Introduction

The C4 grasses such as sugarcane, sorghum, Napier grass and Miscanthus hold much promise as feedstock for bioenergy production due to their high biomass yields. Most of the harvestable biomass in these species is the culm which represents up to 75% of the total aboveground mass.

The main driver for the increase in organ size and organic content is cell expansion and is the dominant trait that is measured during plant phenotyping (Fahlgren et al. 2015; Tardieu et al. 2017; Shameer et al. 2020).

The process by which grasses produce, transport and store carbohydrates underpin all aspects of yield traits. The rate of gain in yield potential for many of the grasses has been slowing despite the overall increase in demand for more biomass. In addition, the yields from the primary crops are predicted to decline over the next two decades in semi-arid regions of the world due to climate change (Lobell et al. 2011).

Biomass production in grasses is directly related to the physiology of sink–source dynamics and whole-plant carbohydrate partitioning (Ho 1988). What constitutes sink strength or the magnitude of the ‘demand function’ have been long-standing arguments. However, it is widely accepted that it is the competitive ability of an organ to import photoassimilates and that this is the product of the sink size and sink activity (Ho 1988; Herbers and Sonnewald 1998; Slewinski 2012; Bihmidine et al. 2013). Sink strength can be defined as the rate of increase in the dry weight of a particular organ/tissue. Accumulation of dry weight is the net result of total nutrient import minus nutrient export and respiration (Doehlert 1993).

In all the C4 grasses, there is a strong correlation between culm length, internode length, and aboveground biomass (Lingle and Thomson 2012; Kebrom et al. 2017). Internode elongation is controlled by genetic (Qiu et al. 2019; Chen et al. 2020), developmental (Lingle 1997; Rae et al. 2006; Lingle and Thomson 2012; Bonnett 2013), and environmental factors such as temperature (Rae et al. 2006) and water availability (Smith and Singels 2007; Bonnett 2013). Cooler temperatures and reduced moisture availability lead to shorter internodes. Temperatures above 36 ℃ also had the effect of reducing internode length (Rae et al. 2006).

Growth by cell expansion is driven by turgor pressure that, in turn, depends on the osmotic and pressure potential. The expansion of the cell depends on the balance between the turgor pressure and the mechanics of the cell wall (Cosgrove 2005). For example, when the cell wall loosens (or relaxes) turgor drops, and this results in a decrease in the internal water potential, which then drives water influx and cell elongation. The opposite will happen when the cell wall becomes more rigid (Cosgrove 2005; Schopfer 2006).

The grasses, and many other monocots, store excess carbohydrates in the form of soluble sugars or sugar polymers within the vegetative tissues (Antony et al. 2008; Davis et al. 2011; Martin et al. 2016). Grass stems are capable of storing substantial amounts of soluble carbohydrates in the storage parenchyma cells within the internodes (Hoffmann-Thomas et al. 1996; Botha and Moore 2013).

Complex hormonal interactions are involved in the control of internode elongation (Kebrom et al. 2017). Various studies have shown that gibberellin, brassinosteroids and auxin are important in promoting internode elongation (Moore 1980; Salas Fernandez et al. 2009; Wei et al. 2019; Chen et al. 2020; Nagai et al. 2020). In contrast, abscisic acid, ethylene and jasmonic acid inhibit internode elongation (Azuma et al. 1995; Heinrich et al. 2013). There is also a strong link between the expression of GA-oxidases and decreased internode elongation (Kebrom et al. 2017). Trinexapac-ethyl (Moddus®) which specifically disrupts GA synthesis is often used to slow up the sugarcane culm growth (van Heerden et al. 2015) with the aim of producing sugarcane culms with a higher sugar content for harvest.

