Metabolite Proles of Energy Cane and Sugarcane Reveal Different Strategies During the Axillary Bud Outgrowth

Sugarcane (Saccharum spp.) is one of the most well-known plants which possesses a large accumulation of sucrose. Another cultivar, energy cane, is an interspecic hybrid with higher ber and lower sugar content than sugarcane. Commercial cultivation of sugarcane and energy cane is carried out by vegetative propagation, through the distribution of culm segments (setts) or pre-sprouted seedlings (PSS). In this context, the metabolism of axillary bud outgrowth is crucial for cultures that use vegetative propagation. In this work, we evaluate the metabolic prole of sugarcane and energy cane in the early hours during the axillary bud outgrowth. Sugarcane showed few metabolic changes, except for the signicant increase in glutamate levels, which may be associated with root formation in the culm. In contrast, energy cane presented signicant changes in amino acid catabolism, increased levels of reducing sugars, lipids, and metabolite activity in the phenylpropanoid pathway. These results together reveal changes in the energy and redox status of the cell, electron transport for the TCA cycle, and an increase in compounds related to cell wall formation and growth in energy cane. Our study provides new insights on the regulation of the axillary bud of species of the Saccharum complex. the setts similar secondary After the the root occurs after However, has to have a different development with development rst and then root formation 10 days after planting chromatogram area of each metabolite. Principal component analysis (PCA) and discriminant analysis (PLS-DA) were performed; mean values were transformed into log and z-score for normalization; the heat map was generated. Statistical analysis occurred within each cultivar, signicantly different using Student’s t-test (p-value ≤ 0.05). A two-way between groups ANOVA was used to evaluate time and variety interaction effects for dependent variables through the aov() function of the R 3.6.2..


Introduction
Sugarcane (Saccharum spp.) stands out among the main crops in the world, especially in countries with tropical and subtropical climates, for the production of sugar and ethanol. It is a crop of great interest, due to its energy and biomass conversion e ciency. In the last decades, breeding programs had as main objective the increase of sucrose in the culms. However, recent advances in industrial technologies and in biotechnology have made possible the fermentation of soluble sugars generated from the deconstruction of biomass (cellulose and hemicellulose), producing the so-called second-generation ethanol 1,2 . In this context, the selection of hybrids with high amounts of biomass resulted in individuals twice as productive as sugarcane. These hybrids, known as energy cane 3 , are an interspeci c hybrid arising from backcrossing two species, S. spontaneum and S. o cinarum, generating a plant with higher ber and lower sugar content than sugarcane 4 . The morphological characteristics of energy cane results in a crop with greater potential for biomass production when compared to sugarcane. Such performance is mainly due to the high density of the plant, high sprouting rate, faster growth and development, tillering rate, root volume, and biomass accumulation 4,5,6 .
Commercial cultivation of sugarcane and energy cane is carried out through vegetative propagation.
Traditionally, the planting of the crop is carried out by the distribution of culm segments (setts) with 3-4 buds in furrows or through the system of pre-sprouted seedlings (PSS) -culm cutting around the node containing one axillary bud 7 . The regulation of axillary bud outgrowth is crucial for cultures that use vegetative propagation. The buds are axillary meristems composed of cells and tissues in a state of latency and with great power of differentiation 8 . In most plant species, the axillary buds are dormant and, although meristems are fully developed, there is no division and cell growth 9 . The state of dormancy can be characterized based on three factors: i) when it is due to the internal state of the bud, known as endo-dormancy; ii) when there is endogenous signaling to bud from other organs, such as hormonal signaling, known as para-dormancy; and iii) when there are external stimuli to the plant such as temperature variation, photo-period, availability of water and nutrients, which is known as eco-dormancy 10,11 . Therefore, the shoot branching pattern depends not only on the initiation of the axillary meristems, but also on the regulation of bud outgrowth which, in turn, is crucial to control shoot architecture and biomass production 12 .
The regulation of the axillary bud is governed by a complex interaction between environmental, genetic and metabolite factors 13 . Changes in metabolite signals from other regions of the plant are detected before axillary bud outgrowth begins 14 . Moreover, several hormones play an important role as the main regulatory molecules during axillary bud outgrowth, among which auxin, cytokinin and strigolactone stand out 11,12,15 . Furthermore, the cellular energy for growth can be provided through glycolysis and amino acids fed in the TCA cycle 16,17 . Sugars and amino acids are part of a sophisticated metabolic network, linking energy status and regulating growth 16 . Studies in plant metabolism have focused on elucidating the function and regulation of certain biosynthetic pathways 18 . In this context, metabolomics has been used as a tool to elucidate the mechanisms involved in metabolic regulation, as well as to understand the interaction between genotype and phenotype 19 . Recently, metabolites in bud and culm portions of 16 sugarcane cultivars have been associated with cultivars with a high sprouting rate 20 . In addition, the metabolic pro le of energy cane setts treated with auxin (bud sprouting inhibitor), revealed metabolites associated with presence of oxygen and energy status during bud outgrowth 21 . However, key markers that regulate axillary bud outgrowth remain largely unknown.
In the present study, we evaluated changes in the metabolic pro le during the axillary bud outgrowth of sugarcane and energy cane and veri ed different strategies used for each cultivar during the rst hours of the axillary bud outgrowth. Our result results demonstrate greater metabolic activity during the axillary bud outgrowth in energy cane compared to sugarcane. Also, the key metabolites contributions to the dormancy process and the mechanism of the sprouting process of species of the Saccharum complex are introduced, providing insights for future molecular and agricultural research.

