Mangrove wetlands are influenced by chronic periodic tidal inundation, which is responsible for the typical characteristics of marsh habitats, such as abundant organic matter and nutrients and a reducing environment (Chen et al. 2010). Such wetlands serve as appropriate habitats for multiple microbes that contribute to the production of NO. We have previously investigated daily variations in NO emission flux in K. obovata wetlands (Chen et al. 2010). Based on this previous work, we designed the current study to unravel the effects of NO on root development and growth of a mangrove plant, K. obovata, using SNP as an exogenous NO donor. We found that NO promoted lateral root growth in a dose-dependent manner (Fig. 1). Under normal physiological conditions, NO is an important signaling molecule for a diverse range of organisms’ physiological processes. However, excess NO produced in the organisms has obvious cytotoxicity. Now, various studies have been widely described its dual roles in the plant kingdom, which might depend on the cellular conditions and NO contents (Beligni and Lamattina 1999, Böhm et al. 2010). In soybean, high amounts of NO functioned as a stress factor resulting the decreasing of root growth that is corelated with cell death. Whereas lower concentration of NO, due to its action as a signaling molecule, could induce soybean root growth and lignification (Böhm et al. 2010). Studies also showed that the high concentration of NO accumulated in roots could effectively inhibit the root elongation and growth of tomato (Correa-Aragunde et al. 2004) and wheat (Groppa et al. 2008). In accordance with these results, some differences in the lateral root development and growth were found depending on the SNP concentration, the low concentrations (0.05–1 mM) stimulated the growth very effectively with the optimum concentration of 0.2 mM, while reverse influence was observed at higher SNP concentrations. However, few genomic resources were available for this genus. A de novo transcriptomic approach was therefore helpful for further investigating the changes in global gene expression that underlie modulation of K. obovata lateral root development by NO.
Changes in global gene expression induced by NO in K. obovata
Transcriptome sequencing was performed on the NovaSeq Illumina platform. After quality filtering, we assembled 122,736 unigenes from NO and CK libraries, and 105,999 of the unigenes showed significant similarity to known proteins in the NCBI Nr database (Table 2). The remaining 13.64% unannotated unigenes may be specific to K. obovata or may be homologous to genes with unknown biological functions in other species. Cellular process, metabolic process, single-organism process, cell part, binding and catalytic activity were the primary GO terms assigned to the unigenes, and these may be considered putative biological processes that respond to NO (Supplementary Fig. S5). This result is consistent with previously published transcriptomic data in which binding and catalytic activities predominated at the transcriptome level in response to NO in Arabidopsis thaliana (Hussain et al. 2016) and Gossypium hirsutum (Huang et al. 2018). KEGG pathway classification identified specific pathways involved in lateral root growth, such as transport and catabolism, signal transduction, translation, carbohydrate metabolism, and environmental adaptation (Supplementary Fig. S6).
Further functional enrichment analysis indicated that starch and sucrose metabolism was significantly up-regulated in response to NO (Figs. 5 and 6). Moreover, DEGs associated with plant hormone signal transduction and cell wall metabolism were also differentially regulated by NO (Fig. 7). Similarly, Li et al. (2016) suggested that sugar metabolism and the auxin and cytokinin signaling pathway primarily contributed to root development in grafted apple. GO enrichment analysis of birch cells treated with SNP also indicated that NO had a significant impact on carbohydrate metabolism and cell wall biosynthesis (Zeng et al. 2014).
The highly represented pathways and significantly enriched GO terms in our study suggested that NO positively enhances lateral root development and growth of K. obovata through the modulation of starch and sucrose metabolism, plant hormone signaling, and cell wall metabolism.
NO modulates genes associated with starch and sucrose metabolism to promote lateral root development and growth of K. obovata
Both internal and external factors impact the root growth of higher plants, and sugar is a particularly important internal factor (Ruan 2014). The mangrove plant K. obovata has a specific reproductive strategy known as vivipary (Chen et al. 1995). It has been demonstrated that hypocotyls of viviparous seedlings contain a considerable amount of starch that is used as an energy source during the early phase of development (Hanashiro et al. 2004). In this study, a key starch metabolic gene encoding β-amylase (BMY) was induced in response to exogenous NO, suggesting that NO may accelerate the process of starch hydrolysis, providing the necessary energy for lateral root development and growth.