Sink strength (sucrose import) into the internodes is dependent on maintaining a low sucrose concentration in the cytosol (Wang et al. 2013). There are three important components of the ‘demand’ function in the internode, namely use of sucrose for biosynthesis (cell wall and other cellular constituents), respiration and storage of sucrose in the vacuole (Botha 2018). Comparing the metabolic changes associated with internode elongation in the presence or absence of Moddus® could provide valuable information regarding the rate and duration of internode elongation, as well as carbon partitioning within the internodes. The study shows that GA metabolism is directly linked to the rate but not the duration of internode elongation.

Material and Methods

The research trial was conducted in a field of sugarcane variety KQ228 at Sugar Research Australia’s Burdekin Station, QLD (19°34′0.80"S, 147°19′30.7"E). Prior to stick planting in August, the soil was nutrient-tested and fertilised according to 6 Easy Steps nutrient recommendations (Schroeder et al. 2010). The sugarcane was furrow-irrigated with a 7-day flood irrigation schedule throughout the cropping cycle. The trial was a completely randomised design including two treatments with four replicate plots. Each replicate consisted of four 10 m rows with a 1.5 m spacing between rows.

Moddus® Treatment

Moddus® is an emulsifiable concentrate containing the active compound Trinexapac-ethyl at a concentration of 250 g L−1 and is readily mixed with water before application as a spray. Moddus® was applied to the crop as a foliar pray at a dose of 50 g active ingredient ha−1 (0.25 label rate) using an agricultural handheld knapsack sprayer. The Moddus® concentrate was diluted in water (0.21 ml L−1), and 6 L applied to each replicate (987 L ha−1). Handling, mixing and application were in alignment with the Moddus® safety datasheets (SDS). Moddus® treatment occurred at 140 and 169 days after planting (DAP). The last application was 26 days prior to sample collection in March (crop age 6 months).

Non-destructive Measurements

Six primary shoots from the inner two rows in each plot were tagged for easy identification. Stalk elongation and phyllochron development were non-destructively measured in-field. This was done to ensure minimal disruption of canopy development by not changing shoot and leaf numbers. Stalk length was measured from ground level to the top visible dewlap (TVD). Leaf numbers and plant height were determined on a two-weekly basis throughout the first six months of crop development.

Identification and Measurement of Individual Internodes

At 197 DAP four primary shoots were randomly selected from the inner two rows of each plot. The length and diameter of the top 12 internodes were measured with digital vernier calipers. Internodes were numbered according to van Dillewijn (1952) (Fig. S2). The volume of the internodes was calculated using Eq. 1.

$${\text{volume}} = \pi *{\text{radius}}^{2} *{\text{length}}$$
(1)

Modelling Growth

The growth of the culm was modelled by applying a logistic growth function

$$\int \left( {{\text{growth}}} \right) = \frac{{\max }}{{1 + e^{{ - k\left( {\left( {{\text{tmid}}^{{ - t}} } \right)} \right)}} }}$$
(2)

where (length) is the change in the phenotype parameter (length, diameter or volume) and (t) represents time or thermal time. The parameters to be fitted are maximum size (max) and tmid the time where half of the maximum size (length, diameter or volume) was reached. The steepness of the growth curve is represented by k. The slope of the curve at tmid was calculated by

$${\text{slope}} = \max *\frac{k}{4}$$
(3)

For internode growth, tmid was substituted by imid where i represents the internode number (Fig. S2). The thermal time for the development of a new phytomer in KQ228 is 30DD18 (Fig. S1) By substituting internode number with the thermal time for internode formation, the thermal time to reach 50% of the maximum length or volume can be calculated. The doubling rate thermal time (DD18)) required to double the length/volume at the tmid was calculated from log2/k.

Thermal Time for Development

Culm elongation and appearance of new internodes (and leaves) are driven by thermal time (degree days (DD)) defined as the sum of daily effective temperatures (Tmean–Tbase) where Tbase is the daily mean temperature below which the process under consideration ceases (Donaldson 2009; Bonnett 2013; Inman-Bamber 2013).

