The importance of UDP-G control and its effect on C partitioning has been indicated in several studies of plant systems, whereby the kinetic properties and abundance of enzymes involved in UDP-G synthesis/utilisation had a significant effect upon the volume of C moving into the hemicellulose, cellulose and sucrose pools [13,14,15,16,17,18,19]. UDP-G metabolism is central to key sources of C deposition in the sugarcane plant, therefore is likely that the expression of genes involved in UDP-G metabolism would have differed expression in relation to how C is utilised in a given tissue/organ. Additionally, most of these genes are known to be in multi-gene families, and many of these multi-gene families likely contain isoforms that overall have a larger effect on metabolism than others. By determining tissue-specific isoforms, gene candidates for altering UDP-G metabolism and by proxy C metabolism could be identified, in order to define the strategies to modify plant biomass. Here, to provide a broad overview of differences in organ-specific expression of genes associated with the UDP-G metabolism, we analysed their expression in 3 major organs of the sugarcane plant including leaves (mature source organ), internodes (young and mature sink organ) and root (meristematic sink organ).
Expression of genes associated with the UDP-G metabolism in internodes
Sucrose synthesis and degradation in internodal samples by the SPP, SPS and SuSy enzymes (Fig. 2), is closely linked to sugarcane internodal maturity, whereby sucrose cleavage and hydrolysis prevails in immature internodes and sucrose synthesis and lack of sucrose utilization into respiration and insoluble components prevails in mature internodes . As indicated in this study and previous studies of SuSy genes, expression and enzymatic activity are significantly higher in immature internodes [47, 48], likely providing C for hemicellulose and cellulose synthesis from UDP-G. Interestingly, within the SuSy gene family, the highest expression was limited to 2 of the 4 SuSy genes (SuSy 1 and 2) indicating that they may code for enzymes that have heightened importance in the cleavage of sucrose into UDP-G (Fig. 2a), as has been reported in Arabidopsis and Sugarcane [30, 48, 49]. As expected, enhanced expression of SuSy in immature internodes, also coincided with higher expression in cell wall synthetic related gene families, including CesA, CSL (A, C, D and F families, see Fig. 3), GALE, UAXS, UGE, UXE and UGD (Fig. 4). Some of the aforementioned genes code for enzymes that directly consume UDP-G, including UGD, CesA and some CSL gene families [50,51,52], in essence having direct competition with sucrose synthetic enzymes. The key genes coding for enzymes that are responsible for the bulk of UDP-G into cellulose and hemicellulose pools are likely coded for by UGD 5 (Fig. 4a), and CesA subunit genes 1–1, 1–2, 3, 5, 7 and 8 (Fig. 2a). In a related study in the UGD gene family in Arabidopsis, an enzyme isoform with high affinity for UDP-Glucose, also had high affinity for the downstream product UDP-xylose, which acts as a feedback inhibitor , The associated genes also had higher expression, suggesting they code for enzymes that hold a primary role in the assimilation of C into the hemicellulose pool in immature tissues. Other UGD gene isoforms with lower volumes of expression likely code for enzymes with low substrate affinity and operate in the background throughout the sugarcane plants lifecycle. This concept could credibly be applied to most gene families in this study.
In conjunction with the heightened sucrose cleavage to UDP-G, cumulative invertase expression in CINV and CWI gene families was significantly higher in immature internodes in comparison to mature internode samples (Fig. 4b and c, respectively). This was expected as sucrose hydrolysis into reducing sugars characterizes the first step of C movement into several pathways, leading into cell wall, protein, respiratory and other secondary metabolite pools . The heightened levels of reducing sugars in immature internodes , indicates the activity of invertases hydrolysing imported sucrose. The heightened expression of the CWI 1 and 7 genes in the immature internodes (Fig. 4c), suggests prominent activity of C fixation into parenchyma tissue from conducting tissues, and the apoplastic/symplastic transfer of sucrose [54, 55]. Lower expression of CWI in mature internodes may suggest that C importation facilitated by the activity of invertases is less pronounced. The heightened expression of genes encoding phosphorylating enzymes, FK and HXK in immature internodes (Fig. 7e and f), particularly genes HX 3 and 5 and FK 2, further supports the notion of high C movement likely toward pentose phosphate pathway and glycolysis, as reviewed by . The indication of enhanced C flux into these pathways that may be mediated by gene expression is important, as results from  suggested very little difference in expression of transcripts associated with these pathways, which may suggest associated enzymes operate during favourable metabolic conditions, i.e. when metabolites are available. This indicates the potential importance of invertases and phosphorylating enzymes in releasing C to the pentose phosphate and glycolytic pathways.
