2368117/v1

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published 08 Feb, 2024

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Background: Spirodela polyrrhiza, a simple aquatic floating plant with great potential in synthetic biology. It has been noted that nitric oxide (NO) stimulates plant development and raises the biomass and flavonoid content in some plants. However, the molecular explanation on the mechanism of NO action is still unclear.

Results: S. polyrrhiza was treated with various concentrations of sodium nitroprusside (SNP) as an NO donor. Physiological and transcriptomic analysis were performed in our study. The results showed that under low concentration SNP conditions, S. polyrrhiza alleviated malondialdehyde accumulation, increased levels of fresh weight, dry weight, starch, soluble protein, and flavonoids, and enhanced antioxidant enzyme activity. 2776 genes were found to have differential expression in the S. polyrrhiza treated with 0.025 mM SNP and control groups by RNA-Seq. Of these DEGs, in comparison to the controls, 1425 genes were up-regulated and 1351 genes were down-regulated. The findings of the qRT-PCR study revealed that the expression of genes involved in flavonoid biosynthesis, NO biosynthesis, as well as transcription factor (TF) was increased, while the expression of photosynthesis related-genes was decreased. Under SNP treatment, S. Polyrrhiza redirects metabolic flux of fixed CO₂ into starch synthesis branch and flavonoid biosynthesis branches.

Conclusions: The results provide new insights into the mechanisms causing the accumulation of starch and flavonoids by SNP treatment, meanwhile, The SNP-regulated genes would make excellent candidates for synthetic biology to increase the flavonoid content in S. Polyrrhiza.

Spirodela polyrrhiza

Sodium nitroprusside

Metabolic flux

Synthetic biology

Duckweed (Lemnacecae family) is the tiniest and simplest flowering aquatic plant in the world, with a size of only a few millimeters. Its development is exceptionally climatically adaptable [1]. It has a long yearly production period with an almost exponential growth rate, producing biomass faster than most other plants. Lemnaceca, a collective term for plants in the Lemnoideae subfamily, refers to monocotyledonous aquatic plants that float freely and have vascular systems. Lemnoideae includes five genera: Landoltia, Lemna, Spirodela, Wolffiella, and Wolffia [2]. The biological structure of duckweed is simple, which the plant body consists of “frond” leaf-like structure, and “root” (except for Wolffiella and Wolffia, which are rootless duckweeds), propagate mainly via asexual reproduction [3], it grows very fast and accumulates biomass with high content of starch [4] and protein [5] and low content of lignocellulose [6]. In recent years, duckweed has made a breakthrough in biosynthesis, with S. polyrrhiza as a chassis plant for synthetic biology. The advantages are as follows: (1) biological structure: S. polyrrhiza, the largest duckweed in North America, is roughly 7 to 15 mm long and has huge fronds, making it useful for laboratory molecular biology research and tissue culture operations [7]. (2) clear genetic background: the duckweed family's first species with a full genome sequence is S. polyrhiza, and a high-confidence genetic map has been created based on the genomic data [8]. (3) platform for genetic modification that is well-established: till now, the genetic transformation of S. polyrrhiza genera has been achieved successfully and the transform efficiency reaches 5%-13% [9–11]. (4) abundant precursor substances: S. polyrhiza contains a variety of beneficial secondary metabolites, such as terpenoids and phenolic compounds [12].

Nitric oxide (NO) is a small diffusible gaseous bioactive substance and an important signaling molecule that has positive effects on mediate diverse plant physiological processes such as germination, root growth, flowering, stomatal closure, and resistance to biotic as well as abiotic stresses [13–16]. NO acts as an antioxidant molecule, may maintain ROS homeostasis by regulating the antioxidant enzyme activities [17]. Sodium nitroprusside (SNP) is a NO donor that can release NO upon dissolution in aqueous solvents. Research has demonstrated that SNP treatment improves the activity of antioxidant in blueberry fruit, tomato, soybean sprouts, okra, chard and alfalfa [18–23]. Recent years, it is becoming more and more clear that NO is a mediator of the synthesis of secondary metabolites in plants. Research has shown that NO of the biosynthetic of secondary metabolites such as alkaloid [24], flavonoids [25], terpenoids [26] and saponin [27] via the donor SNP. The majority of studies, however, concentrated on vegetables and herbaceous plants [28–30] and there were few studies on aquatic plants [31].