Sucrose is the major form of carbohydrate translocated from photosynthetic sources to non-photosynthetic sinks such as the root system. When it reaches a sink, sucrose must be broken down into hexoses or their derivatives for use in multiple metabolic and biosynthetic processes (Ruan 2014). Invertase (INV) and sucrose synthase (SUS) are two major enzymes that are responsible for the cleavage of sucrose in higher plants (Ruan 2014). In this study, five of six SUS genes were up-regulated, whereas three INV genes encoding a typical cell wall invertase (CWIN) were down-regulated in K. obovata roots after exogenous NO treatment (Fig. 7). Claeyssen and Rivoal (2007) demonstrated that CWIN activity coupled with a high hexose/sucrose ratio was commonly associated with high cell division rates during root initiation and expansion. Thereafter, a switch to the later maturation stage was accompanied by a shift from the INV to the SUS pathway of sucrose degradation (Claeyssen and Rivoal 2007, Weber et al. 1997). Increased SUS activity was associated with a transition from cell division to cell differentiation and elongation (Claeyssen and Rivoal 2007, Weber et al. 1997). We therefore inferred that NO may have a role in the promotion of lateral root elongation through up-regulation of SUS expression. This result is consistent with the changes in root morphology observed under exogenous NO addition (Fig. 1).
After the breakdown of sucrose into Glc and Frc, Frc can be irreversibly transformed into fructose 6-phosphate (F6P) by hexokinase (HK) (Galina and da Silva 2000). F6P may enter glycolysis and the tricarboxylic acid (TCA) cycle to produce energy, or it may be converted into UDP-Glc as a glucosyl donor for the synthesis of cell wall β-glucan (Galina and da Silva 2000). In this study, HK expression was markedly increased under exogenous NO addition, suggesting that NO may play a pivotal part in enhancing K. obovata lateral root development and growth by providing a consistent source of energy and cell wall polysaccharides.
Trehalose, a source of energy and carbon, can protect bioactive substances and cellular membranes from inactivation or denaturation under adverse stress conditions (Elbein et al. 2003). Trehalose biosynthesis in plants is a two-step pathway catalyzed by trehalose-6-phosphate (Tre6P) synthase (TPS) and Tre6P phosphatase (TPP) via Tre6P (Elbein et al. 2003). In this study, three genes associated with trehalose biosynthesis were up-regulated (two TPS and one TPP) in response to exogenous NO, whereas a gene encoding trehalase, which breaks down trehalose to form two Glc, was down-regulated. Trehalose metabolism has a fundamental and pervasive role in the life of plants (van Dijken et al. 2004). Overexpression of a Tre6P synthase/phosphatase fusion gene in rice elevated trehalose accumulation and conferred high tolerance to salt, drought, and low-temperature stresses (Garg et al. 2002). In addition, AtTPS1 is central to normal vegetative growth and the switch to flowering in Arabidopsis (van Dijken et al. 2004). In the present study, expression of TPS and TPP was dramatically induced by NO, and that of treA was depressed, implying that trehalose biosynthesis is enhanced by exogenous NO addition and may contribute to K. obovata lateral root development and growth. Taken together, our results suggest that starch and sucrose metabolism is accelerated by NO, thereby supplying more energy to promote lateral root development of K. obovata.
NO modulates genes associated with plant hormone signal transduction to promote lateral root development and growth of K. obovata
Lateral root development and growth—including cell division, differentiation, expansion, and patterning—are tightly modulated by phytohormones (Bao et al. 2004, Ren and Gray 2015). One of the functions of auxin in plants is to regulate transcription by promoting the ubiquitination of auxin/indole-3-acetic acid (Aux/IAA) proteins through the activity of the SKP1-Cullin-F-box (SCF) complex and auxin transport inhibitor response1 (TIR1) or its paralog, auxin receptor F-box protein (ABF) (Deng et al. 2012). The auxin response factors (ARFs) released from the degradation of Aux/IAAs serve as transcriptional repressors or activators of specific genes that contain the auxin-responsive element (AuxRE) promoter element (de Jong et al. 2015, Waller et al. 2002).