Sprouting rate and shoot height
Commercial cultivars of sugarcane (9 month-old CTC 4) and energy cane (9 month-old Vertix 12) were used in this study. The + 3 to + 14 internodes were randomly selected. The setts were planted individually in trays and were evaluated for the progress of emergence (Emergence Velocity Index -EVI) for each bud.
It was considered it to have sprouted when the shoot emerged from the vermiculite, and shoot height was evaluated. After 30 days, the energy cane presented twice the EVI of sugarcane (Fig. 1A) and shoot height of 34.12 ± 4.33 cm compared to 7.43 ± 2.79 cm of sugarcane (Fig. 1B). With only eight days of planting, 83.33% of the energy cane setts had sprouted, with the average shoot height of 8.08 ± 1.69 cm, however, in sugarcane only 25.0% of the buds had sprouted, with average shoot height of 0.36 ± 0.69 cm (data not shown).

Analysis of GC-TOF-MS reveals difference in the metabolism of axillary bud outgrowth of energy cane and sugarcane
To unravel the differential metabolome of the axillary bud outgrowth of energy cane and sugarcane, ve buds from both cultivars were randomly collected at 0 and 48 hours after planting resulting in a total of 20 samples. GC-TOF-MS analysis allowed us to identify a total of 46 metabolites, which included amino acids, sugars, and organic acids (Table S1 and Fig. S2. Supplementary information). The identi ed metabolites are involved in several metabolic pathways, such as the tricarboxylic acid (TCA) cycle, glycolysis, amino acid biosynthesis, organic acid biosynthesis and phenylpropanoid biosynthesis. Multivariate statistical analysis of metabolite data through Principal Component Analysis (PCA) showed clear discrimination among axillary bud outgrowth in energy cane ( Fig. 2A), however, in sugarcane this difference was not so clear (Fig. 2B). Similarly, partial least squares-discriminant analysis (PLS-DA) of metabolites during the axillary bud outgrowth showed separate grouping for each time in energy cane Next, we sought to verify the metabolic changes during the axillary bud outgrowth. In energy cane, 20 metabolites were found to be differentially changed between 0 and 48 hours (p 0.05). Methionine, lysine, arginine, tryptophan, ornithine, glycine, alanine, isoleucine, ra nose, GABA (γ -aminobutyric), and lactate showed reduced levels from 0 to 48 hours. In contrast, nine metabolites increased their levels according to bud outgrowth: pyroglutamate, chorismate, aspartate, quinate, ferulate, fucose, fructose, aconitate and myo-inositol. In contrast, in sugarcane it was possible to observe differential changes only in the levels of glutamate, induced after 48 hours (Fig. 3). Our results provide the rst molecular clues to the sprouting rate and shoot height differences observed between a cultivar of energy cane and sugarcane.
To further investigate the metabolic pathways that modulate the bud outgrowth in energy cane and sugarcane, a pair-wise comparison between growth times 0 and 48 hours for each cultivar were performed. We observed signi cant metabolite changes (p 0.05), as depicted on the metabolic map these results suggest key different strategies during the axillary bud outgrowth of the two cultivars. Moreover, even after 48 hours of sugarcane planting, the axillary bud outgrowth and metabolic changes were not veri ed, which indicates that the axillary bud is in the dormant phase.
In addition, to de ne metabolic changes related to the sprouting process in the energy cane, the pairwise Pearson correlation coe cient was analyzed for each metabolite. Metabolites involved in the phenylpropanoids biosynthesis showed strong positive correlation when paired against each other. On the other hand, we veri ed a strong negative correlation between some amino acids, among them pyroglutamate (Fig. S3). Clearly, these ndings suggest that during the axillary bud outgrowth, there was an increase in sugar metabolism and phenylpropanoids biosynthesis and a decrease in amino acid metabolism.

Major response related to LC-ESI(+)-MS/MS analysis
Untargeted LC-ESI(+)-MS/MS analysis was performed as a complementary approach to describe the changes in metabolism during the axillary bud outgrowth. PCA and PLS-DA of metabolites showed separate grouping in energy cane, however in sugarcane we did not observe the same result (Fig. 5). In total, 35 compounds were detected in the bud's samples ( Fig. 6A) (Table S2 and Fig. S2. Supplementary information). Among these metabolites, eight were common between the two approaches and had the same concentration pro le: phenylalanine, caffeate, tyrosine, asparagine, glutamate, pyroglutamate, leucine, and chorismate ( Fig. 6B).
Next, we performed two-way ANOVA analysis of the metabolic pro le between the two cultivars in relation to time (p-value = 0.00792) and in relation to cultivars only (p-value = 0.00328). The cultivar and time interaction proved to be signi cantly different (p-value = 0.00181), which indicates the relationship of the cultivar and the relative quanti cation of metabolites is time-dependent. Moreover, we can notice two groups of metabolites clearly different in both cultivars where 16 metabolites demonstrated to have higher concentrations in energy cane, which includes organic acids, lipids, choline derivatives, amino acids or derivatives. While a group of 12 metabolites were less concentrated in energy cane than in sugarcane, which mostly includes sugars (Fig. 6A).

