Involvement of lignin biosynthesis in the regulation of stem development and secondary growth
As internodes elongate, stem lignification gradually deepens. Increasing stem maturity accelerates stem lignification and subsequently increases the stem/leaf ratio, resulting in decreased digestibility. Previous studies have shown that the maturity of the stem affects the digestibility of forage, and improving digestibility is one of the primary goals of alfalfa breeding . Therefore, in order to monitor changes in the digestibility of alfalfa, recent research has focused on the expression of key genes involved in the phenylpropanoid biosynthesis pathway [17–18]. Cell wall synthesis, including the synthesis of cellulose, hemicellulose, and lignin, is a complex biological process. Cellulose biosynthesis is performed by a membrane-bound rosette structure of which sucrose synthase is an integral component . The presence of lignin, a complex phenolic polymer formed from three alcohol monomers (coumarin, coniferol, and myrosinol), is the main reason for reduced alfalfa digestibility . Lignin biosynthesis requires the participation of a variety of enzymes, and lignin is one of the essential products of the phenylpropanoid metabolic pathway. The specific biosynthesis of monolignols begins with the production of phenylalanine in the shikimate pathway [21–22] and continues with the formation of 3-p-hydroxyphenyl, guaiacyl, and syringyl units and finally the synthesis of lignin .
An interaction network of 21 proteins was involved in phenylpropanoid biosynthesis (Fig. 7), and most of the key enzymes of phenylpropanoid biosynthesis were identified in the current study. In total, 11 DEPs were mapped onto the phenylpropanoid pathway (Fig. 8a), including phenylalanine ammonia-lyase-like protein (PAL), cytochrome P450 family cinnamate 4-hydroxylase (C4H), caffeic acid O-methyltransferase (CCoAMT), 4-coumarate:CoA ligase-like protein (4CL), monoglyceride lipase-like protein (MGL), HXXXD-type acyl-transferase family protein (HCT), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), cinnamyl alcohol dehydrogenase-like protein (CAD), horseradish peroxidase-like protein (HRP), glycoside hydrolase family 1 protein (GH1), and cytochrome P450 family monooxygenase (F5H). Compared with MS, nine of these proteins were significantly upregulated in FS, whereas HXXXD-type acyl-transferase family protein (HCT) and cytochrome P450 family monooxygenase (F5H) were downregulated. These expression changes provide a reasonable explanation for the higher levels of stem lignification in the F genotype (Supplementary Fig. S1).
Phenylalanine ammonia-lyase-like protein (PAL), an enzyme associated with lignification in primary and secondary tissues, catalyzes the deamination of phenylalanine to initiate phenylpropanoid metabolism . The expression of PAL varies coordinately with condensed tannins (CTs) accumulation during the primary to secondary growth transition in stems, and PAL is mainly expressed in non-lignifying cells of stems, leaves, and roots . In the current study, PAL was downregulated in MS compared with FS, consistent with the maturation of the stem (Fig. 6a), suggesting that PAL played an essential role in phenylpropanoid metabolism as well as in stem development. A previous study has reported that both coumarate 3-hydroxylase (C3H) and C4H are involved in the early steps of monolignol biosynthesis and exert a negative effect on stem digestibility . In the current study, the content of C4H was significantly lower in MS than in FS (Fig. 6a), consistent with lower monolignol biosynthesis and higher stem digestibility in MS.
CAD catalyzes the final step in monolignol synthesis and is therefore a crucial enzyme for the synthesis of S-, G-, and H-lignin . 4CL plays a part in the biosynthesis of lignin monomers, particularly guaiacyl (G) lignin , and there is an overlap in the expression patterns of 4CL family genes . In the current study, CAD, 4CL, and C4H were enriched abundantly in the phenylpropanoid biosynthesis pathway. Furthermore, all of these proteins exhibited consistently lower protein and transcript levels in MS than in FS (Fig. 6a), consistent with greater lignin accumulation in FS. Previous studies have reported that the downregulation of CCoAOMT and CCoAMT leads to lower lignin levels and a reduction in G units . In the current study, CCoAOMT and CCOAMT were significantly downregulated in MS relative to FS (Fig. 6a), consistent with lignin biosynthesis in MS.