In this study, five auxin response genes (IAA16, IAA27, AUX22, SAUR36, and GH3) and four auxin response factor genes including one ARF1 and three ARF9 were differentially expressed in the roots of K. obovata in response to exogenous NO (Fig. 7). The primary auxin response genes can be classified into three major groups: Aux/IAAs, SAURs (SMALL AUXIN UP RNA) and GH3s (Gretchen Hagen3). Aux/IAA proteins are transcriptional repressors, and SlIAA15 down-regulated tomato lines showed increased lateral root formation (Deng et al. 2012). GH3 family proteins promote the conversion of active IAA to its inactive form (Yang et al. 2015). Here, four primary auxin response genes (IAA27, AUX22, SAUR36, and GH3) were differentially expressed in response to exogenous NO, suggesting that the establishment of an appropriate auxin response system is important for NO’s mediation of lateral root development and growth in K. obovata.
BRs are linked to lateral root development and interact with auxin to increase lateral root formation in Arabidopsis (Bao et al. 2004). It was well-known that the BR signal is perceived by the plasma-membrane-localized receptor kinase BRI1 and proceeds through the co-receptor BAK1 and additional downstream positive and negative regulators to mediate the expression of BZR1 and its homolog, BES1, which directly control BR-responsive gene expression (Chung and Choe 2013, Ye et al. 2011). Our results indicated that NO takes role in the BR signaling pathway to modulate root development and growth in K. obovata. The BR receptor kinase BRI1 and the positive regulator BSK were significantly up-regulated, whereas the negative regulator BIN2 and the transcription factors BZI1 and BES1 were significantly down-regulated. Combined with previous studies (Chung and Choe 2013, Ye et al. 2011), our results suggest that NO may promote lateral root development and growth by enhancing the BR perception and signaling pathway.
The action of ethylene in lateral root formation has previously been characterized using mutants in Arabidopsis(Alonso and Stepanova 2004, Negi et al. 2008). In the present study, one gene encoding ACC synthase (ACS) and two genes encoding ACC oxidase (ACO) were significantly down-regulated in response to exogenous NO (Fig. 7). These genes are involved in the two committed steps of the ethylene biosynthesis pathway. Initially, S-adenosyl-L-methionine (SAM) is transformed by ACS into ACC, in what is generally considered the rate-limiting step. Ethylene is then released from ACC by ACO (Park et al. 2018). Previous work has shown that the aco1-1 Arabidopsis mutant has reduced ethylene production in root tips and enhanced lateral root development compared to the wild type (Park et al. 2018). Our results suggest that NO influences ethylene synthesis, potentially affecting lateral root development through the suppression of ACS and ACO. A gene encoding ethylene insensitive 2 (EIN2) was also significantly down-regulated in response to exogenous NO. EIN2 is an important signal transducer in the ethylene signaling pathway, and its functional deficiency in Arabidopsis gives rise to conspicuous ethylene insensitivity and a failure to display known ethylene responses (Miyata et al. 2013, Roman et al. 1995). LjEIN2-1 and LjEIN2-2 from Lotus japonicus together control ethylene signaling to suppress root growth and nodule formation (Miyata et al. 2013). Based on our data and previous work, we concluded that NO may decrease the expression of ACS, ACO and EIN2, therebyreducing ethylene biosynthesis and signaling and enhancing lateral root development and growth.
Accumulated evidence suggests that abscisic acid (ABA), a universal stress hormone, takes part in the regulation of lateral root development (De Smet et al. 2003, Xing et al. 2016). Two genes encoding abscisic aldehyde oxidase (AAO) and abscisic acid insensitive 5 (ABI5) were identified in this study (Fig. 7), both are associated with ABA synthesis and signaling. AAO functions in the final step of ABA biosynthesis by oxidizing ABA aldehyde to ABA (Seo et al. 2000). ABI5, a basic leucine zipper transcription factor, acts as a molecular hub in the NO-mediated balance between early development and stress (Albertos et al. 2015). NO facilitates seed germination in Arabidopsis through a mechanism linked to ABI5 degradation (Albertos et al. 2015). Taken together, these results suggest that decreased ABA levels and expression of ABI5 mediated by NO may contribute to K. obovata lateral root development and growth.