Unexpectedly, in the sucrose synthetic gene families, SPS and SPP, between the mature and immature internodes, expression was higher in immature internodes (Fig. 2b and c), which is in contradiction to several studies that have found an opposite trend in enzymatic activity of SPS [47, 57]. It must be noted that enzymatic activity and gene expression are not necessarily correlated . Further, another study has found similar results, whereby enzymatic activity was higher in immature internodes. The results from this study may indicate that the absence of competition for C in mature internodes by sucrose degradative and downstream enzymes, is the key to sucrose accumulation in mature internodes, as has been indicated in several studies [47, 59]. In a previous study by Botha and Black (2000), it was intimated that there could be an additional kinetic form of SPS that allows for heightened enzymatic activity, which explains the heightened activity of SPS in mature internodes , however, there is no evidence of increased transcription in any of the 5 SPS isoforms in mature internodes in our study. These results suggest enzymatic activity of SPS is determined beyond transcription.
Expression of genes associated with the UDP-G metabolism in roots
Root and immature internodes are both meristematic sinks, moving a large proportion of fixed C into the cell wall, protein and respiratory pools. Despite this, there were large expressional differences between root and immature internode samples likely indicating spatial regulation of specific gene family isoforms. Some of the transcriptional differences between the two meristematic sinks is likely a result of the difference in age the samples were taken from the sugarcane plant (roots from a 3-month-old plant and immature internodes from a 9-month-old plant). However, previous compositional analyses of sugarcane roots display a differing requirement for fixed C than immature internodes [32, 60]. Compositional analyses of 3-month old sugarcane roots indicated differences in simple sugar and hemicellulosic monomer content in comparison to immature internodes . Also, in immature internodes, there is an underlying trend for the accumulation of sucrose that does not exist in roots . Concerning the simple sugar content, i.e. fructose, glucose and sucrose, it was postulated that the reduced levels in the root sample indicate the efficient breakdown and movement of fixed C into the cell wall, protein, organic acid and respiratory pools in roots. This is made clear by the high expression of VINV in roots (Fig. 4d), which has been implicated as a key enzyme negatively affecting the accumulation of sucrose in several plant systems, as reviewed by ; and the heightened expression of SPS 1 and 4 genes (only significant in Q208 genotype, see Fig. 2c). In this study, low VINV expression in all internodes and leaf samples, indicating low VINV activity (Fig. 4d), likely allows the accumulation of sucrose for storage or transportation. Higher observed expression of VINV in roots suggests a lower requirement for sucrose bioaccumulation. Additionally, the high expression of two other invertase families in roots in comparison to immature internodes, including, CINV and CWI may also likely indicate a low inclination for sucrose bioaccumulation in roots (Fig. 4b and c). The higher expression of CWI isoforms 1, 2, 5 and 7 in root samples in comparison to immature internodes suggest a heightened role for CWI in roots, may enable an increase in hydrolysis of apoplastic sucrose, which in turn ensures a steep concentration gradient enhancing sucrose delivery to roots from mature leaves . Interestingly, in sugarcane, the activity of CWI in internodes is correlated with higher sucrose levels [63, 64]. Enhanced invertase activity is counterintuitive to enhanced sucrose levels, however, the sucrose cleavage and resynthesis model as proposed by Glasziou and Gayler , may explain this. Higher expression of CWI genes in roots suggests the intercellular sucrose cleavage and intracellular resynthesis model to not be relevant in the root sample, as there is no evidence for large degrees of sucrose resynthesis to be occurring . This notion is further supported by the lower expression of SPS observed in roots (only significantly different in Q208 genotype, Fig. 2c) in comparison to immature internodes and leaf samples.
Corresponding expression of SuSy genes between immature internodes and root samples likely indicates a high degree of UDP-G formulation from sucrose (Fig. 2a), followed by C utilisation into cellulose and hemicellulose pools. The expression of genes associated with UDP-G into cell wall polysaccharides differed greatly between immature internodes and root samples. Heightened expression of an additional UGD gene family isoform (UGD 4), and the MIOX gene suggests there may be enhanced enzymatic activity indicating the strong demand of C to be moved into the hemicellulose fraction in roots or an organ specific function (Figs. 4a and 5d, respectively). Evidence of heightened expression in a specific UGD isoform has been reported in Arabidopsis seedlings . Additionally, downstream steps of hemicellulose synthesis displayed a significant difference between these two samples, which could be responsible for the heightened amount of arabinose and galactose mixed linkages in roots , particularly GALE 1 and UXE 2 (Fig. 4f and c). Although, as shown in related analyses, there was also higher expression in other hemicellulose related transcripts in roots, that do not result in differences in composition [32, 39]. Interestingly, CesA expression was significantly higher in immature internodes (Fig. 3a). The significant differences in gene expression related to hemicellulose and cellulose synthesis could be related to the presence of specialised cells in both roots and internodes [66,67,68], having different requirements for C, or differing metabolic conditions, i.e. access to substrates or the presence of feedback inhibitory molecules . Notably, compositional analysis of roots and internodes, as presented in , displayed no difference in the ratio of hemicellulose and cellulose, indicating differences in expression of related genes may not affect the fixed nature of the cell wall component ratios.
