Emerging evidence indicates that auxin plays an important role in the regulation of plant elongation in a low R:FR environment. However, the specific mechanisms by which auxin mediates hypocotyl elongation in response to EOD-FR are uncertain. Previous studies found that (1) EOD-FR treatment significantly increased seedling hypocotyl elongation but had no effect on stem diameter, biomass, or seedling index [1, 3], and (2) that the immediate cause of hypocotyl elongation was the expansion of hypocotyl cells . The results of the present experiment confirm these conclusions.
We found that the polar auxin transport (PAT) inhibitor NPA completely abolished EOD-FR-induced elongation and caused significant decreases in stem diameter, dry weight, fresh weight, and hypocotyl IAA content. It is worth mentioning that the level of IAA in NPA-T leaves was significantly higher than that of other treatments. We speculate that EOD-FR treatment increased leaf IAA levels, but this IAA could not be transported to the hypocotyl by PAT and therefore accumulated in the leaves. The increase of free IAA content in hypocotyls after EOD-FR treatment therefore appears to be closely related to PAT activity.
Next, we performed a detailed investigation of the mechanisms by which auxin affects hypocotyl elongation under EOD-FR, using a combination of transcriptome profiling, metabolomics analysis, and physiological measurements.
Auxin is essential for EOD-FR-mediated hypocotyl elongation
As early as 2008, Csukasi et al. had discovered TAA1, a key enzyme in the first step of auxin biosynthesis under low R:FR conditions . Since then, increasing numbers of researchers have performed in-depth studies to demonstrate that auxin plays a vital role in low-R:FR-mediated elongation. However, EOD-FR is a special form of far-red light treatment, and few studies have addressed its relationship to auxin. Here, we have shown that auxin is essential for EOD-FR-mediated hypocotyl elongation. Several lines of evidence support this conclusion.
First, after EOD-FR treatment, the level of free IAA in hypocotyls increased significantly during EOD-FR treatment. However, hypocotyl elongation mediated by EOD-FR was abolished in the presence of the PAT inhibitor NPA. Second, we identified a large number of DEGs related to auxin biosynthesis, transport, and response in EOD-FR-treated plants (Fig. 5). Third, the content of tryptophan, a key auxin synthesis precursor, increased significantly after EOR-FR treatment (Additional file 1). Taken together, these results strongly suggest that auxin plays an essential role in the regulation of hypocotyl elongation in response to EOD-FR.
The pathway of auxin biosynthesis after EOD-FR treatment
There are multiple auxin biosynthetic pathways in plants, and the TAA-YUC pathway (IPyA pathway) appears to be the primary pathway that responds to low R:FR . TAA1 catalyzes the first step in this pathway , and a family of enzymes encoded by YUCCA genes catalyze the second step . YUCCA appears to mediate the rate-limiting step in IAA synthesis [28, 29].
RNA-seq data showed that the expression of one TAA1-related TRYPTOPHAN AMINOTRANSFERASE RELATED 2 (TAR2) gene and two YUCCA genes were upregulated under EOD-FR. Here, we found a significant correlation between YUCC8 expression levels and the abundance of three auxin-related metabolites and previous studies have shown that YUCC8 is closely related to the response of plants to dark environments and that its expression increases significantly in low R:FR environments . Therefore, YUCC8 may be a key gene that specifically responds to EOD-FR. All of these results indicate that the TAA-YUC pathway is the main route for free IAA synthesis in response to EOD-FR treatment.
Increased L-tryptophan content in hypocotyls under EOD-FR also confirms the above conjecture. Tryptophan is an important precursor of auxin synthesis, and its content is closely related to the level of plant auxin. It has previously been shown that IAA levels in rice calli and potato buds with excessive tryptophan synthesis increased 57- and 39-fold, respectively [31, 32].
Moreover, the expression of a gene encoding N-(5'-phosphoribosyl) anthranilate isomerase 1 (trpF) in the tryptophan synthesis pathway and the abundance of the tryptophan precursor indole  also increased significantly after EOD-FR treatment. These may be important mechanisms underlying the increased tryptophan observed in plants under EOD-FR. The trpF appears to be a key gene of tryptophan metabolism induced by EOD-FR.