Previous studies have shown that S. polyrrhiza coped with abiotic stress by fixing CO₂ into the starch biosynthesis pathway, as a result, biomass rich in starch is rapidly accumulated [32]. We're curious how the energy distribution of S. polyrrhiza will change after NO treatment. It remains unknown whether the metabolic flux is primarily directed into the starch branch or the secondary metabolites branch. In our work, S. Polyrrhiza were treated with exogenous NO, the changes in biomass accumulation, starch content, flavonoid content, and antioxidative related enzyme activity dynamics of S. Polyrrhiza were studied. Moreover, transcriptome profiles of S. Polyrrhiza the SNP stress treatment and a control treatment were sequenced. This provided molecular support for the metabolic flux research of S. polyrrhiza under NO treatment and the use of S. polyrrhiza as a bioreactor. This is crucial for the development of S. polyrrhiza as a model plant for synthetic biology.

Effect of SNP on NO accumulation

One of the first reactions of plants to various environmental stressors is the accumulation of NO. The results of present work show that NO levels of the S. Polyrrhiza treated with SNP are increased(Fig. 1), showing that SNP exposure may induce rapid NO generation in the S. Polyrrhiza. The highest NO level is obtained at about 1 day after SNP treatment (Fig. 1), which means that at 0.05 mM SNP, the accumulation of NO was enhanced by 89.9% compared to the control group.

Photosynthetic pigment and chlorophyll fluorescence

Quantification of chlorophyll is frequently used to estimate how plants react to various stresses. The contents of S. polyrrhiza's photosynthetic pigment were found to be significantly different between CK and SNP treatments. However, there was no discernible distinction between the SNP treatment groups. As the SNP concentration increase, the chlorophyll a, b and carotenoids showed a rising trend after downward first. Under SNP stress reduced chlorophyll a by 48%, chlorophyll b by 39.7%, carotenoids by 35.3% at 0.01 mM, compared to plants grown under control conditions (Fig.2 A, B, C). As the SNP concentration increase (Fig.2 D, E, F), the Fv/Fm showed a downward trend, lowest at 0.5 mM SNP and it is decreased by 76.2% higher than the control group. The ABS/RC and DIo/RC change trend was contrary with Fv/Fm, peaked at 0.5 mM SNP and it is increased by 2.94 and 16.9-fold higher than the control group.

Physiological response of S. polyrrhiza to exogenous NO

We measured fresh weight, dry weight, starch, soluble protein, MDA, H₂O₂, proanthocyanidins contents and SOD, POD, CAT enzyme activity to observe the physiological changes in S. polyrrhiza response to NO (Fig. 3).

As the SNP concentration increase, the fresh weigh showed a significant downward trend after rising first, peaked at 0.01 mM SNP and it is increased by 12.58% higher than the control group (Fig. 3A). Application of SNP significantly affected dry weigh accumulation of S. polyrrhiza. Comparing the results to the control samples, the findings indicated that SNP can increase the dry weight yield (Fig. 3B). The dry weight of S. Polyrrhiza with an increase of 27.87% in 0.01 mM SNP treatment samples compared with control group. The starch content change trend was consistent with fresh weigh and dry weigh, it increased and then decreased and peaked at 0.025 mM SNP, which was around 1.5 times more than that of the control (Fig. 3C). The soluble protein change trend was consistent with starch. The highest soluble protein content was demonstrated in plants grown on medium with 0.025 mM SNP, which increased 42.06% over the control group (Fig. 3D). The MDA content was lower in the SNP treatment group than in the control group, with the lowest significant MDA concentration (0.3 mM SNP) decreasing by 55.63% (Fig. 3E), which implied that exogenous NO at a specific concentration effectively inhibit the accumulation of MDA. The H₂O₂ content of 0.5 mM SNP-treat group increased compared with control group, from 19.56 μmol/g FW to 29.92 μmol/g FW, indicating that the high concentration of SNP caused an excessive accumulation of ROS, which aggravated membrane lipid peroxidation (Fig. 3F) .

The SOD activity showed a significant downward trend after rising first, compared to the control group, the SOD activity of 0.01 mM SNP treatment group increased quickly, from 149.67 U/g FW to 201.67 U/g (Fig. 3G). The CAT activity showed a downward trend after rising first with the increase of SNP concentration, however, there was no distinction between SNP and the control group (Fig. 3H). The POD activity showed a significant downward trend after rising first with the increase of SNP concentration, and POD activity was significantly higher in the 0.1 mM SNP treatment group (3860.03 U/g) than in the control group (1015.43 U/g) (Fig. 3I).

