Organ-specific expression of genes associated with the UDP-glucose metabolism in sugarcane (Saccharum spp. hybrids)

doi:10.21203/rs.3.rs-1452946/v1

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Research Article

Organ-specific expression of genes associated with the UDP-glucose metabolism in sugarcane (Saccharum spp. hybrids)

https://doi.org/10.21203/rs.3.rs-1452946/v1

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published 13 Jan, 2023

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Background: The importance of uridine 5′-diphosphate glucose (UDP-G) synthesis and degradation on carbon (C) partitioning has been indicated in several studies of plant systems, whereby the kinetic properties and abundance of involved enzymes had a significant effect upon the volume of C moving into the hemicellulose, cellulose and sucrose pools. In this study, the expression of 136 genes belonging to 31 gene families related to UDP-G metabolism was studied in 3 major sugarcane organs (including leaf, internode and root) at 6 different developmental stages in 2 commercial genotypes.

Results: Analysis of the genes associated with UDP-G metabolism in leaves indicated low expression of sucrose synthase, but relatively high expression of invertase genes, specifically cell-wall invertase 4 and neutral acid invertase 1-1 and 3 genes. Further, organs that are primarily responsible for sucrose synthesis or bioaccumulation, i.e. in source organs (mature leaves) and storage sink organs (mature internodes), had very low expression of sucrose, cellulose and hemicellulose synthesis genes, specifically sucrose synthase 1 and 2, UDP-G dehydrogenase 5 and several cellulose synthase subunit genes. Gene expression was mostly very low in both leaf and mature internode samples; however, leaves did have a comparatively heightened invertase and sucrose phosphate synthase expression. Major differences were observed in the transcription of a large number of genes between immature sink organs (roots and immature internodes). Gene transcription favoured utilisation of UDP-G toward insoluble and respiratory pools in roots. Whereas there was comparatively higher expression of sucrose synthetic genes, sucrose phosphate synthase 1 and 4, and comparatively lower expression of many genes associated with C flow to insoluble and respiratory pools including myo-Inositol oxygenase, UDP-G dehydrogenase 4, vacuolar invertase 1, and several cell-wall invertases in immature internodes.

Conclusion: This study represents the first effort to quantify the expression of gene families associated with UDP-G metabolism in sugarcane. Transcriptional analysis displayed the likelihood that C partitioning in sugarcane is closely related to the transcription of genes associated with the UDP-G metabolism. The data presented may provide an accurate genetic reference for future efforts in altering UDP-G metabolism and in turn C partitioning in sugarcane.

UDP-Glucose metabolism

Sugarcane

Sucrose Synthase

UDP-Glucose Dehydrogenase

Invertase

Sucrose Phosphate Synthase

myo-Inositol Oxygenase

Internodes

Roots

Leaves

In plants, uridine 5′-diphosphate glucose (UDP-G) is a nucleotide sugar that is consumed irreversibly to produce the major components of the cell wall, cellulose and hemicellulose [1–4]. UDP-G can also be degraded into sucrose then resynthesized [5–7]. As UDP-G is directly synthesised into/or degraded from sucrose, it is likely that this nucleotide sugar’s consumption is also linked to lignin production, which occurs via the hydrolysis of sucrose leading into the glycolytic and oxidative pentose phosphate pathways [8]. Other roles for UDP-G in plants include its role as a glucose residue donor in callose formation, glycoproteins, glycolipids and sulpholipids, and its essential role in the glycosylation of steroids, flavonoids, betalains, glucosinolates and terpenoids [9]. Further, several studies have suggested a central role for UDP-G as a signal molecule regulating growth and development, biomass accumulation, and programmed cell death [8, 10–12]. The synthesis and degradation of the UDP-G metabolite are central to the amount of carbon (C) moving into the major pools within the sugarcane plant, specifically sucrose, cellulose and hemicellulose pools [7]. 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 insoluble and soluble pools [8, 13–19], see Fig. 1 for a summary.