Auxin transport is enhanced by EOD-FR treatment
Based on changes in plant morphology and auxin levels after NPA treatment, we believe that PAT is necessary for hypocotyl elongation mediated by EOD-FR. PAT is accomplished through the concerted action of PIN-FORMED (PIN) efflux carriers, P-GLYCOPROTEIN/ATP-BINDING CASSETTE B4 (PGP/ABCB) family members, and auxin influx AUXIN1/LIKEAUXIN1 (AUX/LAX) transporters, all are important for increasing IAA transport to specific cell types .
Polarly distributed PINs have been shown to play an important role in plant elongation and growth under low R:FR . Here, we found that the expression of PIN4-related genes was significantly upregulated after EOD-FR treatment, consistent with the findings of Takemura et al in a study of Platycodon grandiflorum . PIN4 may therefore be a crucial efflux carrier protein for EOD-FR-mediated elongation.
In addition, a gene encoding PIN-like (PILS) protein, which is structurally similar to PIN, was also identified in our transcriptome data. PILS localizes to the endoplasmic reticulum and participates in the dynamic balance of auxin in that organelle . We found that the expression of PILS5 was significantly downregulated 7.7-fold in response to EOD-FR. Decreased PILS5 expression seems to suggest a cumulative decrease in IAA in the ER, causing more IAA to flow into the nucleus and influence various biological processes , but further studies are needed to identify specific underlying mechanisms.
Unlike PINs, ABCB proteins are involved in the ATP-dependent influx and efflux of auxin and are located uniformly throughout the plasma membrane . In the sav4 mutant with a basic auxin transport deficiency, auxin efflux mediated by ABCB is blocked, and the plant's ability to respond to low R:FR is significantly impaired , indicating that ABCB is necessary for the low R:FR response. Here, two genes encoding proteins similar to ABC transporter B family member 19 (ABCB19) were significantly upregulated under EOD-FR. It has been reported that ABCB19 can affect the elongation of hypocotyls  and stabilize PIN proteins on the plasma membrane to coordinate the regulation of auxin efflux . In addition to auxin efflux carriers, AUX1-LIKE (LAX) auxin influx carriers were also significantly upregulated after EOD-FR treatment. Studies on AUX/LAX in other plants have shown that hypocotyl length is significantly reduced in the PaLAX1 mutant compared to that of wild type , and there is a close relationship between PIN and LAX . At present, there are few studies of AUX/LAX genes under low R:FR conditions, but it is likely that they play an indispensable role in EOD-FR-mediated hypocotyl elongation. Here, we identified six LAX-like genes that may have a role in the EOD-FR response (Fig. 5). We also identified a gene related to vacuolar auxin transport, WALLS ARE THIN 1 (WAT1), whose encoded protein is involved in maintaining the dynamic balance of intracellular auxin .
In addition to carriers that directly participate in auxin transport, two CBL-interacting serine/threonine protein kinase (CIPK) related genes were significantly up-regulated after EOD-FR treatment. CIPKs can participate in auxin transport by regulating gene expression; for example, the tobacco high-expression CIPK mutant CaCIPK6T1878-90 shows increased basic auxin transport .
Here, we showed that many genes related to auxin transport are significantly upregulated after EOD-FR treatment; this may enhance the transport of free auxin to the hypocotyl, thereby increasing its IAA level. Synergy among various auxin transport carriers is probably required to maintain the auxin concentrations necessary for regulation of plant growth and hypocotyl elongation in response to EOD-FR. At present, the specific functions of these DEGs remain to be studied in pumpkin.
EOD-FR significantly affects the component of auxin signal transduction in hypocotyl cells
The cellular response associated with free auxin was transduced through a specific signal transduction pathway . In the nucleus, auxin regulates the expression of downstream genes by specifically activating auxin response factors (ARFs) through the SCFTIR1/AFB-AUX/IAA pathway , in turn regulating the expression of downstream genes. It has been reported that the AUX/IAA, GH3, and SAUR gene families are the main downstream response genes induced by auxin in the nucleus , and low-R:FR-mediated elongation is closely related to their expression in Arabidopsis [48, 49].