For KQ228, the base temperature for phyllochron formation is 8 ℃, and 18 ℃ for internode elongation.

Sampling Specific Internodes

Internodes 2, 4 and 6 were removed from the stalk and a 30-mm-long section was cut from the bottom of the internode. Approximately 8 mm Ø cylindrical cores were bored off-centre (avoiding the pith) and vertically down using a 12-mm cordless drill and Diamond Drill Bit. The cylindrical samples were placed in a labelled 2-mL screw cap tube and snap frozen in liquid nitrogen and stored at − 80 ℃. The drill bit borer was sprayed with 70% (v/v) ethanol and wiped between samples.

Sugar Concentration

Sucrose content was determined using the standard enzymatic method (Bergmeyer 1974) with a spectrophotometer (BMG LABTECH, FLUOstar Omega) and 96-well UV-clear plate (Thermo Fisher, UV Microtiter). Glucose composition was determined using Amplex@ Red/glucose oxidase enzyme assay (Life Technologies) in a 96-well plate (Thermo Fisher, Microtiter) with a spectrophotometer (BMG LABTECH, FLUOstar Omega). Fructose content was determined using a BioVision Fructose Flurometric assay kit in a 96-well plate Thermo Fisher, Microtiter) with a spectrophotometer (BMG LABTECH, FLU-Ostar Omega). A 1/10 dilution of the OxiRed probe and a running temperature of 37 ℃ was optimal for this assay.

Data Analyses

Statistical analyses were performed in R (version 3.61) using the package Agricolae (De Mendiburu and Reinhard 2015). One-way ANOVA tests were used to make multiple comparisons followed by a least significant difference test (LSD) (Steel et al. 1997). The Tukey’s HSD post hoc tests were used to compare the group means shown in the graphs with different letters and corresponding colours. All graphs in the boxplot format were prepared in R using the package multcompView, in which the default is to present the upper and lower sides of the box as the first and third quartiles.

Results

Culm Growth and Development

The growth rate of KQ228 is present in Fig. S1. The data showed that maximum rate of culm elongation occurs around 1160 degree days (DD18)(Table S1). The growth doubling time peaked at approximately 300 DD18. New leaves appear approximately every 70 DD8. This translates to approximately 30 DD18. These values were used to calculate internode growth rates in thermal time.

The rate of internode expansion markedly accelerated by the time the leaf attached at its base (leaf 1) has fully expanded. Elongation was completed in the internode attached to leaf 6 (Fig. 1A). Moddus significantly reduced internode length (Fig. 1A) but had little effect on the culm diameter (Fig. 1B). Because of the strong suppression of internode elongation, the volume of individual internodes was significantly reduced (Fig. 1C).

Fig. 1
figure 1

Effect of Moddus on internode growth. A Internode length, B internode diameter and C internode volume

Full size image

The lines in Fig. 1 represent the modelling of internode size by applying the logistic growth function Eq. (1). Important to note that in this document internode numbering is based on leaf number where leaf 1 represents the leaf with the top visible dewlap. Botanically internode number 1 is probably internode 3 or 4.

The time to double size (drate) was not significantly different between the control and Moddus treatments (Table 1).

Table 1 Logistic sugarcane growth in the presence of Moddus. The fitted logistic models were used to predict the growth rates of internodes 2, 4 and 6
Full size table

The modelled changes in internode length and volume are presented in Table 1. An analysis of variance (ANOVA) showed that only the maximum length (max) and slope (rate of growth) were significantly affected by Moddus (p < 0.05). In contrast the presence of Moddus had no effect on the duration of internode elongation or the rate of increase in volume (Table 1 and Fig. 2). The time to reach the mid-point (tmid of growth) and the time to double size (drate) were not significantly different between the control and Moddus treatments (Table 1).