Previous studies have reported that shikimate/quinate HCT plays an essential role in lignin biosynthesis, and there is a strong correlation between HCT accumulation and lignin properties . In the current study, protein and transcript levels of shikimate/quinate HCT were lower in MS than in FS, consistent with lower lignin accumulation in MS. Moreover, four lipid-transfer proteins, eight serine proteases, and six fasciclin domain proteins (including 3 fasciclin-like arabinogalactan protein, FLA) were also enriched in stems, and these proteins have been reported to participate in cell wall maturation and secondary growth [31–32].
Taking all of the genes mentioned above into consideration, DEPs were involved in lignin biosynthesis and the regulation of stem development and secondary growth. Although the M genotype had a thicker stem (Fig. 1a), the expression of DEPs associated with lignin synthesis was lower in M than in F, suggesting that M accumulated less lignin. This indicates that the M genotype may have higher biological yields and a higher nutritional value than the F genotype (Fig. 1b, Supplementary Fig. S1).
Differences in metabolism-related DEPs between leaves of two alfalfa genotypes
Leaves originate from the SAM and ultimately become flat organs specialized to facilitate light capture and photosynthesis. Leaf morphogenesis, which gives rise to a wide variety of sizes and shapes, is an intricate process that is regulated by many genes from multiple pathways [33–36]. Early leaf development is arbitrarily divided into three stages: the initiation of the leaf primordium, the establishment of leaf adaxial-abaxial polarity, and the expansion of the leaf blade [37–38]. Previously, KANADI and YABBY transcription factors have been reported to be responsible for the development of abaxial tissues [39–42]. Initial asymmetric leaf development is regulated by polar YABBY expression . Furthermore, gain-of-function alleles of KANADI and YABBY3 (YAB3) result in radial abaxialized organs [40–41] and abaxial tissue differentiation, respectively [39,42].
Leaf development is regulated by internal genetic mechanisms and external environmental cues. Phytohormones, especially auxin, regulate the entire process of leaf development . Plant auxin homeostasis is mainly maintained by three processes: de novo IAA biosynthesis, IAA degradation, and IAA conjugation/deconjugation. In addition, IAA carboxyl methyltransferase has been reported to have an essential role in the regulation of auxin homeostasis through the conversion of IAA to methyl-IAA ester (MeIAA) . In the current study, indole-3-acetaldehyde oxidase, which is involved in the biosynthesis of IAA, was lower in ML than in FL, suggesting that less auxin was produced in ML . In addition, the trend in indole-3-acetaldehyde oxidase levels was consistent with that of ARP and AIR (Fig. 6b), which are involved in auxin homeostasis and play vital roles in leaf development.
Photosynthesis is a process used by plants and other organisms to convert light energy into chemical energy that can later be released to fuel the organisms' activities. Photosystems are functional and structural units of protein complexes involved in photosynthesis that together carry out the primary photochemistry of photosynthesis: the absorption of light and the transfer of energy and electrons. LHCI influences the capture of light energy by photosystem II, which is the key step in the light reactions . The chloroplast NAD (P) H dehydrogenase (NDH) complex, as an electron donor, mediates photosystem I (PSI) cyclic and chlororespiratory electron transport [48–49]. Efficient operation of NDH requires supercomplex formation via minor LHCI in Arabidopsis, and both play essential roles in photosynthesis . In the current study, LHCI and NDH were downregulated in ML relative to FL (Fig. 6b). The reduced protein abundance may have resulted in a decreased photosynthetic rate per unit leaf area in ML. However, compared with F genotype alfalfa, larger leaf area in M genotype alfalfa (Fig. 1b) might have compensated for the decrease of photosynthetic rate in ML, which provided a reasonable explanation for higher leaf biomass in ML.
Green plants obtain most of their energy from sunlight via photosynthesis by chloroplasts (including chlorophylls a and b), which gives them their green color. Interestingly, DEPs associated with carbon fixation metabolism and photosynthesis were significantly enriched in leaves, which supply energy for plant development. Chlorophyll, the primary pigment in plant leaves, mainly participates in photosynthesis, and chlorophyll biosynthesis plays an essential role in leaf development. The insertion of magnesium into the chlorophyll molecule is primarily controlled by the activity of magnesium chelatase subunit CHLI, which performs a critical step in the chlorophyll biosynthetic pathway [51–52]. In the current study, six DEPs were mapped onto the porphyrin and chlorophyll metabolism pathway, including porphobilinogen deaminase (PBGD), uroporphyrinogen decarboxylase (UPOD), bacteriochlorophyll synthase (BchG), red chlorophyll catabolite reductase (RCCR), CHLI, and magnesium-protoporphyrin IX monomethyl ester cyclase (MPEC), which were significantly enriched in leaves (Fig. 8b). Moreover, both CHLI and MPEC  were significantly upregulated in ML relative to FL, suggesting that chlorophyll biosynthesis and photosynthetic efficiency were higher in ML (Fig. 6b). In addition, a higher content of granule-bound starch synthase I in ML also suggested the accumulation of more photosynthetic products , consistent with higher biomass in ML than in FL.