NO modulates expression of genes associated with cell wall metabolism to promote K. obovata lateral root development and growth
Plant cell growth is restricted by the cell walls, dynamic and complex structures that consist of polysaccharides (mainly cellulose, hemicellulose, and pectin), highly glycosylated proteins, and lignin (Somerville et al. 2004). In the present study, exogenous NO altered the expression of 42 DEGs associated with cell wall biosynthesis and modification (Fig. 7).
As the major component of plant cell walls, cellulose is necessary for plant morphogenesis. Cellulose synthase (CESA) was first identified in cotton fibers (Pear et al. 1996), and its central role in the biosynthesis of crystalline cellulose was confirmed using the rsw1 mutant of Arabidopsis (Arioli et al. 1998). Decreased CESA in primary walls is correlated with inhibited cell elongation and was revealed in some of these mutants to be associated with elevated levels of ethylene and jasmonate (Ellis et al. 2002). Genetic studies have identified a number of genes that contribute to the overall process of cellulose biosynthesis, including genes that encode endoglucanase 25 (KOR) and COBAR-like protein (COBL). KOR participates in cell wall assembly during the processes of cell plate maturation and cell elongation in cytokinesis, and it is necessary for the formation of cellulose microfibrils and the secondary cell wall (SCW) in the developing xylem (Zuo et al. 2000). COBL, encoding a glycosyl-phosphatidyl inositol-anchored protein, is primarily responsible for SCW biosynthesis. It affects cellulose crystallinity status and the orientation of cell expansion (Niu et al. 2015). A previous study revealed that GhCOBL9A and GhCOBL13 are predominantly expressed during SCW biosynthesis in fiber development and are co-expressed with GhCESA4, GhCESA7, and GhCESA8 in Gossypium hirsutum (Niu et al. 2015). In the present study, three CESA, two COBL, and one KOR gene were up-regulated in response to exogenous NO (Fig. 7), suggesting that NO may induce the expression of thesegenes to promote cell elongation and cellulose deposition during lateral root development and growth.
Xyloglucan is a polysaccharide that makes up about 20–25% (dry weight) of the primary cell wall in dicotyledons (Fry 1989). Xyloglucan endotransglucosylase/hydrolases (XTHs), key enzymes in xyloglucan metabolism, promote cell expansion by catalyzing the cleavage of xyloglucan molecules and assembling new raw materials into the cell wall matrix (Pan et al. 2017). Two of three XTHs were significantly up-regulated in response to exogenous NO and may have promoted cell wall extension during lateral root development and growth. Other DEGs were also involved in hemicellulose metabolism, such as genes encoding cellulose synthase-like protein(CSL) and UDP-glucuronate:xylan alpha-glucuronosyltransferase 2 (GUX2) (Chou et al. 2015, Mortimer et al. 2010). AtCSL superfamily genes can be classified into six subfamilies and are thought to encode the catalytic subunits of enzymes that synthesize hemicellulose backbones (Chou et al. 2015, Richmond and Somerville 2000). Three genes encoding CSLs were differentially expressed in response to exogenous NO, indicating that NO may influence the biosynthesis of hemicellulose backbones.
Xylan, the principal hemicellulose in many plant secondary cell walls, has a backbone of β-(1,4)-linked xylosyl residues that is variably substituted with side chains, including methylglucuronic acid (MeGlcA) and glucuronic acid (GlcA) (Lee et al. 2012). GUX2, a xylan glucuronosyltransferase, is required in order to substitute the xylan backbone with MeGlcA (Mortimer et al. 2010). Deficiency of GlcA and MeGlcA side chains in the gux1/2/3 triple mutant led to reduced secondary cell wall thickening and decreased plant growth in Arabidopsis(Lee et al. 2012). In the present study, GUX2 was up-regulated in response to exogenous NO (Fig. 7), suggesting that NO may influence secondary cell wall deposition in K. obovata roots.