A gene family is a set of functionally similar genes formed via whole genome duplication and derived originally from a single ancestral gene [20]. The value for plant systems having developed multiple gene isoforms coding for the same enzyme is that often small alterations to enzyme structure can confer differing affinities to substrates and feedback inhibitory molecules, meaning the flux through this point in the pathway can continue in a variety of cellular conditions [4, 21, 22]. The value in understanding the full array of gene family isoforms was shown in an attempt to downregulate an important consumer of UDP-G into the hemicellulosic precursor UDP-glucuronic acid, via the activity of UDP-G dehydrogenase (UGD) in sugarcane. A sugarcane genetic knockout mutant of a single UGD gene was created in [23]. Cessation of UGD activity was expected to have a reductive effect on the hemicellulose fraction, however, no differences in composition were observed. Upregulation of the myo-inositol pathway, which provides an alternative pathway for C to enter hemicellulose synthesis, was detected in the mutant, which was used to explain the lack of compositional difference in comparison to the control [23]. However, based on previous characterisations of the UGD gene family in other plants [4, 21], it is highly unlikely that there is a single UGD gene in the sugarcane genome, which may also have contributed to the reasons why no difference in cell wall composition was recorded in the UGD single mutant. Therefore, in sugarcane, successful alterations of UDP-glucuronic acid synthesis is likely contingent on changes to genes associated with the myo-Inositol pathway, but also the identification and alteration of additional UGD genes. In a related study within the Arabidopsis genome [21], of the 4 UGD genes, only double genetic knockout mutants of UGD 2 and UGD 3 genes combined led to reduced UDP-glucuronic acid synthesis. Suppression of UDP-glucuronic acid synthesis caused a significant decrease in the hemicelluloses arabinose, xylose, apiose and galacturonic acid, in turn causing dwarfism and other developmental defects in the double mutants [14]. Single knockout mutants of UGD 2 or UGD 3 did not affect the cell wall composition, which indicates the activity of only one of these genes is sufficient to make up for the lack of expression in the other, whilst the other isoforms likely had organ-specific functions. Studies of multi-gene families related to the UDP-G metabolism have reported similar results, whereby knockout mutants of 1, 2 or even 3 isoforms were found to have a disproportionate effect on the physiology of the mutant, particularly alterations to the cell wall [15, 24–29].

Modern high throughput technologies such as next-generation sequencing (NGS) allow a platform to look broadly at differences in gene expression in different tissues and organs [30]. In this study, to study the organ-specific expression of genes associated with the UDP-G metabolism in the sugarcane plant, we used mature sugarcane leaves (dedicated source organ), young internodes (sink organ where the bulk of UDP-G and hexose phosphates are utilised in cellulose, hemicellulose, protein synthesis and respiration), maturing and mature internodes (sink organ where respiration is limited and UDP-G is utilised in sucrose storage) and root (a meristematic sink that has similar C requirements to immature internodes) [31].

Plant material collection, data generation and transcript profiling

The plant material used in this experiment is derived from two commercial sugarcane genotypes, KQ228 and Q208 provided by Sugar Research Australia (SRA). Leaf and internode samples of both genotypes were taken from 9-month old commercial stands. Root samples were obtained from 3-month old research plants in ‘soft’ above-ground pots. For sampling, schematic see [31]. Briefly, a total of 6 samples were collected at different developmental stages from three major sugarcane organs, including internodes, leaves and root. Hereafter, these samples are referred to as TI (Top Internode), MI (Middle Internode), BI (Bottom Internode), L1 (1st Visible Dewlap Leaf), L5 (5th Visible Dewlap Leaf) and R (Root). All samples were collected in triplicate and ‘snap frozen’ in liquid nitrogen within a minute of excision, put on dry ice in transit and kept in a -80°C freezer before preparation. Before RNA extraction samples were homogenized in cryogenic conditions. RNA extractions were conducted using the combined Trizol kit and RNeasy Plant minikit, as described in [32]. Samples were prepared with Illumina TruSeq™ stranded total RNA library prep kit, using 1000ng of the submitted RNA, by QBI (Queensland Brain Institute, University of Queensland, St Lucia QLD, 4072). Each sample was sequenced in 3 lanes using an Illumina HiSeq2000 instrument to obtain 125bp paired-end reads. Read data was assessed using FastQC [33], to determine the quality and adapter sequences. A full-length sugarcane Iso-seq transcriptome database (SUGIT) developed by Hoang et al. 2017, was utilised as the reference sequence [34], in the assessment of transcript specific gene expression. The pipeline used to profile the transcriptome of the multiple samples in this experiment is based on the protocol ‘Expression Analysis using RNA-seq’ by QIAGEN bioinformatics [35] in the CLC Genomics Workbench version 12 (CLC-GWB, CLC Bio-QIAGEN, Aarhus, Denmark). The CLC-GWB analyses were conducted on a CLC Genomics Server, the CLC server, nodes and CLC-clients which are part of the Robert Henry Bioinformatics infrastructure at QAAFI, The University of Queensland, Australia. All CLC infrastructure was hosted by the Research Computing Centre (RCC), based at the University of Queensland, Australia [36]. For further details of plant collection and data generation, details see [37].

