Leaf Senescence
Leaf senescence is the last stage in the life history of red maple leaves, from maturation tosenescence, where the most obvious external change is yellowing. Our previous research revealed that the yellowing of the senescing leaves of red maple is primarily attributed to the degradation of chlorophyll, not the emergence of yellow plant-based pigments such as carotenoids[45]. We used ultrahigh-performance liquid chromatograph Q extractive mass spectrometry (UHPLC-QE-MS) to analyze the metabolism of non-senescing leaves (fully expanded non-senescing leaves) and senescing leaves (100% yellowing of the leaf surface; ~95% chlorophyll loss) (Fig. 1B). The test results showed that there are many different metabolites in the metabolic pathways of amino acids, lipids, and other biological macromolecules in the non-senescing leaves compared to senescing leaves. (Fig. 1C, Supplementary Table S4).
Leaf senescence is a genetically regulated recycling process in the lifecycle of plants in functional organs with photosynthesis to the degradation stage. To evaluate the leaf senescence process in Acer rubrum, we performed combination sequencing with non-senescing leaves and senescing leaves. Based on the sequencing results, we compared the numbers of differentially expressed genes in related pathways such as amino acid metabolism, lipid metabolism, carbohydrate metabolism, energy metabolism, the metabolism of cofactors and vitamins, and nucleotide metabolism in non-senescing and senescing leaves. For the comparison (non-senescing vs senescing leaves), the greatest number of DEGs were found in carbohydrate metabolism (1623 up- and 1530 down-regulated). Conversely, the metabolism of cofactors and vitamins represented the smallest group, with 269 up-and 163 down-regulated unigenes (Fig. 1D, Supplementary Table S5).
Ethylene
The phytohormone ethylene is extensively involved in many plant processes, where previous studies have found that ethylene levels were linked to leaf senescence. In many species, ethylene treatment can initiate alterations in aging characteristics including promoting the reduction of chlorophyll, chlorophyll–protein complexes, and other macromolecular substances, while increasing the activity of proteases and other metabolic enzymes[46, 47]. Considerable research has summarized the synthesis pathway of ethylene as the conversion of methionine to S-adenosylmethionine (SAM), SAM to 1-aminocyclopropane-1-carboxylic acid (ACC), and ACC to ethylene[48]. As there is alimited quantity of methionine in plants, the methylthio group, is recycled to replenish methionine levels and sustain ethylene biosynthesis. The detected ethylene and related compounds in the metabolic pathway are arranged in corresponding positions (Fig. 2). As displayed, S-Adenosyl-L-methionine, S-Methyl-5-thio-D-ribose 1-phosphate, 2,3-Diketo-5-methyl thiopentyl-1-phosphate, and ethylene were shown to be most abundant in senescing leaves, whereas S-Adenosylmethioninamine, 5’-Methylthioadenosine, 5-Methylthio-D-ribose, 1,2-Dihydroxy-5-(methylthio)pent-1-en-3-one, 4-methylthio-2-oxobutanoic acid, and 1-Aminocyclopropane-1-carboxylic acid were shown to be the more abundant in non-senescing leaves.
Of the 43 differential expression genes (DEGs) involved in ethylene biosynthesis, 39 are highly expressed in senescing leaves (Fig 3). ACC synthase (ACS) and ACC oxidase (ACO) are rate-limiting enzymes in the ethylene biosynthesis pathway. In our study, no detectable DEGs of ACS were observed. Seven DEGs of ArACOs were detected, where most (5/7) exhibited higher expression in senescing leaves. All of the 27 ET-signaling genes are up-regulated in non-senescing leaves.