Flavonoids biosynthesis affected by exogenous NO

Previous studies have been reported that duckweed species S. polyrrhiza have been widely utilized as folk medicine in China, Korea and a few European nations [33]. Duckweeds are medicinal herbs that don't have severe side effect [34]. Flavonoids are the main pharmacological effects components of duckweed [35] , such as apigenin, orientin, vitexin, and luteolin-7-O-glucoside. Orientin, vitexin and luteolin-7-O-glucoside has anti-inflammatory, antioxidant, anti-tumor and other effects [36-38]. We determined the contents of three bioactive compounds of the flavonoids biosynthesis pathway, orientin, vitexin, and luteolin-7-O-glucoside. In comparison with the CK, the three compounds levels were significantly increased by all groups of SNP elicitation（Fig. 4 A, B, C）. The treatments with 0.025 mM SNP were most efficient, among the three compounds has the highest content. Proanthocyanidins are a kind of polyphenols. Their special structure determines their strong antioxidation, and they have anti-cancer, antimutagenic, cardiovascular protection and other effects [39]. The proanthocyanidins contents showed a downward trend after rising first with the increase of SNP concentration. Reach the maximum value in 0.025 mM SNP (Fig. 4D). This result is consistent with the observation at 0.01, 0.025 mM SNP leaf appeared chlorosis and accumulation of purplish red pigment on the back mentioned above (Additional file 1: Figure S1). Hence, SNP acted as an effective elicitor to positively, modulate flavonoids biosynthesis and 0.025 mM SNP might be the optimal concentration for further analysis.

Global gene analysis

In total, six cDNA libraries were constructed and sequenced. While robust data were collected, after data filtering, 43.50, 44.84, 44.32, 39.85, 48.41, 39.08 million high-quality reads were obtained at the CK and SNP treatment, respectively. Subsequently, these clean reads were de novo assembled into 33,684 genes, with a mean length of 1,360 bp and a N50 length of 2,791 bp (Table 1).

The unigenes mapped to the database searches are shown in Table 2. Using the NCBI NR database, 15,127 unigenes were mapped to ten identified plant species and ‘other’ plant species (Additional file 2: Figure S2). Using SwissProt database, 11,816 unigenes were annotated and reviewed with the UniProt Knowledgebase. Using the KOG database, proteins deduced from the identified RNA sequences were matched with 9,314 unigenes identified and classified into 25 functional categories. Using the KEGG database, 14,966 unigenes were enriched in 137 biochemical pathways, such as plant-pathogen interaction, carbon metabolism, plant hormone signal transduction and phenylpropanoid biosynthesis. Transcriptome profiles were compared between CK and SNP-treated group. A total of 2,776 DE genes were identified, of which, 1,425 was up-regulated, while 1,351 was down-regulated respectively (Fig. 5).

DEGs related to transcription factors (TFs)

Transcription factors (TFs) are key regulators that temporarily and spatially turn on or off the transcription of their target genes through binding certain upstream elements [40]. A total of 208 significant differences TFs (FDR < 0.05 and |log2FC| > 1) were identified regulated S. Polyrrhiza response between CK and SNP and divided into 36 categories, including 125 up TFs and 83 down TFs (Fig. 6A). In this study , several TFs responded significantly to SNP stress (Fig. 6B), including the ERF (22), bHLH (20), NAC (17), WRKY (15) (Additional file 3: Table S1). The ERF that are specific to plants as well as play a role in a variety of developmental processes, such as flowering [41], seed development [42], and fruit ripening [43]. Among these TFs, the most abundant were the ERF family, which accounted for 11.7% of total TFs. Gene expression changes a log2-fold, between-5.42 and 10.88. BHLH is involved in stress tolerance, pathogen defense, and nutrient uptake but also plays an important role in the production of secondary metabolites such as flavonoids and anthocyanins [44,45]. Twenty members of the bHLH TFs were differentially expressed between SNP conditions, ten of these genes were up-regulated and ten were down-regulated. A crucial part of plant biological and abiotic stress responses is played by the TFs NAC, which is unique to plants [46]. Seventeen members of the NAC TFs family, thirteen of these genes were up-regulated and four were down-regulated. The WRKY take part in the response to injury, aging, development, and disease [47]. Additionally, WRKY controls how plants grow and develop as well as how they react to stress. Fourteen of the fifteen members of the WRKY-TFs family were up-regulated, while one gene was down-regulated.

DEGs identified at photosynthesis

Based on their functions, the genes involved in photosynthesis were selected for this study and classified into two groups (Fig. 7). Most of the photosynthetic genes belong to the first class, which is involved in the light reaction, such as photosystem I and II reaction center genes PsbP, PsbQ, PsbS, PsbW, PsbY, Psb27, PsaD, PsaE, PsaF, PsaG, PsaL, PsaO and Cytochrome b/6 complex PetC were expressed lower levels in SNP treatment (Additional file 4: Table S2). The second category consists of antenna proteins, which are light-harvesting chlorophyll a/b binding (LHC) proteins. Most of the genes (Lchas, Lchbs), which are light-harvesting protein genes, showed similar expression patterns to those light reaction genes (Additional file 4: Table S2).