Discussion
Sprouting is an important stage of development which is regulated for axillary bud outgrowth being a central process for establishing the crops that are planted through vegetative propagation 22 . We veri ed the sprouting capacity of a cultivar of energy cane and sugarcane. The emergence velocity index, followed by a faster shoot development, was clearly higher in energy cane (Fig. 1). Energy cane demonstrates greater capacity and homogeneity of sprouting of all internodes 6 , and a higher sprouting rate when treated with auxin previous to planting 21 . The faster shoot development of energy cane ( Fig. 1B) can be associated with two factors: (i) early shoot development followed by the roots formation, which suggests an early onset of the photosynthetic process while the opposite is observed in sugarcane; and (ii) the higher rate of nocturnal growth of energy cane compared to sugarcane 6 . These data can suggest a differentiated metabolism in the axillary bud of energy cane, which contributes to a rapid axillary bud outgrowth.
Metabolomics has been used as a great molecular tool, able to help unravel mechanisms of plant metabolism during growth. Integration of both GC-TOF-MS and LC-ESI-MS/MS allows to identify a set of metabolites involved in several aspects of plant development, plant pathogen interaction, and abiotic stress conditions 23 . Here, we present the metabolites' relative abundance during the axillary bud outgrowth (0 and 48 hours) of energy cane and sugarcane, and associate these data with metabolic pathways. We found a prevalence of reducing sugars (glucose and fructose) in energy cane while in sugarcane a trend in the reduction of the levels of these sugars was veri ed. During the axillary bud outgrowth, there is a decrease in the concentration of sucrose and an increase of hexoses 24 . Interestingly, a greater performance of a sprouting process is associated with low levels of sucrose present in the culms 25 . In energy cane, the low levels of sucrose and consequent high levels of reducing sugars suggest that glucose and fructose play an important role in regulating bud outgrowth when they are rapidly synthesized and metabolized. Furthermore, axillary buds are centers of meristematic activity, which exhibits a high rate of metabolism, thus requiring a range of soluble carbohydrates for the sprouting process. These metabolites can be translocated to buds through source organs, such as culms, causing a progressive tissue increase 26 . Culm metabolism is extremely important in determining axillary bud outgrowth or dormancy status 27 . The metabolic pro le of sugarcane culm and bud revealed that the metabolic network of culm is more coordinated than that of bud. Besides that, glutamate and serine are metabolites that present a clear connection between the two tissues 20 .
The role of amino acids in plant metabolism is fundamental for a multitude of metabolic reactions related to various physiological processes, such as plant growth and development, generation of metabolic energy or redox power, and resistance to abiotic and biotic stress 28,29,30 . We found differences in abundant levels of amino acids during the axillary bud outgrowth and overall were highly correlated. We identi ed changes in levels of amino acids associated with ASP-famliy pathway (AFP) such as lysine, isoleucine, and methionine of which aspartate is a precursor 16,17,31 . Lysine and isoleucine catabolism in the TCA cycle allows continuous operation of the mitochondrial electron transport chain under limited energy or carbon availability 31 . Moreover, methionine is a fundamental metabolite in plants, once the majority of this amino acid is converted to S-adenosylmethionine (SAM) which regulates a range of biological processes, such as the formation of cell wall, syntheses of ethylene, vitamin, and polyamines 17,31 . Ethylene has demonstrated a great activator for the development of axillary bud in sugarcane 8 . In energy cane, we veri ed an increase in aspartate and a reduction in the levels of the lysine, isoleucine and methionine, which suggest the catabolism of these amino acids to supply electrons for the TCA cycle and for the formation of SAM during axillary bud outgrowth. In sugarcane, we see an increase in the levels of lysine, threonine, and methionine, that is a consequence of the high levels of glutamate. In Arabidopsis thaliana, high concentrations of lysine cause a delay in breaking seed dormancy 32 , which corroborates to our results in sugarcane.