Abscisic Acid
Abscisic acid (ABA) was once thought to be the most effective hormone in the promotion of leaf senescence in addition to ethylene. We investigated the enrichment of metabolites and genes involved in ABA synthesis, and the ABA-signaling pathway (Fig. 3). Our results showed that the content of abscisic acid was high in senescing leaves and exceeding that in non-senescing leaves. At the ABA synthesis stage, three genes demonstrated differential expression. Among them, ArZEP had a higher expression level in senescing leaves, while ArNCED and ArABA2 were highly expressed in non-senescing leaves. Of the 23 ABA-signaling genes, 12 were up-regulated in senescing leaves (ArPYL2, ArPP2C4/5/6/7, ArSNRK2-3/4/5/7/8/9, ArABF3) and 11 were down-regulated (ArPYL1, ArPP2C1/2/3, ArSNRK2-1/2/6, ArABF1/2/4/5) (Fig 3).
Cytokinins
Cytokinins (CKs) are phytohormones, which play an important role in plant growth and development. The administration of exogenous cytokinins on ex vivo and living leaves can prevent leaf senescence[52]. By analyzing the levels of cytokinins in leaves prior to and following senescence, it was revealed that there was a negative correlation between cytokinins levels and aging processes in various tissues and plants[7, 53, 54]. Furthermore, genetic manipulation of cytokinin production in transgenic plants has provided very solid evidence for the inhibition role of cytokinins in leaf senescence [7, 55-57].
In this study, the content of cytokinins in senescing leaves was far above that of non-senescing leaves, and genes related to various aspects of CK homeostasis were collected (Fig 4). Cytokinin signaling is based on two-component system (TCS) that is achieved by the continuous transfer of phosphate groups between major components. Our results showedthat all differentially expressed genes in this signal transduction pathway were down-regulated in senescing leaves (Fig. 4).
Auxin
Early study revealed that exogenous application of auxin was inversely related to leaf senescence, and that the endogenous auxin levels of many plants decreased with advancing age[58]. Plants achieve the synthesis of tryptophan to auxin through three pathways, named after the intermediate products, these are include the indole-3-pyruvate pathway, the tryptamine pathway, and the indole-3-acetonitrile pathway[59]. At present, these two of three synthetic pathways have not been fully investigated, and key genes controlling the relevant synthetic steps have yet to be discovered. Many of these steps are still potential models based on existing experiments.
In senescing leaves of red maple, the tryptophan concentration is higher than in non-senescing leaves. Indole-3-acetaldehyde (IAAld), indole-3-acetonitrile (IAN), and indole-3-acetic acid (IAA) have a higher content in non-senescing leaves (Fig. 5). A total of 22 key differential expression genes were obtained in the auxin synthesis pathway, including the YUCCA, ALDH, and TAA1 family genes. There are seven YUCCA gene expression levels in senescing leaves that are significantly down-regulated (indole-3-pyruvate pathway). The indole-3-pyruvate pathway is the first completely elucidated auxin biosynthetic pathway, and the most fundamental and important auxin synthesis pathway in plants[60, 61].
Many studies have shown that YUCCA inactivation in this pathway reduces the synthesis of auxin in plants, whereas overexpression of the YUCCA gene leads to the overproduction of auxin, which causes developmental defects[62]. Therefore, YUCCA is a rate-limiting factor for auxin synthesis, and its gene expression pattern plays a crucial role in auxin synthesis. In this study, the expression of ArYUCCA genes in senescing leaves was down-regulated, suggesting its key role in auxin synthesis. Through differential gene screening, 35 differentially expressed genes, including TIR1, IAA, ARF, and SAUR family genes, were found in the auxin signal transduction pathway. The analysis of each family gene expression pattern revealed that the TIR1, IAA, and ARF gene families were up-regulated in non-senescing leaves, while the SAUR gene family had both up-/down-regulated genes in non-senescing leaves (Fig 5).
Jasmonic acid
Jasmonic acid and its derivatives are an important class of plant hormone that is required for plant growth and development and assists with enduring stress and completing the life cycle. The first documented function of jasmonic acid was the promotion of senescence in isolated oat leaves[63, 64]. Therefore, we investigated the metabolite content and the expression patterns of DEGs in jasmonic acid biosynthesis and signal transduction related pathways of red maple (Fig. 6). The content of α-Linolenic acid and methyl jasmonate in non-senescing leaves was higher than that in senescing leaves, and the content of phosphatidylcholine and jasmonic acid was higher in the latter.