DEGs identified at starch metabolism

Transcriptome data indicate that carbon fixation pathways are significantly affected by 0.025 mM SNP treatment. Following SNP treatment, the expression of a few genes related to the Calvin cycle was reduced. The SNP down-regulated genes include those encoding sedoheptulose-bisphosphatase (SEBP), fructose-bisphosphate aldolase (ALDO), fructose-1,6-bisphosphatase (FBP), ribose 5-phosphate isomerase A (RPI), ribulose-bisphosphatecarboxylase (RBCS), glyceraldehyde-3-phosphate dehydrogenase (NADP⁺) (GAPA), phosphoribulokinase (RPK). Starch metabolism is important for the supply of energy during S. Polyrrhiza development. Twenty-three DEGs were enriched in starch and sucrose metabolism pathways (Additional file 5: Table S3). A number of important enzymes are involved in starch biosynthesis such as ADP-glucose pyrophosphorylase (AGP). Transcripts encoding AGPase were significantly up-regulated under SNP (Fig. 8). Results showed that the expression of transcript-encoding AGPase was up-regulated from 35 to 90 FPKM under SNP. The degradation of starch is catalyzed by α-amylase and β-amylase [48], which responsible to the degradation of starch to smaller hydrocarbons, β-amylase down-regulated under SNP, while α-amylase was no significant differences. Transcript-encoding β-amylase exhibited an expression level of 69.8 FPKM under the untreated samples and decreased to 28.5 FPKM under SNP.

DEGs identified at ROS

ROS are chemically reactive molecules that contain oxygen, carbohydrates, lipids, proteins and DNA are all damaged by the over-production of ROS [49,50]. The data from our transcriptome analysis revealed that transcript abundance of genes coding for different components of the ROS scavenging machinery does differ dramatically between control and SNP treatement (Fig. 9). A total of 34 putative genes were found for encoded proteins with antioxidant properties (Additional file 6: Table S4). There were 24 up-regulated unigenes that encoded proteins such as glutathione s-transferases (GST), glutathione peroxidase (GPX), APX, POD, lipoxygenase (LOX) , monodehydroascorbate reductase (MDAR), and alternative oxidase (AOX). There were 14 down-regulated unigenes that encoded proteins such as SOD, POD and GST. However, there were no appreciable variations in the expression of CAT-encoding transcripts during SNP treatment compared to the untreated samples.

DEGs identified at flavonoid biosynthesis

The phenylpropanoid biosynthesis route in plants contributes to a number of different biosynthetic branches, such as flavonoid biosynthesis and anthocyanin biosynthesis (Fig. 10). Analysis of the S. Polyrrhiza flavonoid synthesis pathway identified 13 differentially expressed structural genes (Additional file 7 : Table S5 ). Phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-hydroxycinnamoyl-CoA ligase (4CL) are the universal factors involved in flavonoid biosynthesis [51]. The expression of transcript-encoding PAL, C4H and 4Cl was up-regulated by 3.5, 1.8 and 2.8-fold, respectively, under SNP. Chalcone synthase (CHS), chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H) the enzymes that catalyze the first three reactions of flavonoid biosynthesis branch. The expression of transcript-encoding CHS, CHI and F3H was up-regulated by 1.5, 1.4 and 1.8-fold, respectively, under SNP. Meanwhile, the expression of Flavonoid-3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and anthocyanidin 3-O-glucosyltransferase (UFGT) was up-regulated by 1.1, 3.2, 2.6 and 2.8-fold, respectively, under SNP.

Quantitative real-time PCR (qRT-PCR) validation

To further confirm the veracity and dependability of the DEGs data generated by RNA-Seq, 17 DEGs associated with exogenous NO response were picked out for qRT-PCR verification, including genes related to flavonoid biosynthesis pathway, NO biosynthesis pathway, photosynthesis and transcription factor. The qRT-PCR results were shown in Fig. 11. The results showed a strong association between the transcriptome data and the expression levels of the chosen genes.

It has been reported that SNP stress triggers the NO biosynthesis pathway, resulting in a high endogenous NO level [21, 52]. The previous studies were in accordance with our result. So far, the main NO synthesis pathways identified are the NOS (nitric oxide synthase, NOS) and NR (nitrate reductase, NR) pathways [53]. The NOS pathway is mainly dependent on NO synthase (NOS), which generates NO and L-citrulline by oxidizing L-arginine [54], For the NR pathway, nitrate reductase (NR) can reduce NO₃⁻ to NO₂⁻, and then nitrite reductase (NiR) can reduce NO₂⁻ to NO, and NR can also oxidize NO to nitrate to scavenge NO, which shows that NR is an important regulator of NO homeostasis in plant [53]. In transcriptome analysis of the key enzyme for NO synthesis, the expression level of NRT and NR gene exhibited increased expression under SNP stress (Additional file 8: Table S6). There is evidence that NO also increases cytosolic free Ca²⁺ through Ca²⁺ channels, transporter proteins, cGMP and cADPR up-regulation [55, 56]. In transcriptome analysis of calmodulin expression, the genes encoding calmodulin, calcium-binding protein and cyclic nucleotide gated channel were up-regulated, indicating that Ca²⁺ was regulated in S. polyrhiza under SNP stress. It has been noted that every signaling pathway engages protein kinases, which can activate a variety of downstream TFs family proteins [57]. In this study, several TFs responded significantly to SNP stress, including the ERF (22), bHLH (20), NAC (17), WRKY (15). But more research is needed to determine whether these TFs work independently or in concert. A series of downstream NO-responsive genes are expressed as a result of TF activation, and their transcription further controls the equilibrium of cellular metabolism.