Fig. 2
figure 2

Effect of Moddus on the rate of internode elongation (A) and increase in volume (B)

Full size image

The fitted models describing internode size were then used to predict the dimensions of all the internodes. The thermal time required for the appearance of a ‘new’ internode is 30 DD18 (Fig. S1). This number was used to substitute internode number for thermal time (DD18), and the rate at which the internode length and volume change was then calculated (Fig. 2).

The rate of increase in internode length and volume was significantly different between the Moddus and control treatment (P < 0.0001). These data demonstrate that culm elongation was only driven by the growth of the top six internodes. Internodes 1–3 accounted for most (> 80%) of the culm growth (Figs. 1 and 2). Evidently, Moddus had no effect on the pattern, but only on the rate of internode growth.

Biomass Accumulation and Sugar Content

Three internodes (2, 4 and 6) representing different stages of growth were selected for further analyses.

An ANOVA of internode results showed that the fresh weight and dry weight increased significantly during internode development (Fig. 3). In each of the development stages, the weight of the internodes from the Moddus treatment was significantly lower than in the control.

Fig. 3
figure 3

Internode weight on the fresh A and dry weight B at different stages of development. Tukey’s HSD (p = 0.05)

Full size image

The pattern of change differs between the Moddus and control treatments (Fig. 3). The control treatment continues to increase both fresh and dry weights across the three internodes, while the Moddus treatment slows significantly between internodes 4 and 6 in both fresh and dry weights.

In order to calculate the metabolite concentrations, the differences between the fresh weight and dry weight of the internodes were used to calculate the total water volume of the internode.

The sugarcane internode represents a unique system where sucrose is distributed between the cell and the apoplast throughout development. The concentration of sucrose in the cytosol and apoplast changes in a tightly coordinated way (Welbaum et al. 1992; Welbaum 2013). The apoplast volume in sugarcane internodes increases with maturity and ranges between 10 and 30% of the total volume (Welbaum and Moore 1990). However, for young internodes the range is narrow (10 and 15%). For the purpose of this work, we made no attempt to correct for the contribution of the apoplast volume to total volume.

There are marked differences in the sugar composition and levels of individual sugars between the control and Moddus treatments (Table 2). The content of the reducing sugars glucose and fructose on a dry mass basis decreased significantly (P < 0.05) during internode development in the control tissue (Table 2). In contrast, the sucrose content significantly increased more than fourfold between internode 2 and 6 in the control tissue. The total sugar content in the Moddus-treated internode 6 tissue was significantly higher than in the control tissue.

Table 2 Concentration of sucrose, glucose, fructose in sugarcane internodes at different stages of development following a Moddus treatment. Concentration is expressed on both a fresh and dry weight basis
Full size table

By taking the total weight of the internodes into consideration, the total sugar content per internode was calculated (Fig. 4). Contrasting patterns in total sugar content per internode were evident between the control and Moddus treatments. The data showed that the contribution of glucose and fructose to total biomass significantly decreased in the control tissue (Fig. 4 A, B). In the Moddus treatment, the contribution of the reducing sugars to total dry mass did not decrease. Sucrose content as a percentage of total internode mass increased significantly in both the control and Moddus treatment (Fig. 4C).

Fig. 4
figure 4

Effect of Moddus on the ratio of specific sugars to the total dry mass internodes during development. A glucose, B fructose, C sucrose and D total sugar. Tukey’s HSD (p = $0.05)

Full size image

Because sucrose, glucose and fructose played such a dominant role in the biomass composition of the internodes (30% in the control and up to 60% in the Moddus treatment), we considered them as a single dominating osmolyte and treated the internode as a single cell. We therefore adapted the equation in Shameer et al. (2020) to the following:

$$\sum n_{i} m_{i} = C_{{{\text{internode}}}} V_{{{\text{internode}}}}$$
(4)
$$\frac{{\sum n_{j} m_{j} }}{{\sum n_{k} m_{k} }} = \frac{{V_{v} }}{{V_{c} }}$$
(5)

where n1 and m1 are the van’t Hoff factor and the number of moles of the osmolyte i, respectively, in the internode (Eq. 8). The distribution of the osmolyte between the cytosol (j) and vacuole (k) is reflected by Eq. 5. At steady state, the ratio of the osmotic content of the vacuoles and cytosol must be equal to the ratio of their volumes, Vv and Vc, respectively (Shameer et al. 2020).