Relevant studies have shown that the stability of AUX/IAA proteins is enhanced in the PhyA mutant, weakening the low-R:FR-mediated elongation reaction . Furthermore, the expression of AUX22 is significantly lower in auxin-insensitive mutants than in wild-type plants under low R:FR conditions . Here, we identified four AUX/IAA genes (Fig. 5) , including two AUX22 genes, suggesting that AUX22 may affect hypocotyl elongation in response to EOD-FR treatment. Two AUX/IAA genes (IAA11 and IAA14) were also significantly up-regulated and may be important candidate genes in the EOD-FR response.
The GH3 gene family is related to the formation of auxin conjugates and regulates plant auxin homeostasis. Hypocotyls of Arabidopsis with GH3.17 deficiency accumulated more IAA and exhibited increased sensitivity to a low R:FR environment, resulting in longer hypocotyls . In our study, the expression of GH3.6 decreased significantly 9.35-fold after EOD-FR treatment. Recent research shows that overexpression of GH3.6 in the Arabidopsis mutant dwarf in light 1-D (dfl1-D) produces strong developmental phenotypes such as short hypocotyls . Here, up-regulation of ILR1 and down-regulation of GH3.6 suggest that the mechanisms underlying increased free IAA in hypocotyls under EOD-FR treatment are complex, although the regulation of IAA homeostasis clearly plays a certain role.
In recent years, SAURs have been show to play an important role in the auxin-related regulation of apoplast acidification . Specifically, SAUR proteins inhibit the activity of phosphatases, thereby preventing plasma membrane H+ATPase dephosphorylation and activating H+ATPases, which in turn leads to apoplast acidification and the promotion of cell wall protein activity . We found that several SAUR genes were significantly up-regulated after EOD-FR treatment (Fig. 5). SAUR71 has been shown to regulate hypocotyl stele growth in Arabidopsis , and SAUR50 has been shown to promote hypocotyl elongation in the dark . The increased expression of SAUR genes likely contributed to apoplast acidification in hypocotyl cells after EOD-FR treatment, in turn affecting plant growth. At present, the functions of many SAUR proteins remain unknown, but as the largest family of auxin early response genes, SAURs are undoubtedly very important for EOD-FR-mediated hypocotyl elongation.
In addition to the typical nuclear auxin signal transduction pathway, some auxin-regulated processes are controlled by the independent TIR1/AFB-AUX/IAA pathway . The related molecular basis has not yet been clarified, but auxin binding protein 1 (ABP1) is involved in some of these processes [57, 58]. In response to low R:FR, the expression levels of several genes regulated by auxin and shading in abp1-5 mutants were 3–5 times lower than those in the Col wild type, indicating that ABP1 is also an important factor in FR response . We found that the expression of auxin receptor abp19a-related genes increased significantly after EOD-FR treatment. Although many studies have shown that ABP1 has an important role in plant growth, more in-depth studies are needed to analyze its role in the EOD-FR response. In summary, after EOD-FR treatment, there were significant differences in the expression of different auxin response genes in cells, which presumably led to physiological phenomena such as cell expansion.
EOD-FR treatment enhances the activity of cell wall proteins
Hypocotyl elongation induced by EOD-FR is closely related to cell wall relaxation and activity. In this process, cell wall proteins play an important role in cell expansion . Research shows that XTHs act on xylan chains, relax the cell wall, accelerate cell wall remodeling, and regulate cell expansion . EXPs cause wallloosening by disrupting non-covalent interactions between cellulose microfibrils and matrix polysaccharides .
Previous studies have shown that the expression levels of XTH22 in the doc1/BigArabidopsis mutant are significantly lower than those in the wild type under low R:FR , indicating that increased auxin levels in EOD-FR-treated plants may be an important reason for upregulated XTH expression. Moreover, it has been shown that XTH9 expression increases significantly under low R:FR in Arabidopsis .
At present, XTHs are thought to be the main cell wall modifiers during plant response to low R:FR, but studies on Arabidopsis show that EXPs can enhance or supplement the function of XTHs . Recent research shows that the expression of several EXP-related genes is upregulated after far-red light treatment in Brassica napus, further confirming the interaction between FR and EXPs . Here, we identified 13 XTH genes and 12 EXP genes (Fig. 3), including XTH22 and XTH9. All were significantly up-regulated after EOD-FR treatment, perhaps in response to apoplast acidification  induced by SAUR.