The most significant photosynthetic pigment in plants' chloroplasts is chlorophyll (Chl), which is crucial for absorbing light and transmitting light energy in the antenna systems. [58]. The findings of this study showed that exogenous SNP caused the chlorophyll content of S. polyrhiza to decrease, and similar findings were seen in other investigations, such as torreya grandis [59], chard [22] and syobean seedlings [60]. This may be due to the damage of photosynthetic pigments caused by SNP stress or the degradation of chlorophyll induced by NO [61]. In addition, there are also reported indicating that chlorophyll content increases under SNP treatment, such as wheat seedlings [62] and barley seedlings [63]. The measurement of chlorophyll fluorescence characteristics, which is practical, precise, and sensitive, is frequently employed to reflect the photosystem alterations of plants under environmental stress [64]. Generally, plants grown under stress have lower Fv/Fm than non-stressed plants [65], which is consistent with the performance of S. polyrhiza (Fig. 1D ). Compared with CK-treated seedlings, SNP treatment caused an increase in PSII activity in S. Polyrhiza, which is indicated by lower Fv/Fm and higher ABS/RC and DIo/RC. The increase in ABS/RC in duckweed under SNP treatment can be explained by a decrease in the number of active reactive centers (RC) of PSII, which might serve as a defense mechanism to reduce the burden of its systems when stress occurs. These findings are consistent with previous reports on torreya grandis [59]. The antenna proteins are essential for absorbing solar energy and for photoprotection in stressful situations [66]. These proteins assume a conformation that allows them to release the extra energy from excitation as heat when plants are exposed to stressful circumstances that could cause photo-oxidative damage [67]. We found that SNP treatment down-regulated the expression level of LHCA4, LHCA5, LHCA6, LHCB2.1, LHCB5. Similar results were also reported by a previous study in alfalfa seedlings [23]. Therefore, we propose that SNP reduced the photosynthesis by down-regulating the expression levels of LHCA4, LHCA5, LHCA6, LHCB2.1, LHCB5.

Starch is one of the major storage compounds of duckweed and usually accumulates under stressed conditions, which is an adaptive strategy gained during the long evolutionary process to enable the plant to survive the adverse environment [32]. The starch content reached 4.57 mg/g of fresh weight in the treated samples and 1.83 mg/g of fresh weight in the control samples (Fig. 2C). As a result, the total starch that accumulated in the treated samples was 1.5 times higher than that in the control samples. Starch accumulation is closely related to starch synthesis and metabolism. Commonly, Two common tactics are used to increase starch accumulation: increasing the amount of substrate from other metabolic pathways used for starch and sucrose metabolism or decreasing the pace at which starch is degraded [32]. AGPase is essential for controlling the accumulation of starch in plants [68]. In our study, the RNA-seq results showed enhanced expression of AGPase key enzyme genes in starch biosynthesis pathway under SNP stress in S. Polyrrhiza. In agreement with the results of previous studies, under the condition of nutritional starvation, the starch content and AGPase activity of Landoltia punctata significantly increase [69], suggesting that the high accumulation of starch was prompted by enhancement of the starch synthesis pathway in S. polyrhiza under SNP. We believe that starch accumulation is also related to the decreased starch degradation in S. polyrhiza, β-amylases are a group of hydrolases that are involved in starch degradation in plant tissues. The expression of key genes involved in the starch degradation pathway was down-regulated, including β-amylases. Previous studies have shown that β-amylase gene was down regulated in Landoltia punctata, L. Turionifera, kiwifruit and alfalfa [23, 70–72], which is consistent with our results. Calvin cycle is responsible for the biosynthesis of sugars from photosynthetic carbon. In our study, the expression of genes related to the calvin cycle was down-regulated affected by SNP treatment. However, the expression of genes related to the calvin cycle was up-regulated after MH treatment, the starch accumulation showed a similar trend [48]. The above results were inconsistent with our result. We speculated that NO may inhibit the calvin cycle by lowering the expression level of calvin cycle-related genes, thereby reducing carbon flow from photosynthesis to the starch biosynthesis branch. Taken together, we speculate that The increased expression of genes related to starch synthesis and the decreased expression of genes linked to starch metabolism under SNP resulted in the accumulation of starch by S. polyrhiza.