Using the sugar data and the above assumptions, the osmotic potential (ψs) of the internodes was calculated using the following:

$$\varphi _{s} = - miRT$$
(6)

where m is the concentration of the osmolyte (in this case total sugar), i is the dissociation constant (1 for sugar), R is the gas constant and T is the absolute temperature.

In a living tissue, ψs results in the development of osmotic pressure with a positive value equal to ψs. The description osmotic pressure is used in the next sections.

Two important aspects can be seen from the calculated ψs in the control and Moddus-treated internodes (Fig. 5). Firstly, the osmotic pressure increases as internodes get older. Secondly, the osmotic pressure in the Moddus-treated internodes is significantly higher than in the control tissue. Despite the lowering in the ψs in the internodes their elongation slows up and terminates around internodes 6 and 7 (Figs. 1 and 2). This lack of further expansion despite the increase in osmotic pressure implies that the decrease in ψs is offset by a build-up of a pressure potential (ψp) that negates the osmotic pressure. The observation that the Moddus-treated internodes cease expansion, despite their much higher osmotic pressure, strongly suggests that ψp of these internodes was much higher than in the control tissue.

Fig. 5
figure 5

Calculated changes in the osmotic potential of the internodes with and without a Moddus treatment. Tukey’s HSD (p = 0.05)

Full size image

Sink Strength and Volume

Sink strength is the net increase in dry weight of individual internodes. Accumulation of dry weight in the internode is the net result of total sucrose and nutrient import into the internode, minus the carbon and nutrient export, and respiratory carbon loss due to respiration. The top 6 elongating internodes represent a carbon and nutrient sink and not a source. Hence, the carbon and nutrient export has been set as zero.

There are three important aspects to biomass accumulation during growth. Firstly, there is the requirement to accumulate osmolytes in the vacuole to facilitate water uptake. Secondly, the synthesis of extra cell wall, cell membrane and proteins to maintain cell functions, and thirdly the energy (respiratory) cost of the first two elements.

Carbon accumulation in the internodal tissue was therefore viewed to be comprised of the cell wall, sucrose and ROPAL. ROPAL is defined as the two reducing sugars (glucose and fructose), organic acids, protein, amino acids, and all other carbon compounds. All the carbon accumulation in the internode is through sucrose input from the phloem.

$${\text{Sucrose}}_{{{\text{import}}}} = {\text{sucrose}} + {\text{cellwall}} + {\text{ROPAL}} + {\text{respiration}}$$
(7)

The cell wall component as a percentage of total dry mass does not change significantly during the internode elongation phase and reported values vary between 40 and 45% (Botha et al. 1996; Lingle 1997; Lingle and Thomson 2012). After elongation has stopped, the percentage contribution of the cell wall to total biomass decreases because of the volume being constant, and dry mass continuing to increase. In calculating the cell wall component, it was assumed that cell wall content is a function of total volume.

Respiration to support cell expansion and biomass accumulation can be higher that 50% of the total carbon input (Shameer et al. 2020). Because the bulk of the energy requirements is linked to solute accumulation and non-growth associated maintenance respiration (Shameer et al. 2020), respiratory carbon loss was not considered and assumed to be directly related to the rate of biomass accumulation, and hence would represent a similar proportion of growth between the control and Moddus treatments.

The distribution of carbon between the cell wall, the sucrose and the rest of metabolism was calculated by applying Eq. (7) without respiration (Table 3). In addition, all the carbon not in sucrose reflect the net breakdown of sucrose entering the internode. This would be indicative of the hydrolysis by invertases and cleavage by sucrose synthase (SuSy).