Another amino acid with an important role in regulating plant metabolism is glutamate, being a primary product of nitrogen assimilation 33 . Glutamate plays a very important role in the plant signaling and in the construct of root architecture in arabidopsis 34,35 and is a metabolite that acts as a crosstalker between bud and culm of sugarcane 20 . Our data show a signi cant increase in glutamate during the axillary bud outgrowth in sugarcane. Taken together, we hypothesized that increased levels of glutamate in the bud may act as a signaling molecule in the culm for root formation on the setts (morphologically similar to secondary roots). After planting the sugarcane, the root formation occurs after 24 hours 36 . However, energy cane has shown to have a different development strategy, with shoot development rst and then root formation 10 days after planting 6 .
In addition, it is well known that glutamate is a precursor to the synthesis of proteins, polypeptides, and organic compounds. These organic compounds include proteinogenic amino acids, such as glutamine, proline, arginine, histidine and non-proteinogenic amino acids, such as GABA 37,38,39 . The synthesis of GABA can occur through two alternative routes: (i) glutamate can be transported to the cytoplasm, where is degraded into GABA, which is subsequently imported into the mitochondria to be converted as a nal product into succinate in the TCA cycle, regulating the redox balance and metabolism energy 28,40 ; (ii) ornithine and arginine catabolism results in the formation of putrescine, through enzymes decarboxylases. Putrescine is then converted into spermidine and spermine, and then into GABA in the cytoplasm 41 . GABA can act as signaling molecule in plant growth and development 42,43,44 and plays an important role during seed germination, providing, through the TCA cycle, building blocks for metabolic reorganization, prior to the degradation of energy-demanding storage reserves 39 . In energy cane, we observe an increase in the catabolism of GABA, ornithine, arginine, and ASP-family amino acids, which taken together results in an increased activity in the TCA cycle, providing increased electron transport and energy during the axillary bud outgrowth. The reduction in GABA levels has also been reported in bud outgrowth of setts of energy cane under water soaking 21 .
Moreover, in energy cane we observe a reduction in pyruvate, alanine and lactate levels. In sugarcane, we observed a reduction only in lactate levels. Lactate synthesis is controlled by the pH of the cytosol under hypoxia where it is initially converted to lactate but, as the pH decreases, an accumulation of ethanol occurs in the cell 45 . The formation of alanine diverts carbon to neutral amino acids, preventing cell acidi cation and carbon loss 28 . Energy cane setts under the auxin stimulus showed an increase in lactate levels resulting in a delay in the bud sprouting process, possibly caused by cell acidi cation 21 . The low levels of lactate may be an indication that this metabolite is a possible repressor for the axillary bud outgrowth, since cellular acidity can cause hypoxia 46 . Furthermore, oxygenation may be a rst clue to the transition from breaking dormancy and developing the axillary meristem 47 . Molecular oxygen is essential for the formation of ATP, serving as a terminal electron acceptor for the transport chain in the TCA cycle and providing energy for growth.
Metabolites associated with cell growth and expansion are related to the phenylpropanoid pathway. In energy cane, we veri ed an increase in the levels of p-coumarate, caffeate, and ferulate during the bud outgrowth. Phenylpropanoids are necessary for the biosynthesis of a large number of metabolites, including avonoids, hydroxycinnamate esters (HCEs), wall-linked hydroxycinnamic acids (HCAs) and lignins 48,49 . In most plants, the rst step in the pathway of phenylpropanoids is the deamination of phenylalanine in cinnamate by L-phenylalanine ammonia lyase (PAL; EC 4.3.1.24). However, it has been shown that the grasses of the Poaceae family (or Gramineae, also known as true grasses) and to a lesser extent to some orders of dicotyledonous plants, such as Fabales, Malvales, Asterales, Caryophyllales, ferns, and Solanales use a more e cient route than dicots. In this pathway, tyrosine is directly transformed into p-coumarate by phenylalanine / tyrosine (bifunctional) ammonia lyase (PTAL; EC 4.3.1.25) 49,50,51 . Caffeate and ferulate are precursors to most lignins, lignans, and are associated with suberin and cutin polymer matrix waxes 51,52,53 . Ferulate is found as a component of the