Overproduction of ROS during abiotic stress is typically noted, and this might result in some damage that eventually leads to oxidative stress [61]. In this study, we further explored the effects of SNP on oxidative damage of S. Polyrhiza. As a result of the SNP treatments, MDA levels in S. polyrhiza increased and ROS levels accumulated, indicating oxidative damage. This is because elevated ROS may result in oxidative stress, which causes membrane damage and lipid peroxidation, and MDA has been used as a marker for damage [73]. The ROS scavenging enzymes are crucial components of the plant defense mechanism in response to ROS production. Antioxidant enzymes and antioxidants synthesized in plants can remove excess ROS in response to adversity. When it comes to enzymatic antioxidants, the enzyme SOD serves as the first line of defense by converting O²⁻ into H₂O₂, which is then metabolized by the enzymes APX, POD, and CAT [74]. In our study, the SOD activity of S. Polyrhiza increased but not the gene expression of the two types of SOD. Similar situations have occurred in Lemna minor under NH₄ ⁺ toxicity [75]. This may indicate a lag from gene transcription to enzyme action under SNP stress treatment. Research demonstrates that SNP treatment increases SOD activity in chard, blueberry fruit and okra [18, 21, 23]. In our study, activity of POD and CAT increased in S. Polyrhiza treated with SNP when compared to the control plants, and therefore, increased expression of the POD genes is very understandable. Study of S. polyrhiza has indicated that through enhancing antioxidative enzyme activity such as POD and CAT to resist salt stress [76, 77]. In addition, the expression of genes related to antioxidant-related such as GPX, GST, MDAR, APX, LOX, and AOX was up-regulated affected by SNP treatment. Research has shown that the expression of genes like GPX, GST and APX was up-regulated in the NO-treated ganoderma lucidum mycelia and birch cells [49, 78]. In fact, many previous studies showed a close connection between NO and genes related to antioxidant enzyme [79].

Apart from alleviating the toxic effects caused by ROS, NO is also related to secondary metabolite synthesis [80, 81]. In our study, the content of flavonoids such as orientin, vitexin, luteolin-7-O-glucoside and proanthocyanidins showed a significant downward trend after rising first under SNP treatment. We speculate that SNP concentration range from 0 mM to 0.025 mM usually promoted the accumulation of cellular biomass and enhanced the production of secondary metabolites, however NO may shown to be toxic to plant growth in SNP concentration ranges from 0.025 mM to 0.5 mM [49, 82–85]. We performed further transcriptome analysis on S. polyrhiza samples treated with 0.025 mM SNP and control samples. After SNP treatment, it was discovered that DEGs had a high enrichment in phenylpropanoid metabolism (Fig. 9 ). Naringenin is one of the crucial substrates for the synthesis of flavonoids. Moreover, DEGs that catalyze the key enzymes in naringenin synthesis, such as PAL, C4H, 4Cl, CHS, and CHI had high expression levels under a 0.025 mM SNP. Therefore, we speculate that these genes were up-regulated, which may then lead to an increase in naringenin biosynthesis. Naringenin can be catalyzed by F3′H to produce flavonoids such as orientin, vitexin, and luteolin-7-O-glucoside. Meanwhile, the expression of F3′H was higher than 70 FPKM. This possibly explains why S. polyrhiza under 0.025 mM SNP has accumulated flavonoids such as orientin, vitexin, and luteolin-7-O-glucoside. In addition, naringenin can be catalyzed by F3H, DFR and ANS to produce proanthocyanidins. Simultaneous, the expression of genes like F3H, DFR and ANS was also up-regulated. This may be the main reason why S. polyrhiza treated with 0.025 mM SNP would accumulate proanthocyanidins. Taken together, we speculate that the flavonoids were accumulated through the high expression of flavonoids related genes, which was consistent with the Landoltia punctata under nutrient starvation [86]. Furthermore, it was discovered that 0.01, 0.025 mM SNP accumulated purple coloration on the underside of the frond, whereas the control group showed no discernible changes (Additional file1 : Figure S1). It has been reported that the reddish-purple tint on the underside of its fronds is a result of anthocyanin production [86]. In our study, the genes encoding anthocyanins synthesis such as F3H, DFR, ANS and UFGT were up-regulated. We speculate that SNP treatment may have contributed to the anthocyanin accumulation in S. polyrhiza, which was consistent with the Landoltia punctata under nutrient starvation [86]. Finally, we summarized a possible molecular mechanism of NO-responsiveness in S. polyrhiza (Fig. 12)