Chlorophyll degradation occurs during leaf senescence, the final stage of leaf development that is regulated by transcription factors and receptor kinases through signal perception and transduction [55–56]. Recent research has shown that chlorophyllase, magnesium-chelating substance, and RCCR participate in chlorophyll breakdown [57–58]. In the current study, RCCR, a major inducer of cell death , had consistently lower protein and transcript levels in ML than in FL (Fig. 6b), probably resulting in RCCR accumulation in FL. Enhanced chlorophyll degradation likely further reduced photosynthetic rate, contributing to the differences in biomass observed between the alfalfa genotypes.
Comparative proteomics analysis of DEPs in leaves and stems
Substance synthesis and energy metabolism provide an essential nutrient supply for plants and are indispensable for the completion of normal growth and development. Previous studies have reported that carbohydrate metabolism directly affects plant growth status . Interestingly, carbon metabolism and energy metabolism showed similar trends in stems and leaves. Likewise, the largest group of DEPs between the alfalfa genotypes was also related to metabolism in leaves and stems. DEPs involved in glycolysis/gluconeogenesis, the pentose phosphate pathway, and pyruvate metabolism were identified in leaves (Fig. 5b). Similarly, a large number of DEPs involved in the pentose phosphate pathway, the citrate cycle (TCA cycle), and glyoxylate and dicarboxylate metabolism were identified in stems of the alfalfa genotypes (Fig. 5a). In addition, most of the DEPs identified and upregulated in MS and ML were involved in carbohydrate metabolism and amino acid biosynthesis, indicating that primary metabolism was enhanced to facilitate leaf and stem development and promote increased biomass in the M genotype.
Stem and leaf tissue provide necessary nutrients and material reserves for flower development at the budding stage. In the current study, linoleate 13S-lipoxygenase 2-1 and seed linoleate 9S-lipoxygenase involved in the inositol phospholipid signaling pathway were identified in stems and leaves. Previous studies have shown that various components of the inositol phospholipid signaling system participate in vacuolar changes during pollen development and in vesicle transport during pollen tube growth . The decreased protein abundance of linoleate 13S-lipoxygenase 2-1 and seed linoleate 9S-lipoxygenase in the M genotype suggested that this genotype could maintain longer vegetative growth and avoid earlier reproductive growth, which was conducive to the accumulation of more biomass.
Anthocyanoin biosynthesis plays an indispensable role in pollen development, especially for alfalfa that is cross-pollinated. To avoid self-pollination in alfalfa, floral pigmentation is conducive to attracting insects and transmitting pollen. Dihydroflavonol 4-reductase (DFR), an essential enzyme for anthocyanin biosynthesis that catalyzes the reaction of dihydroflanovol to leuco- cyanidin (-delphynidin and -pelargonidin) , was significantly downregulated in M genotype alfalfa, further demonstrating that M genotype alfalfa could maintain longer vegetative growth. Moreover, as the final storage form of photosynthetic products, dark storage protein was upregulated and enriched in the M genotype, providing a further explanation for the increased biomass of the M genotype.
The biological functions of 302 DEPs identified only in stems and 212 DEPs identified only in leaves were further analyzed. In terms of lignin biosynthesis and phenylalanine metabolism, although the DEPs were identified in leaves and stems, almost all the enzymes associated with phenylalanine pathways were significantly enriched in stems, whereas few accumulated in leaves, suggesting that lignin synthesis and phenylalanine metabolism mainly play a role in stem development. However, the differences in biomass between the two genotypes derived from differences in photosynthetic efficiency. Several DEPs involved in porphyrin and chlorophyll metabolism (including PBGD, UPOD, BchG, ChlI, and MPEC) and granule-bound starch synthase I were only identified in the leaf proteome and were upregulated in ML, providing a reasonable explanation for the higher biomass of the M genotype.