cell wall and is considered important to increase the rigidity and strength of the cell wall and conduction vessels in grasses 54,55 , dicots 56 and gymnosperms 57 . In these cell walls, ferulate serves as an initiation site for ligni cation, acting as a crosslinking system for arabinoxylans and lignins 49,54,58,59,60 . It is proposed in grasses that the formation of cross-linked between ferulate, arabinoxylans and lignin, is the mechanism by which cells end the elongation process, alternating from the development of the primary to the secondary wall 58,61 . Monocotyledons have a unique cell wall composition, showing high proportions of type S lignin, hydroxycinnamates (ferulate and p-coumarate) attached to the cell wall and the presence of tricin avonoid 62 . Furthermore, ferulate is the main hydroxycinnamic derivative of young cell walls while p-coumarate is an indicator of cell wall maturity, since it is esteri ed mainly in side chains of S units and its incorporation follows the same deposition pattern syringe units 63,64,65 . In addition to their role in the plant wall, phenolic compounds are often associated with improved redox status and antioxidant protection of cells, acting in the elimination of reactive oxygen species (ROS) 49,66 .
Breaking dormancy in the axillary meristem is a process that requires energy to trigger cell division and elongation 12 . For such processes, ascorbate biosynthesis plays a fundamental role 67 . However, myoinositol and galacturonate are metabolites that interconnect ascorbate biosynthesis, promoting cell division and elongation 67 . Myo-inositol is an important cellular metabolite that forms the structural basis of a series of lipid signaling molecules that function in several pathways, including responses to stress, regulation of cell death, biosynthesis of secondary metabolites and phytohormones 68 . Among the phytohormones, cytokinins play an important role in the regulation of axillary bud outgrowth, which in turn are controlled by auxins through the process of apical dominance (para-dormancy) 10,11 . Our data demonstrate that during the axillary bud outgrowth in the energy cane, there is a signi cant increase in the levels of myo-inositol and metabolites of the phenylpropanoid pathway, which together may be correlated to the process of division, elongation and cell growth.
Growth and development processes are closely linked to lipid biosynthesis. During cell growth, there is an increase in lipid metabolism 69 . Lipids are vital cellular constituents because they provide the structural basis for membranes and energy storage for metabolism 70 . Phospholipid bilayers maintain the structure and functionality of all membrane systems 70 . In this context, choline is a key metabolite precursor to phospholipid and plays an important role as osmoprotectant, improving the plant growth under stress conditions 71,72 . Choline biosynthesis is derived from lipid metabolism (Kennedy pathway) where phosphocholine is dephosphorylated to form choline 72 . Exogenous application of choline results in increased tolerance to abiotic stress in several plants 70,73,74 . In addition, it is well understood that energy cane has greater resistance to drought than other conventional cultivars 4,5 . Our LC-ESI(+)-MS data demonstrated that the metabolic pro le of energy cane showed a higher relative concentration of lipid metabolism compounds and choline derivatives, which may be related to the rapid growth and greater resistance of these cultivars to abiotic stress.
In conclusion, the metabolic pro le of a diverse set of compounds, such as sugars, amino acids, lipids, and organic compounds was quite evident, especially in energy cane which demonstrated a faster metabolism from sugarcane. The changes in metabolic pathways during the axillary bud outgrowth in energy cane and sugarcane is consistent to the sprouting speed observed. Remarkably, energy cane demonstrated changes in sugar levels, catabolism of amino acids, increased relative concentration of organic compounds, and greater abundance of lipids and choline derivatives which demonstrate energy production and formation of membrane and wall cells for growth. On the other hand, sugarcane showed an increase in the levels of glutamate and other amino acids, which may be related to bud dormancy and root formation in the culm. Together, our data demonstrate, for the rst time, differences in the axillary bud outgrowth strategy of a cultivar of energy cane and sugarcane (Fig. 7), which may be the basis for new studies and targets for breeding programs.