0.025mM SNP can effectively promote vegetative growth, the results show S. polyrrhiza growth attributes (fresh weight, dry weigh) and antioxidant enzymes activity (SOD, POD, CAT) increased and at the same time induce starch and flavonoid accumulation. Insights into the potential molecular pathways behind the starch and flavonoid accumulation in S. Polyrrhiza during SNP were gained from the transcriptome data. These results suggest that after treatment of S. Polyrrhiza with 0.025mM SNP, the metabolic flux may be partly directed to the starch biosynthesis branches and partly into the flavonoid biosynthesis branches. The regulated genes by SNP treatment would provide good candidates for improving the flavonoid content by synthetic biology in S. Polyrrhiza.

Plant materials and treatments

Spirodela polyrrhiza（L.）Schleid were grown aseptically on Datko medium as described by Wang and Kandeler [87] under long-day conditions (16-h light and 8-h dark) with a light intensity of ~ 45 µmolm⁻²s⁻¹ at the plant level. The temperature of the culture room was kept at 22 ± 2 °C. S. polyrrhiza plants were transferred to fresh medium every 15 days, to minimize the effect of nutrient shortage.

S. polyrrhiza will be pre-cultured for 8 days with consistent growth were divided into eight groups as follows: (1) control (CK), without any treatment; (2) 0.01 mM SNP treatment; (3) 0.025 mM SNP treatment; (4) 0.05 mM SNP treatment; (5) 0.1 mM SNP treatment; (6) 0.3 mM SNP treatment; (7) 0.5 mM SNP treatment; (8) 1.0 mM SNP treatment. SNP was dissolved in distilled water, filter-sterilize and add to datko medium. The SNP treatment occurred for three successive days, leaf samples were collected, frozen in liquid nitrogen and stored at −80 ℃.

Photosynthetic pigments and chlorophyll fluorescence

Extraction of leaf pigments was done in absolute ethanol under dim light according to Arnon [88] . A 0.05 g sample was macerated in 3 mL of absolute ethanol and place the dark for 24 h at 28 ℃, and centrifuged at 5000×g for 5 min. The supernatant was collected and absorbance of chlorophyll a, b and carotenoids recorded at 663, 645, and 470 nm, respectively. The PSII parameters were analysed using a pulse-amplitude modulation chlorophyll fluorometer (Handy-PEA, Hansatech，England ). Measurements were done in the centre of the well-developed and different concentrations of SNP treatment leaves, with three measurements per replicate. Each leaf were measurements after dark-adapted for 30 min.

Assays of physiological parameters

Fresh and dry weight were measured with a digital balance; The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were determined using enzyme activity kits (Beijing SolarBio Science & Technology Co., Ltd., Beijing, China). The content of starch, soluble protein, malondialdehyde (MDA) and H₂O₂were determined using content detection kit (Beijing SolarBio Science & Technology Co., Ltd., Beijing, China); Proanthocyanidin content was determined by Luthar and Kreft method [89] 0.05 g sample was macerated in 5 ml 60% ethanol and ultrasonication at 30 ℃ for 30 min, centrifuged at 5000×g for 10 min at 25 ℃. An aliquot of 1 ml supernatant was mixed with 2 mL 4% vanillin solution and 1 ml concentrated hydrochloric acid, 20 min reaction at room temperature and absorbance measured at 530 nm. NO content was measured with Plant NO ELISA Kit (Catalog number: A013-2-1, purchased from Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd).

Determination of orientin, vitexin and luteolin

The dry materials were ground into fine powder. Approximately 50 mg of powder was extracted in 5 mL of methanol by ultrasonication at 50 ℃ for 50 min, followed by centrifugation. Samples were separated using ultra high-performance liquid chromatography (UHPLC) with a ACQUITYUPLCBEH-C18 (2.1 mm × 50 mm, 1.7 μm, Waters). In the solvent system, mobile phase A was 0.1% (v: v) formic acid and mobile phase B was methyl-alcohol. The gradient conditions were applied as follows: 0 ~ 7 min, 25% ~ 40% B; 7 ~ 10 min, 40% ~ 50% B; 10~11 min, 50% ~ 40% B; 11 ~ 12 min, 40% ~ 25% B; 12 ~ 14 min, 25% B. The flow rate was set at 0.3 mL/min, and the injection volume was 1μL.