Table 3 Biomass components of sugarcane internodes following a Moddus treatment. All components are expressed as glucose equivalents. The cell wall and ROPAL were calculated (see text for details)
Full size table

The total mass of the internodes from both the control and Moddus treatments was significantly higher at each subsequent stage of development (Table 3). The sucrose content of the control internodes increased between each stage of development, but in the Moddus treatment there was no significant difference between internodes 4 and 6.

Despite the significant difference in total biomass between the two treatments at each stage of development, sucrose was only significantly different between the internode 6 samples. A similar pattern was evident in total sugar except for the internode 6 samples. In contrast to sucrose, the total sugar content was not significantly different between the internode samples (Table 3). This is due to the much higher reducing sugar content in the Moddus treatment than in the control (Table 2).

The partitioning of carbon between the different metabolic components was significantly different between the control and Moddus treatments (Fig. 6). Sugars dominate the biomass profile in the Moddus treatment (Fig. 6C), and the importance increased during internode development. Both sucrose and the reducing sugars increased during development in the Moddus treatment. In the control tissue, sucrose increased but the reducing sugars decreased significantly during development. The partitioning of carbon to the OPAL component was significantly higher in the control treatment at all three stages of development (Fig. 6). Very little partitioning to this fraction was evident in the Moddus treatment in internodes 4 and 6 (Fig. 6B, C).

Fig. 6
figure 6

Relative contribution of the different metabolic components to total internode biomass in control and Moddus-treated sugarcane. A Internode 2, B internode 4 and C internode 6. Reducing = contribution of fructose plus glucose and OPAL = rest of cellular components (see text for details)

Full size image

The data presented in Table 3 were used to calculate the flux of carbon into sucrose, the cell wall, and the rest of metabolism in internodes 4 and 6 and expressed as a rate thermal time. The base temperature for partitioning is not known so thermal time without base correction was used.

For example, the net flux into the rest of metabolism (ROPAL) was derived from

$${\text{flux}}_{{{\text{sucrose}}}} = ~\frac{{{\text{molesROPAL~DD}}2 - {\text{molesROPAL~DD}}1}}{{{\text{DD}}2 - {\text{DD}}1}}~~$$
(8)

where moles are expressed as moles glucose equivalent in the fraction, and DD2–DD1 = 140DD.

The importance of internode volume as a driver for sink strength was evident (Table 4). The net flux of carbon increased significantly between internodes 4 and 6 in the control tissue but not the Moddus treatment (Table 4). The net flux of sucrose (glucose equivalents) was almost 3 times higher in the control versus Moddus treatment. The flux into the cell wall increased in the control internodes but remains constant in the Moddus treatment. A major difference between the control and Moddus treatments was the flux into the cellular components other than the sugars and cell wall, where the ROPAL flux was significantly reduced in the Moddus treatment.

Table 4 Calculated flux (glucose equivalents) into the different fractions of internodes 4 and 6 of sugarcane treated with Moddus. Total biomass of the internode was converted to moles of glucose
Full size table

Discussion

The data presented here confirm previous reports that culm growth in sugarcane is driven by the expansion of only the top 5 to 6 internodes (Lingle 1997, 1999; Rae et al. 2006, 2013; Lingle et al. 2009). The expansion is primarily in length.

When Moddus is applied to the leaves, internode elongation is significantly reduced. Once absorbed, the active compound in Moddus inhibits the conversion of an inactive precursor (GA20) of the plant hormone gibberellic acid (GA) into one of its main bioactive forms (GA1) and this leads to a reduction in internode length (van Heerden et al. 2015).

It is important to differentiate between the duration, and rate, of internode elongation. The duration of internode elongation is controlled by degree days. The cessation of internode elongation is associated with secondary cell wall deposition (Martin et al. 2016).