Plant Material
Field-grown plants were harvested from Sugarcane Technology Center (CTC) and on Luiz de Queiroz Higher School of Agriculture (ESALQ), and used for preparing cane setts (one-node and single-budded). Commercial cultivars of sugarcane (9 month-old CTC 4) and energy cane (9 month-old Vertix 12) were used in this study. Setts were harvested between 9:00 and 11:00 from + 3 to + 14 internodes. The internode + 1 is connected to the rst leaf with a visible dewlap, the internode count proceeds downward.
The internodes were randomized to ensure similarity in natural conditions.

Growth conditions and Measurements
Sprouting and growth of setts occurred in a greenhouse (plastic-covered equipped with an exhaust fan) under natural conditions of light and temperature (22º49'09.7"S, 47º04'16.4"W). Experiments occurred in November and December, 2018. Internodes were planted in trays (38 cm length, 29 cm width, and 6.5 cm height) with vermiculite (approximately 987.9 ± 0.3 g). Bud sprouting counts were made daily through 30 days after planting (DAP) to determine the Emergence Velocity Index (EVI) using the formula described by Maguire 75 . Shoot height was measured from the ground level to the tip of the longest leaf (fully or not fully expanded) with a ruler (cm). Irrigation occurred daily so that there was no water stress. Another experiment was carried out to analyze the metabolic pro le, buds from both cultivars were collected at 0 and 48 hours after planting with the aid of a scalpel (Fig. S1, Supplementary information) and immediately frozen with liquid nitrogen for analysis of the GC-TOF-MS and LC-ESI(+)-MS/MS. A total of ve biological replicas were collected, with each replicate corresponding to a pool of ve different buds. The material was macerated and stored at − 80 ºC.