Expression of genes associated with the UDP-G metabolism in leaves
The expression of genes related to the UDP-G metabolism in sugarcane leaves is connected to the status of this organ as a net exporter of C in the form of sucrose. As expected, cell wall-related genes that directly synthesize or consume UDP-G, including SuSy, CesA, CSL and UGD gene families, had insignificant amounts of expression in the leaf samples, indicating a transcriptional regulation as a means of ceasing C flow into the cell wall pool in this organ. Interestingly, some CSL gene family groups (Fig. 3) had significant expression in leaves including CSLE, CSLG and CSLH, specifically CSL6–2, CSLG2 and CSLH1–1 genes (Fig. 3e, g and h, respectively). CSL enzymes are responsible for the transfer of UDP-G to 1–3 and 1–4 β-glucan, or the transfer of other nucleotide sugars to form other β-linked backbones, within the hemicellulose fraction. Most CSL genes had significantly higher expression in both meristematic/immature sink samples, which was expected due to the requirement for hemicellulose synthesis. It is unclear why there was significant expression of some CSL gene family groups in leaf samples. Of the CSL gene groups with significant expression in leaves, CSLE and CSLG have an unknown function (Fig. 3e and g), as reviewed by , although it is likely still associated with hemicellulose synthesis, whereas the CSLH group (Fig. 3h) encodes mixed linkage glucan synthases . These CSL groups may have a heightened requirement in leaf sample during development constructing leaf specific structures, with different arrangements of cell wall compounds. The data would also suggest that expression of these genes are retained throughout maturity exclusively in leaf samples, and may also have a role in maintenance.
Unlike most cell wall-related gene expression, high expression of sucrose synthetic genes SPS and SPP (Fig. 2b and c), equivalent to both meristematic samples were observed in leaf sample. Expression of SPP 1 and SPS 1 was most prominent in the leaf (although not significantly different from other samples), which may suggest these specific genes have an enhanced role in leaf sample. This trend has been hypothesised to be due to pronounced role of SPS and SPP in the synthesis of sucrose in source tissues [71, 72]. Relevant to sucrose biosynthesis in leaves is the production of UDP-G which is likely primarily derived from the activity of UGPase which transfers glucose-1-phosphate to UDP-G, whereas in sink samples UDP-G synthesis is derived primarily from sucrose cleavage by SuSy . In support of this notion, transcription of SuSy related genes (Fig. 2a) was significantly higher in meristematic sinks. However, despite the primary the role of UGPase in sucrose biosynthesis in leaves, there was not higher expression in related gene family isoforms (Fig. 7a). This suggests the pronounced role of UGPase in source samples is not determined at the transcriptional level, but at the metabolic level, likely via the availability of hexose phosphates . As indicated by the low expression of SuSy and high expression of SPS and SPP in leaves (Fig. 2c and b), this suggests a bias toward sucrose synthesis in this source organ, which will then be transported to various sink organs. However, the high expression of some invertase genes in the CWI (CWI 4), and ANINV (ANINV 1–1 and 3) gene families suggest sucrose hydrolysis to be a major competing sink for C (Fig. 6c and a, respectively). In a previous experiment of photosynthetic regulation by sugars in sugarcane leaves, fed radiolabelled sucrose was rapidly converted into hexoses, which was stipulated to be due to the activity of SuSy and invertase enzymes . However, based on the low expression of SuSy genes in source organs in this study, the rapid conversion of sucrose may be mostly derived from invertase activity in leaves. The heightened activity of some invertases in sugarcane leaves suggests a major role in the regulation of sugar levels, especially due to the inhibitory nature of high sucrose concentrations on photosynthetic activity [76, 77]. In the case of the CWI gene family, most expression in leaves was contributed by the CWI 4 gene (Fig. 6c), which was also significantly higher than other organ types, which suggests an organ-specific function for this gene, potentially as a regulator of sucrose levels. It must be noted that as leaf organ is highly metabolically active and is a protein-rich organ , there is likely still an underlying requirement for C to be moved into respiratory and protein fractions. It is possible that invertase activity is contributing C to these pools.