Extraction of transcripts of interest and subsequent matching to gene family isoforms

A search for all transcripts associated with gene families related to the consumption/synthesis of the UDP-G metabolite was conducted in the sugarcane SUGIT transcriptome. Using CLC-GWB, relevant search terms, i.e. “sucrose synthase”, were queried against the SUGIT transcript annotations, to derive lists of relevant transcripts, which were extracted as FASTA files. Additional mapping was undertaken using the large-gap mapping tool in CLC-GWB (length fraction of 0.8 and similarity fraction of 0.5), whereby genes of interest from the Sorghum bicolor and Zea mays genomes were mapped against the SUGIT transcriptome, to ensure any mislabeled transcripts were included in downstream analysis. All extracted transcripts were blasted against the S. bicolor genome (accession number NC_012875.2) using NCBI’s nucleotide Basic Local Alignment Search Tool (BLAST) [38], with default settings in blastn, to group transcripts into their respective gene family homologues. The top BLAST hit based on query coverage and per cent identity was used to define the transcripts gene identity.

Genomic location and gene length determination using the sugarcane STP monoploid genome

The sugarcane monoploid genome, a 382-Mb high-quality single-tiling-path (STP) sequence [39], enabled the genomic location and gene length to be determined in most of the genes of interest. The sequences of the genes of interest were also extracted and mapped to the extracted SUGIT transcripts (in CLC-GWB) to ensure paralogous genes (derived from recent genome duplications) were assigned the correct transcripts. To find the genes of interest within the sugarcane monoploid genome, the coding region of equivalent sorghum genes were blasted against the STP sequence. Sequences with high degrees of similarity were extracted then blasted against the sorghum genome to ensure the manual annotation was correct.

Collapsing transcripts into single expression value

The large degree of alternative splices and redundancy within the SUGIT reference transcriptome could reduce the accuracy of expression comparisons between the sugarcane organ types included in this study. Collapsing the normalised read counts (normalised using transcript per million (TPM) calculation) of all transcripts related to a gene, into a single value was deemed to be a far better option for determining the importance of specific gene family homologues, as the more isoforms a specific gene has in a reference transcriptome the less accurate differential gene expression comparisons will be [40]. The RNA-seq application within CLC-GWB assigns reads exclusively to the reference, which means in the case of a read sharing the same identity with two reference sequences it will be assigned to one of the references randomly. Due to this, quantifying the expression of a gene that has multiple isoforms, was simply done by adding the TPM values together to form a single TPM value (Figure S1).

Data processing of gene family expression values

The statistical significance of the mean of the biological replicates was calculated utilising the one-way ANOVA function, with the additional Tukey’s post-hoc t-test (honestly significant difference, HSD test) within IBM SPSS Statistics v27 (SPSS Inc, Chicago, IL, USA). The null hypothesis was accepted at a p-value of > 0.05. Minimum and maximum outliers (two-sided) were removed from some datasets using the modified Thompson Tau test [41]. For one-way ANOVA and Tukey t-test values, see Table S1 and Figures S2-S10.