RNA-seq and bioinformatics analysis

Total RNA samples were extracted. The libraries were prepared using mRNA was isolated from total RNA using Dynabeads oligo (dT). Then, the enriched mRNA fragments were randomly interrupted into short fragments of 200 nt-700 nt by adding fragmentation buffer, and the obtained short fragment mRNA was used as the template. First-strand cDNA was generated using random hexamer primers. The second-strand cDNA was generated using buffer, dNTPs, RNaseH, and DNA polymerase I (Invitrogen). The cDNA fragments were then purified with QiaQuick PCR extraction kit, repairing the ends, adding poly (A), and ligated to Illumina sequencing adapters. Finally, the ligation products were size-selected using agarose gel electrophoresis, PCR amplified, and RNA-seq applied using an Illumina platform from Gene Denovo Biotechnology (Guangzhou, China).

Raw reads obtained from the Illumina sequencing were further filtered to obtain high quality clean reads by removing reads containing adapters, reads containing N ratio greater than 10%, reads all containing a base, and low-quality reads. De novo assembly of clean reads was performed using Trinity [90]. The expression level of each transcript was calculated and normalized to reads per kb per million reads (RPKM). In this study, the level of differential expression for each transcript, with a criterion of |log2(fold-change)| >1 and FDR < 0.05 at different growth stages, was employed to identify DEGs.

Unigenes were annotated against the databases NCBI non-redundant protein (NR), Swiss-Prot protein, Kyoto Encyclopedia of Genes and Genomes (KEGG), euKaryotic orthologous groups of proteins (KOG) and gene ontology (GO) using a BLASTx program with an e-value ≤ 10^-5_.

Quantification of gene expression levels

Total RNA was isolated from samples at the CK and 0.025 mM SNP treatment duckweed using a Eastep super total RNA Extraction Kit (Promega, shanghai) according to the manufacturer’s protocol. The integrity of total RNA was examined using 1.0% agarosegel electrophoresis. Primer sequences of selected DEGs (Additional file 9. Table S7) were designed using Primer premier 5 and synthesized by Sales. Genomics.CN (Tianjin, China). First-strand cDNA was synthesized using a FastKing RT Kit containing with gDNase (TIANGEN Biotech, China) with one cycle at 42 ℃ for 15 min and then 95 ℃ for 3 min. QRT-PCR gene expression was carried out using a SuperRealPreMix Plus (SYBR Green) (TIANGEN Biotech, China) with one cycle at 95 ℃ for 15 min, followed by 40 cycles at 95 ℃ for 10 s and 60 ℃ for 30 s. Actin was used as a reference control gene. The 2^−ΔΔCtmethod [91] was used to calculate relative gene expression.

Statistical analysis

All measurements were performed using three biological replicates. Statistical analysis was performed with Graph-Pad Prism 8.0 software, and significance analysis was conducted with one-way ANOVA multiple comparison (P < 0.05 or P < 0.01).

Ethics approval and consent to participate

Our research did not involve any human or animal subjects, material, or data. We declare that the plant material in the experiment was collected and studied in accordance with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its Supplementary information files. The data used to support the findings of this study are available from the corresponding author on reasonable request.

Competing interests

All authors have declared that they have no conflicts of interest.

Funding

This work was financially supported by National Natural Science Foundation of

China (82074120) for the design of the study and the collection, analyses, and interpretation of data.

Authors' contributions

BBX contributed to the experimental design and manuscript review. YH organized

the experiment process. YMZ modified the picture and wrote the paper. RJ, TYH, YW and WJW participated in the experiments. YW, YRZ and LY also reviewed this manuscript All authors have read and approved the final manuscript.

Acknowledgements

We are grateful to Guangzhou Genedenovo Biotechnology Co., Ltd for assisting in sequencing.

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Table 1 Characteristics of de novo assembled transcriptome in S. Polyrrhiza

Genes Num	GC percentage	N50 number	N50 length	Max length	Min length	Average length	Total assembled bases
33684	48.9927	5252	2791	18736	201	1360	45841369

Table 2. Database searches for collected S. Polyrrhiza is nucleotide sequences

BLASTx search against specific platforms

Values

SwissProt

KOG

KEGG

15127

11816

9341

14966

No competing interests reported.

Supplementarymaterial.zip
Additional file 1: Figure S1. Color change of frond underside under SNP treatment Additional file 2: Figure S2. Species distribution of Nr annotation Additional file 3: Table S1. The DEGs were identified as transcription factor Additional file 4: Table S2. The DEGs were identified as photosynthesis Additional file 5: Table S3. The DEGs were identified as starch metabolism Additional file 6: Table S4. The DEGs were identified as antioxidant Additional file 7: Table S5. The DEGs were identified as flavonoids biosynthesis pathways Additional file 8: Table S6. DEGs involved in NO biosynthetic and signal transduction Additional file 9: Table S7. Primers used for qRT-PCR analysis

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Transcriptomic and physiological analysis of Spirodela polyrrhiza responses to sodium nitroprusside

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