The expression of related genes showed a clear pattern, and the gene expression profiles of the same gene family, except for allene oxide cyclase (AOC), were consistent. Among them, one ArAOC gene and five lipoxygenase (LOX) genes were down-regulated in senescing leaves. Seven biosynthesis genes were up-regulated in senescing leaves, including allene oxide synthase (AOS), allene oxide cyclase (AOC), oxophytodienoate reductase (OPR), and jasmonate O-methyltransferase (JAT), which catalyzed a series of reactions in the JA-biosynthetic pathway. Jasmonate-resistant 1 (JAR1), coronatine insensitive 1 (COI1), jasmonate ZIM (JAZ) proteins, and the transcription factor MYC2 are important constituent elements of the JA-signaling pathway. The first two are down-regulated in senescing leaves, whereas the latter two are up-regulated.
Salicylic Acid
Salicylic acid (SA) is a phenolic plant hormone that can regulate seed germination, cell growth, respiration, stomatal closure, stress response, nitrogen fixation, and the seed setting rate of various plants[65]. The role of salicylic acid in leaf senescence has also been recognized in recent years[66]. Therefore, we investigated the metabolite content and the expression of related genes in the salicylic acid SA synthesis, and the SA-signaling pathway in red maple (Fig. 7). The phenylalanine pathway was the first discovered salicylic acid synthesis pathway. We detected that the phenylalanine content was almost equivalent in both senescing and non-senescing leaves. However, the content of salicylic acid in non-senescing leaves of red maple is higher, although the expression of the core enzyme phenylalanine ammonia lyase (PAL) gene in the senescing leaves is much higher than that of non-senescing leaves. Interestingly, this result was different from those of other studies. In Arabidopsis, the content of endogenous salicylic acid increased with leaf senescence[67].
Studies have shown that salicylic acid signal transduction pathways are primarily NPR1-dependent pathways[68]. Following the depolymerization of NPR1 into a reactive monomer and transfer to the nucleus, it can interact directly with some members of the transcription factor TAG family to induce the expression of downstream disease resistance genes. The expression levels of ArNPR1 gene in senescing leaves and non-senescing leaves were similar; however, 15 members of the 16 TGA genes were up regulated during senescence. Therefore, we hypothesized that the role of SA in the leaf senescence of red maple was dependent on the efficient expression of related genes in signal transduction pathways.
Brassinosteroid
The plant hormone brassinosteroid (BR) play a role in a variety of developmental processes including leaf senescence. Some Arabidopsis mutants of BR biosynthesis (e.g., det2)[69], or with the loss of a signal transduction pathway (e.g., BRI1) have delayed leaf senescence phenotypes and associated aging characteristics[70-73]. However, this phytohormone was not detected in our metabolome test results. Previous studies have shown that the content of brassinosteroid in plant tissues varies greatly[74]. Pollen, immature seeds, and fruits have the highest BR content. Young growing tissue contains higher BR levels; however, mature leaves have lower BR concentrations[75]. .
The transmission of the BR signal from BRI1 located on the membrane to the transcription factor BZR1 in the nucleus is accomplished through a series of protein phosphorylation and dephosphorylation reactions. This series of phosphatases or phosphokinases, including BAK1, BSK, BIN2, and BZR1/2, are down-regulated in senescing leaves (Fig 8).
Gibberellin
Gibberellin (GAs) are a class of phytohormones that belongs to the biguanide compounds, which play an important role in the entire life cycle of plants. The precursor of GA biosynthesis in higher plants is geranylgeranyldiphosphate (GGPP), which is synthesized from isopentenyl pyrophosphate. The impeding effect of gibberellin on leaf senescence has been discussed in many previous studies[76]. Senesent leaves have double the amout of GGPP than non-senescent (GL/YL=0.5; Fig. 9). Of all the genes involved in the gibberellin signaling, only two differentially expressed genes (ArDELLA1 and ArDELLA2) were found, all of which were highly expressed in non-senescing leaves.