Identification of gene families associated with UDP-glucose metabolism in sugarcane

A total of 136 genes from 31 gene families associated with the UDP-G metabolism, were identified within the STP sugarcane monoploid genome (see Fig. 1 for a depiction of the associated pathway). Most of these genes were also represented by 560 transcripts in the long-read SUGIT transcriptome. It must be noted that some of the transcripts represented in the SUGIT transcriptome were not represented in the STP genome, which accounted for 27 genes (Table 1). Comparisons to equivalent genes in the S. bicolor genome identified several likely gene duplications in ADP-glucose pyrophosphorylase (AGP), cellulose synthase-like (CSLE), cytosolic invertase (CINV), sucrose synthase (SuSy), and UDP-G-4-epimerase (GALE) gene families in the sugarcane genome, otherwise known as paralogues [42, 43]. Among the 136 genes, 10 gene homologues/isoforms belonged to the AGP gene family. Within the cellulose synthase (CESA) and cellulose synthase-like (CSL) subfamilies, 10 genes belonged to the CESA subgroup, and 5, 4, 4, 7, 2, 1 and 2 genes in CSL sub groups A, C, D, E, F and H, respectively. Of the four invertase (INV) sub-families, we identified 3 CINV genes, 5 alkaline/neutral (AN) INV, 1 vacuolar (V) INV and 8 cell wall (CW) I. Note that CINV and ANINV are both known as “neutral invertases”, however, they have been annotated as different genes in the S. bicolor genome. Gene families associated with the myo-inositol pathway including glucuronokinase (GluK), myo-inositol phosphatase (MIP) and myo-inositol oxygenase (MIOX) each only had a single gene within the monoploid genome, whilst the MIPS had two gene family members. Gene families associated with hemicellulose synthesis had varying numbers of associated gene family members. A single gene was identified for uridine diphosphate glucose apiose/xylose synthase (UAXS), whilst multiple genes were identified in the UGD, GALE, UDP-xylose synthase (UXS), UDP-xylose epimerase (UXE), UDP-G 4,6-dehydratase (RHM) and uridine diphosphate glucuronic acid epimerase (UGE) gene families, with 3, 5, 6, 3, 3, 4 members, respectively. Single chloroplastic, cytoplasmic and 2 bi-functional phosphoglucomutase (PGM) genes were identified, while 5 UDP-G pyrophosphorylase (UGPase) genes, 3 glucose-6-phosphate isomerase (G6PI), 8 hexokinase (HXK) and 3 fructokinase (FK) genes were also identified. Some genes had large numbers of transcripts represented in the SUGIT transcriptome with MIPS 2, SuSy 1, SuSy 2, SuSy 4, UGD 4, UGD 5, UGPase 2, CesA 5, CSLE 6 − 2, FK 2, and GALE 1 all had over 15 representative transcripts each.