GC-TOF-MS analysis and Data processing
Analyses of the GC-MS of the buds were performed at the Metabolomics Laboratory (LabMet) of the National Biorenovable Laboratory (LNBR). Metabolites were extracted from 50 mg of fresh samples.

Statistical analysis
The analysis of metabolomics data was performed using the MetaboAnalyst 4.0 software 83 . Normalization of the obtained data was carried out to remove systematic as well as replicates variation within the samples. GC-TOF-MS data were normalized using metabolite concentrations; LC-ESI(+)-MS/MS using the chromatogram area of each metabolite. Principal component analysis (PCA) and discriminant analysis (PLS-DA) were performed; mean values were transformed into log and z-score for normalization; the heat map was generated. Statistical analysis occurred within each cultivar, signi cantly different using Student's t-test (p-value ≤ 0.05). A two-way between groups ANOVA was used to evaluate time and variety interaction effects for dependent variables through the aov() function of the R 3.6.2..   Metabolites identi ed by GC-TOF-MS during the axillary bud outgrowth of energy cane and sugarcane.

Declarations
Heatmap was built using log2 followed by the normalization of metabolites by z-score. Blue scale indicates major and minor red, relative concentration. A total of ve biological replicas were used, each replica was composed of a pool of ve buds. Circle with an asterisk represents a signi cant difference between the times of 0 and 48 hours for each cultivar, using the Student T-test (p ≤ 0.05).   Metabolites identi ed by LC-ESI(+)-MS/MS during the axillary bud outgrowth of energy cane and sugarcane. Heatmap was built using log2 followed by the normalization of metabolites by z-score. Blue scale indicates major and minor red, relative concentration. A total of three biological replicas were used, each replica was composed of a pool of ve gems. Two-way ANOVA analysis demonstrate signi cant difference between cultivar and time interactions (p-value = 0.00181).

Figure 7
Schematic representation of the main changes during the axillary bud outgrowth of energy cane and sugarcane In cane energy, an increase in hexoses, a reduction in pyruvate levels and the catabolism of some amino acids was observed, resulting in an increase in the electron transport chain in the TCA cycle.
There was an increase in the levels of lipids and compounds related to the phenylpropanoid pathway, which led to an increase in membrane and cell wall formation. In sugarcane, a reduction in the levels of hexoses, an increase in the levels of pyruvate and amino acids was observed. There was an increase in glutamate, which acts as a signaling molecule between the bud and culm tissues, and leads to the formation of root in the setts. In both cultivars there was a reduction in lactate levels (most evident in energy cane), which leads to an increase in cellular oxygenation, which may be an indication for rapid shoot development in energy cane, while in sugarcane occurs root formation in the setts, which may be related to high levels of glutamate.

Supplementary Files
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