Evidently, a disruption of GA synthesis has no impact on the duration of internode elongation (Fig. 2 and Table 1), but it impacts the rate of internode elongation. As such the application of Moddus presents a good experimental system to differentiate between duration and rate of internode elongation.

The accepted model is that the duration of internode elongation is controlled by secondary cell wall synthesis and lignification of the cell wall

(Komor 1994; Rae et al. 2006). Lignification is already evident in the vascular bundles of young internodes (Bottcher et al. 2013) and starts expanding to the epidermis and storage parenchyma later in development (Jacobsen et al. 1992; Casu et al. 2007; Bottcher et al. 2013). The lignin content increases throughout the period of internode elongation and levels off in internode 6 (Lingle and Thomson 2012; Bottcher et al. 2013). This pattern of lignification of the culm appears to be similar between sugarcane, switchgrass and maize (Riboulet et al. 2009; Shen et al. 2009; Jung and Altpeter 2016). This lignin accumulation pattern is coherent with the fact that lignification begins before the cessation of the elongation process, with the deposition of secondary cell walls in specific cell types (Bottcher et al. 2013).

Internode elongation is dependent on the turgor pressure within the internodal cells, and the latter is dependent on water and carbon availability. Water uptake into the internodal cells is only possible if the cells have a lower water potential than the surrounding tissue. A low water potential inside the cell would requires high concentrations of osmotically active substances (negative osmotic potential) and plasticity of the cell wall (low pressure potential). As the vacuole occupies more than 90% of the total internodal cellular volume (Komor 1994; Rae et al. 2006), it also indicates that the osmotic potential of the vacuole is a major factor in controlling internode elongation.

If the cell wall exerts a resistance (positive pressure potential) equal to the negative water potential created by the water-soluble metabolites in the vacuole water uptake and then cell elongation will stop. Data shown here indicate that by internodes 6 to 7 the water potential gradient between the parenchyma cells and surrounding tissue is zero.

A Model for the Control of Duration and Rate of Internode Elongation

The carbon partitioning and growth of the internodes are consistent with a model that sucrose loading of the storage parenchyma cells occurs through symplastic offloading from the phloem, and that partitioning is primarily controlled by the supply and demand of carbon for growth (Fig. 7). During the elongation phase, the internodes have the capacity to breakdown the bulk of the sucrose that is offloaded (R2, R3 and R4, Fig 7). This probably fulfils three functions, namely maintaining a sucrose gradient between the phloem and the parenchyma cells, contributing to the generation of an osmotic potential gradient, and providing carbon skeletons for cellular metabolism. The large capacity for sucrose breakdown is evident from the dominance of glucose and fructose over sucrose despite the only carbon import being sucrose and the “drainage” of the hexoses to support the rest of metabolism.

Fig. 7
figure 7

A model of the reactions that are important in regulating sink strength and growth of sugarcane internodes. There are two main demand functions in the growing internode (highlighted in red) and the magnitude of this demand is directly linked to the volume of the internode

Full size image

For the internodes to grow/expand a water potential gradient is required that allows water entry into the cytosol and vacuole. At steady state, the ratio of osmolytes in cytosol vs that in the vacuole is equal to the ratio between the volume of the vacuole and cytosol, respectively. Taken that the vacuole volume if at least nine times that of the cytosol (Komor 1994; Rae et al. 2006), it follows that the capacity for loading the vacuole coupled to hydrolysis of sucrose (R10,R4, R11) is important in the establishment and maintenance of the water potential gradients required for internode expansion.

Not only is there a breakdown of sucrose during the internode elongation stage but significant resynthesis of sucrose occurs (Botha 2018). This apparent futile cycle between sucrose breakdown and synthesis is probably important to ensure maintenance of the required water potential gradients and provision of carbon skeletons for growth.