Table 1

**Ordered list of gene families and homologues found within the sugarcane monoploid genome associated with UDP-glucose metabolism.** The No. of transcripts in SUGIT transcriptome column numbers with an asterisk indicates shared transcripts as the sequences were too similar to tell apart. “Not in STP only found in BAC sequences” indicates the presence of a said gene in BAC sequences although it was not included in the STP sequence.
Gene family	Gene name	Gene identifier in sugarcane genome (Garmeur et al. 2018) [39]	Position in the sugarcane genome	Length	Sorghum NCBI transcript sequence accession No. & link	No. of transcripts in SUGIT transcriptome
ADP-Glucose pyrophosphorylase	AGP 1–1	Sh03_t015880	Sh03:28291165..28294172 (+ strand)	3008	XM_002455967.2	2*
	AGP 1–2	Sh03_t015870	Sh03:28260610..28265076 (+ strand)	4467	XM_002455967.2	2*
	AGP 2 − 1	Sh09_t019370	Sh09:34396677..34402313 (+ strand)	5637	XM_021448378.1	12*
	AGP 2–2	Sh01_t024650	Sh01 Sh01:42078537..42080613 (- strand)	2077	XM_021448378.1	12*
	AGP 2–3	Sh01_t024670	Sh01 Sh01:42115922..42117998 (- strand)	2077	XM_021448378.1	12*
	AGP 3	Sh02_t005750 & Sh02_t005760	Sh02 Sh02:9491664..9495697 (- strand)	3784	XM_021452431.1	0
	AGP 4	Not found in monoploid genome	n/a	n/a	XM_021465103.1	6
	AGP 5	Not found in monoploid genome	n/a	n/a	XM_002462095.2	6
	AGP 6	Not found in monoploid genome	n/a	n/a	XM_002463876.2	4
	AGP 7	Not found in monoploid genome	n/a	n/a	XM_021465103.1	2
Cellulose Synthase	CesA 1–1	Sh09_t005560 & Sh09_t005570	Sh09:9351086..9355687 (- strand)	6842	XM_002440649.2	11
	CesA 1–2	Sh03_t003840	Sh03:6782646..6790013 (+ strand)	7368	XM_002455055.2	7
	CesA 2	Not found in monoploid genome	n/a	n/a	XM_021446681.1	3
	CesA 3	Not found in monoploid genome	n/a	n/a	XM_021453750.1	14
	CesA 4	Sh03_t021020 & Sh03_t021030	Sh03:36782684..36788213 (+ strand)	5529	XM_002456316.2	6
	CesA 5	Sh10_t013650	Sh10:25483024..25488997 (+ strand)	5974	XM_021450574.1	19
	CesA 6	Sh02_t006050	Sh02:10077014..10082433 (+ strand)	5420	XM_002459590.2	8
	CesA 7	Sh01_t017850	Sh01:28983708..28987579 (- strand)	3872	XM_002467064.2	6
	CesA 8	Sh_229N08_t000100	Not in STP only found in BAC sequences	5414	XM_021452948.1	4
	CesA 9	Sh02_t013200	Sh02:25312245..25316453 (+ strand)	4209	XM_002460184.2	8
Cellulose Synthase-Like A	CSLA 1	Sh04_t005720	Sh04:8606968..8613408 (- strand)	6441	XM_002453415.2	2
	CSLA 2	Sh01_t020080	Sh01:32953742..32958781 (+ strand)	5040	XM_021451851.1	0
	CSLA 3	Sh02_t009410	Sh02:16752941..16756026 (+ strand)	3086	XM_002459829.2	0
	CSLA 6	Sh04_t016980	Sh04:29898814..29901757 (+ strand)	2944	XM_021458643.1	0
	CSLA 7	Sh02_t028710	Sh02:48277289..48281992 (+ strand)	4704	XM_002461027.2	1
Cellulose Synthase-Like C	CSLC 1	Sh03_t022070	Sh03:38066914..38070773 (- strand)	3860	XM_021457183.1	0
	CSLC 2	Sh_029O19	Not in STP only found in BAC sequences	2475	XM_021454662.1	1
	CSLC 7	Sh09_t015100	Sh09:28563626..28566886 (+ strand)	3261	XM_021447983.1	1
	CSLC 9	Sh01_t005210	Sh01:7634941..7643711 (+ strand)	8771	XM_021448173.1	3
