Leaf Senescence Syndrome
Leaf senescence is the last stage in the life history of red maple leaves, from maturation to attrition, 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. 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. The catabolism of most macromolecular substances in senescing leaves is replaced by anabolic metabolism (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 considered in carbohydrate metabolism (1623 up- and 1530 down-regulated). Conversely, the metabolism of cofactors and vitamins represented the smallest groups, with 269 up-and 163 down-regulated unigenes (Fig. 1D, Supplementary Table S5).
Ethylene
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[45, 46]. 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. A limited 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 of these genes are highly expressed in senescing leaves. 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. Multiple studies have confirmed that ACC-oxidase antisense can delay senescence in ethylene-deficient leaves. It is likely that ArACOs played a key role in the accumulation of ethylene in the senescing leaves of red maple. ET signal transduction is performed according to the "linear" approach, which is initially a membrane-bound receptor with TFs at it ends and multiple elements in its midsection[47]. Three ET receptors (ArETRs), one downstream protein (CONSTITUTIVE TRIPLE RESPONSE1; ArCTR1), and negative regulators (ETHYLENE INSENSITIVE2; ArEIN2), TFs (ETHYLENE INSENSITIVE3; ArEIN3) are up-regulated in non-senescing leaves. Three downstream components, ArEBF1/2, are down-regulated in non-senescing leaves. Previous studies have indicated that CTR1 is a kinase that represses EIN2, such that when the ethylene content is low, the active receptor can bind to the protein and activate it[48]. This implies that ArCTR1 is a key element for the ethylene signal transduction pathway of red maple during leaf senescence. In non-senescing leaves ArCTR1 may inhibit the expression of ArEIN2, which prevents the latter from being dephosphorylated and further cleaved. Thus, the ArEIN2 fragment cannot be released into the nucleus, which subsequently blocks the ethylene signal transduction pathway.
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 in ABA synthesis, and the -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 high 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 and 11 were down-regulated. Much genetic evidence suggests that phosphatases 2C (PP2C) are negative regulators of ABA signaling[49], and of seven negative regulators, four PP2Cs are up-regulated in senescing leaves.
Cytokinins
Cytokinins (CKs) are one of the most classic 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[50]. 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. Furthermore, gene regulated cytokinins synthesis in transgenic plants has provided very solid evidence for the inhibition of cytokinins in leaf senescence.
In this study, the content of cytokinins in non-senescing leaves was far above that of senescing leaves, and genes related to various aspects of CK homeostasis were collected. Cytokinins signaling is based on two-component system (TCS) that is achieved by the continuous transfer of phosphate groups between major components. The key components of this signal transduction pathway are histidine kinase (AHKs), histidine-phosphotransporter proteins (AHP), as well as B-type and A-type nuclear response regulators (B-ARRs and A-ARRs). Previous studies have shown that a high expression of components such as ARR2 in the cytokinin signal transduction pathway also delays Arabidopsis dark-induced leaf senescence. Our results indicated that differentially expressed genes in this signal transduction pathway were down-regulated in senescing leaves (Fig. 4), which was identical to what was expected, and reinforced the proposed inhibitory effect of cytokinins on senescence.
Auxin
Early studies revealed that exogenous auxin levels were inversely related to leaf senescence, and that the endogenous auxin levels of many plants decreased with advancing age. Plants achieve the synthesis of tryptophan to auxin through three pathways, and according to the intermediate products, they include the indole-3-pyruvate pathway, the tryptamine pathway, and the indole-3-acetonitrile pathway[51]. At present, these 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 richer 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 complete auxin biosynthetic pathway, and the most fundamental and important auxin synthesis pathway in plants.
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[52]. 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 severely down-regulated, suggesting its key role in auxin synthesis. Through differential gene screening, 35 differential 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. In general, the fold change of each gene in the auxin signaling pathway is not large, which suggests that ubiquitin-mediated proteolysis during auxin signal transduction is less affected during leaf senescence in the red maple.
Jasmonic acid
Jasmonic acid is 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[53, 54]. 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 senescing leaves was higher than that in non-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. The role of salicylic acid in leaf senescence has also been recognized in recent years[55]. Therefore, we investigated the metabolite content and the expression of related genes in the salicylic acid metabolic pathway in red maple (Fig. 7). The phenylalanine pathway was the first discovered salicylic acid synthesis pathway, where 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 high, 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.
Studies have shown that salicylic acid signal transduction pathways are primarily NPR1-dependent pathways[56]. 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 steroid hormone brassinosteroid (BR) plays a role in a variety of developmental processes including leaf senescence. Some Arabidopsis mutants with BR synthesis (e.g., det2)[57], or the loss of a signal transduction pathway (e.g., BRI1) have delayed leaf senescence phenotypes and associated aging characteristics[58-61]. 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. Pollen, immature seeds, and fruits have the highest BR content. Young growing tissue contains higher BR levels; however, mature leaves have lower BR concentrations. This confirmed that BR is synthesized more profusely during the vigorous growth phase, which was consistent with our research results.
There are two differentially expressed genes in the brassinosteroid synthetic pathway, and both are down-regulated in senescing leaves (Fig. 8). 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, BKI1, BSK, BIN2, and BZR1/2, are down-regulated in senescing leaves. These results suggested that the content of brassinosteroid in senescing leaves likely did not accumulate further.
Gibberellin
Gibberellin is 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[62]. However, our results indicated that most of the metabolites in this pathway in red maple have not been identified, and the content of GGPP in senescing leaves of red maple is high (Fig. 9).
Of all the genes involved in the gibberellin synthesis and metabolic pathway, only four differentially expressed genes were found, three of which were highly expressed in non-senescing leaves. GA-related mutants have seldom been mentioned in the latest research. Either these mutations did not exhibit a phenotype associated with leaf senescence, or the phenotype was neglected and is still unknown. Therefore, research regarding the relationships between gibberellin and leaf senescence in red maple has yet to be further deepened.