Pectin represents up to one-third of the cell wall dry mass and is crucial for the control of cell elongation (Hocq et al. 2017). Several DEGs were associated with pectin metabolism, such as a galacturonosyltransferase 8 gene (GAUT8), pectin methylesterase genes (PMEs), and pectin methylesterase inhibitor genes (PMEIs). GAUTs are pectin biosynthesis enzymes, and GAUT4-silenced tomato fruits have reduced starch accumulation and lower pectin levels, which contribute to greater fruit firmness (de Godoy et al. 2013). In this study, GAUT8 was up-regulated in response to exogenous NO (Fig. 7), suggesting that NO treatment may alter pectin content and solubility in K. obovata. PME modifies cell walls by demethylesterification of the homogalacturonan (HG) backbone. PMEI, a specific proteinaceous inhibitor, is in charge of fine regulation of PME activity in vivo (Roeckel et al. 2008, Wolf et al. 2003). For example, Roeckel et al. (2008) reported that the interactions between PMEs and PMEIs are of vital importance to the cell wall stability of the tobacco pollen tube tip. In addition, inhibition of PME activity by two Arabidopsis PMEIs is a crucial means of controlling pectin esterification (Wolf et al. 2003). In our study, PME and PMEI were differentially expressed in response to exogenous NO (Fig. 7), suggesting that NO may affect the interaction between PME and PMEI, thereby promoting cell wall expansibility and leading to increased lateral root development and growth.
Additional DEGs were also involved in cell wall metabolism. These included genes encoding L-ascorbate oxidase (AO), leucine-rich repeat extensin-like protein 4 (LRX4), glucuronokinase 1 (GlcAK1), UDP-glucuronic acid decarboxylase 2 (UXS2) and UDP-arabinose 4-epimerase (MUR4). AO plays a significant role in redox maintenance and oxidative bursts in apoplasts, thereby controlling cell division and expansion (Xin et al. 2016). Two of three AO genes were up-regulated in response to exogenous NO (Fig. 7), suggesting that NO may promote AO activity and thereby modulate cell division and expansion in K. obovata. The importance of LRX for cell wall formation has been reported previously (Baumberger et al. 2003, Draeger et al. 2015). For example, lrx1/lrx2 mutants showed impaired root hair cell wall structure and growth (Baumberger et al. 2003), and numerous alterations in the cell wall structure of lrx3, lrx4, and lrx5 mutants confirmed the important role of LRX proteins in cell wall development (Draeger et al. 2015). In this study, LRX4 expression increased in response to exogenous NO, suggesting that NO may mediate cell wall formation and development through its effects on LRX4 expression. A. thaliana GlcAK is a key kinase that catalyzes the formation of UDP-GlcA and promotes the formation of cell wall polymers by supplying the required sugar donors (Pieslinger et al. 2010). GlcAK was up-regulated in response to exogenous NO, suggesting that NO positively regulates GlcAK expression, thereby promoting the synthesis of cell wall polymers for lateral root development and growth. UXS has been studied in a wide range of plants because of its vital role in sugar nucleotide interconversion and therefore in plant cell wall biosynthesis (Seifert 2004). The expression of UXS2 increased markedly in response to exogenous NO, providing further evidence that NO influences cell wall biosynthesis in K. obovata. Arabinose (Ara) is an important constituent of various plant cell wall polymers and is essential to plant development and growth (Rautengarten et al. 2011). The MUR4 mutant of Arabidopsis, which has a defective UDP-xylose 4-epimerase, exhibits impaired cell wall growth (Burget and Reiter 1999) associated with reduced synthesis of Ara-containing wall polymers. In the current study, MUR4 expression was sharply elevated in response to NO, suggesting a potential role for NO in cell wall Ara biosynthesis. Taken together, our results demonstrate that NO is likely to play an important role in the metabolism of cellulose, hemicellulose, pectin and other cell wall components, thereby possibly promoting the root development and growth of K. obovata.