Cellulose Synthase-Like E	CSLE 2	Sh04_t018310	Sh04:31810139..31814041 (- strand)	3903	XM_021460485.1	0
	CSLE 6 − 1	Sh02_t015890	Sh02:30256283..30259442 (- strand)	3160	XM_002462490.2	7
	CSLE 6-2a	Sh02_t015810	Sh02:30148511..30154022 (- strand)	5512	XM_002462489.2	15*
	CSLE 6-2b	Sh02_t015820	Sh02:30158577..30163879 (- strand)	5303	XM_002462489.2	15*
Cellulose Synthase-Like F	CSLF 1	Sh02_t024280 & Sh02_t024290	Sh02:42020371..42021308 (- strand)	1226	XM_002462951.2	0
	CSLF 3 − 1	Sh02_t024300, Sh02_t024310 & Sh02_t024320	Sh02:42028378..42030117 (- strand)	2191	XM_021452870.1	2*
	CSLF 3 − 2	Sh02_t024330	Sh02:42047848..42050979 (- strand)	3132	XM_021452421.1	2*
	CSLF 3–3	Sh02_t024380, Sh02_t024390 & Sh02_t024400	Sh02:42134676..42135020 (- strand)	2754	XM_021454665.1	2*
	CSLF 6	Not found in monoploid genome	n/a	n/a	XM_002445057.2	5
	CSLF 8	Not found in monoploid genome	n/a	n/a	XM_021454445.1	2
	CSLF 9	Sh02_t024360 & Sh02_t024370	Sh02:42109930..42111354 (+ strand)	3286	XM_021452834.1	0
Cellulose Synthase-Like D	CSLFD 1	Sh01_t021550	Sh01:36986329..36990074 (- strand)	3746	XM_002467380.2	2
Cellulose Synthase-Like D	CSLFD 2	Not found in monoploid genome	n/a	n/a	XM_002436311.2	2
Cellulose Synthase-Like G	CSLG 2	Sh03_t033750, Sh03_t033760, Sh03_t033770 & Sh03_t033780	Sh03:54594123..54601044 (- strand)	6921	XM_021455434.1	1
Cellulose Synthase-Like H	CSLH 1–1	Not found in monoploid genome	n/a	n/a	XM_021463400.1	1
Cellulose Synthase-Like H	CSLH 1–2	Not found in monoploid genome	n/a	n/a	XM_021463399.1	1
Cytosolic Invertase	CINV 1–1	Sh04_t018360	Sh04:31829368..31832935 (+ strand)	3568	XM_002452587.2	5*
	CINV 2	Sh04_t002850	Sh04:3999533..4003239 (- strand)	3707	XM_002453920.2	1
	CINV 1–2	Sh02_t022210	Sh02:39433217..39439275 (- strand)	6059	XM_002452587.2	5*
Alkaline/ Neutral Invertase	ANINV F1	Not found in monoploid genome	n/a	n/a	XM_002450357.2	0
	ANINV F2	Sh04_t003010	Sh04:4348910..4351981 (+ strand)	3072	XM_002451407.2	3
	ANINV 1–1	Sh01_t031210	Sh01:52365269..52368792 (+ strand)	5203	XM_002465314.2	1
	ANINV 1–2	Sh03_t010910	Sh03:18378830..18382742 (+ strand)	3913	XM_002455539.2	7
	ANINV 3	Sh04_t011120	Sh04:20023276..20026209 (+ strand)	2934	XM_002452150.2	3
Cell Wall Invertase	CWI 1	Sh_226D11	In peripheral contigs (Sh_226D11:88740..93318)	4579	XM_021459751.1	6*
	CWI 2 − 1	Sh06_t003700	Sh06:8281155..8284931 (+ strand)	3,777	XM_002489067.2	1*
	CWI 2–2	Sh06_t003690	Sh06:8271558..8274605 (- strand)	3048	XM_002489061.2	1*
	CWI 2–3	Not found in monoploid genome	n/a	n/a	XM_002489065.2	1*
	CWI 3	Sh01_t007690 & Sh01_t007700	Sh01:10946034..10947984 (- strand)	1895	XM_021451676.1	0
	CWI 4	Not found in monoploid genome	n/a	n/a	XM_021455138.1	3
	CWI 5	Not found in monoploid genome	n/a	n/a	XM_002448667.2	1
	CWI 7	Not found in monoploid genome	n/a	n/a	XM_002448668.2	1
Vacuolar Invertase	VINV	Sh06_t010830	Sh06:19959815..19964282 (+ strand)	4468	XM_002446812.2	1
Fructokinase	FK 1	Sh03_t028370	Sh03:47224065..47227404 (- strand)	3340	XM_002458864.2	2
	FK 2	Sh_206L06_t000080	Not in STP only found in BAC sequences	3072	XM_021464799.1	16