The much higher concentrations of sugars in the Moddus-treated internodes show that the main control of internode elongation is not linked to the creation of an osmotic pressure potential but rather to the maintenance of plasticity and extensibility of the cell wall. This is aligned with the data showing that down-regulation of DELLA proteins (a negative regulator of GA signalling) increases internode elongation (Tavares et al. 2018). It would appear that the application of Moddus did not suppress the ability of the tissue to break down sucrose.

The main driver of biomass input into the system (R1) is the volume of the tissue. Not only does a larger volume require more osmolytes to maintain the required water potential gradients but the expanding cells must synthesise more cell wall, cell membrane, proteins and all the precursors to maintain cellular functions. This additional biosynthesis requirement is captured in two main demand functions highlighted in Fig. 7. The flux to meet these demands are captured in (R7, R8) and extra energy demand with (R9, Fig. 7). The source to meet these demands is the hexose/hexose-P pools.

Because of the significantly smaller demand driven flux through R7, R8 and R9 in the Moddus-treated internodes, hexose and sucrose levels dominate. In contrast, application of GA induce expression of many genes associated with R7, R8 and R9 (Chen et al. 2020). Previous work has shown that disruption of the sucrose hydrolysis (Rossouw et al. 2010) or flow through glycolysis (R8) (Groenewald and Botha 2008) leads to sucrose accumulation but stunted growth.

From previous work, it is known that there is also a significant “futile cycle” of carbon between the hexose phosphate and triose phosphate pools (Bindon and Botha 2002; van der Merwe et al. 2010; van der Merwe and Botha 2013). These apparent futile cycles probably are important in ensuring, carbon partitioning into the other competing metabolic sinks including the respiratory pathways, and synthesis of organic acids and amino acids, proteins and cell walls (Whittaker and Botha 1997; Bindon and Botha 2002).

The flow of carbon towards the two metabolic sinks dominates carbon partitioning and hence sucrose accumulation. R10 primarily functions to maintain osmotic equilibrium between the cytosol and vacuole. Because of the symplastic loading of sucrose (R1), sucrose will continue to enter the cytosol of the parenchyma tissue if a sucrose gradient exists between the parenchyma cells and the phloem. The phloem sucrose concentrations can exceed 700 mM. In internode 6 of the Moddus treatment, the sugar concentration is much higher than in the control treatment and would explain the significantly lower flux (R1) between the treatments.

As internode expansion decreases, the flux of sucrose to the apoplastic space (R11) will increase driven by the concentration gradient and turgor-driven homeostatic leakage.

In the stem, as sucrose begins to accumulate, some of it is moved to the apoplast of stem parenchyma cells by turgor-driven homeostatic leakage. The apoplastic sucrose concentration can reach approximately 400–700 mM (Moore and Cosgrove 1991; Welbaum et al. 1992; Patrick et al. 2013; Welbaum 2013). The sucrose accumulation in the apoplast creates additional demand to increase sink strength. Sucrose backflow into the phloem is prevented by the lignified and suberized cell walls of the inner, mestome sheath (a ring of thick-walled cells that surrounds the vein internally to the bundle sheath cells (Welbaum and Moore 1990; Patrick et al. 2013; Welbaum 2013).

The results from this study demonstrate that sink size is a key driver for culm elongation and biomass accumulation. There are two processes that determine the growth of the internodes. The first is the rate in which the internode expand and evidently GA is an important factor in the control of this parameter. The second is the duration of internode elongation which seems to be independent of GA control, related to thermal time and probably strongly linked to the deposition of lignin and the secondary cell wall. Once the secondary wall is laid down, pressure builds up in the tissue and this curtail further cell expansion.

The data suggest that breeding strategies to increase sugarcane biomass should therefore focus on two issues: firstly, extending the duration of internode elongation, i.e., slow down secondary cell deposition and lignin biosynthesis; secondly, increasing the rate of internode elongation to achieve larger internodes prior to the deposition of a rigid secondary cell wall structure.