	FK 6	Not found in monoploid genome	n/a	n/a	XM_002436715.2	1
Glucuronokinase	GlcK	Sh08_t000060 & Sh08_t000070	Sh08:47728..50793 (+ strand)	2983	XM_002441606.2	11
Glucose-6-Phosphate Isomerase	G6PI (Chloroplastic) 1	Not found in monoploid genome	n/a	n/a	XM_002462464.2	2
	G6PI (Chloroplastic) 2	Sh02_t015090, Sh02_t015100, Sh02_t015110, Sh02_t015120 & Sh02_t015120	Sh02:28752694..28762708 (+ strand)	10014	XM_002462464.2	5
	G6P1 (Cytoplasmic) 1	Not found in monoploid genome	n/a	n/a	XM_021448861.1	9
Myo-inositol oxygenase	MIOX	Not Found in monoploid genome	n/a	n/a	XM_021449433.1	12
Myo-inositol phosphatase	MIP 3	Sh01_t014550	Sh01:22505310..22508087 (- strand)	2778	XM_002466826.2	8
Myo-inositol phosphate synthase	MIPS 1	Sh01_t020350 & Sh01_t020360	Sh01:33923258..33924805 (- strand)	3661	XM_021462678.1	0
Myo-inositol phosphate synthase	MIPS 2	Not Found in monoploid genome	n/a	n/a	XM_021453593.1	27
Phosphoglucomutase	PGM (chloroplastic) 1	Sh03_t015170	Sh03:27010632..27018435 (- strand)	7804	XM_002466531.2	4
	PGM (cytoplasmic) 2	Sh01_t009050	Sh01:13231372..13239619 (+ strand)	6964	XM_002458121.2	12
	PGM Bi-Functional 1	Not found in monoploid genome	n/a	n/a	XM_021452820.1	6
	PGM Bi-Functional 2	Not found in monoploid genome	n/a	n/a	XM_002459927.2	2
Sucrose Phosphate Phosphatase	SPP 1	Not found in monoploid genome	n/a	n/a	XM_021459652.1	12
	SPP 2	Sh09_t003980	Sh09:6497383..6499722 (+ strand)	2340	XM_021447001.1	5
	SPP 3	Sh09_t003990	Sh09:6500269..6502971 (+ strand)	2703	XM_021447003.1	0
Sucrose Phosphate Synthase	SPS 1	Sh03_t030240	Sh03:49867902..49873435 (- strand)	5534	XM_002458946.2	12
	SPS 2	Sh04_t004940	Sh04:7514628..7521887 (+ strand)	7260	XM_021459119.1	2
	SPS 3	Sh10_t014400	Sh10:26819177..26833006 (- strand)	13830	XM_021449299.1	2
	SPS 4	Sh_230M24_p000030	Not in STP only found in BAC sequences	30575	XM_002441477.2	9
	SPS 5	Sh_254P01_p000060	Not in STP only found in BAC sequences	6027	XM_002449248.2	1
Sucrose Synthase	SuSy 1–1	Sh10_t006690	Sh10:10530379..10537554 (+ strand	7176	XM_021449494.1	34*
	SuSy 1–2	Sh10_t006710	Sh10:10605935..10613992 (- strand)	8058	XM_021449494.1	34*
	SuSy 2	Sh01_t026370 & Sh01_t026380	Sh01:44996485..45001411 (+ strand)	4816	XM_021456935.1	22
	SuSy 4	Sh01_t029560, Sh01_t029570 & Sh01_t029580	Sh01:49748737..49751357 (+ strand)	2600	XM_002465258.2	15
	SuSy 6	Sh04_t027720	Sh04:45363593..45367165 (+ strand)	3573	XM_021459722.1	0
	SuSy 7	Sh10_t019090 & Sh10_t019080	Sh10:34312395..34316604 (- strand)	4189	XM_021449504.1	3
UDP Apiose/Xylose Synthase	UAXS	Sh03_t033730	Sh03:54573981..54577001 (- strand)	3021	XM_002459126.2	3
UDP-glucose 4,6-dehydratase (trifunctional)	RHM 1–1	Sh01_t033530	Sh01:55740390..55744433 (- strand)	4044	XM_021450789.1	10
	RHM 1–2	Sh01_t033540	Sh01:55748951..55751423 (- strand)	2473	XM_021449119.1	12
	RHM 3	Sh09_t007030	Sh09:13224450..13225765 (- strand)	1316	XM_021447354.1	0
UDP-glucose Dehydrogenase	UGD 2	Sh01_t013840	Sh01:21392670..21394244 (+ strand)	1575	XM_021463640.1	0
	UGD 4	Sh01_t037600	Sh01:62131181..62134997 (- strand)	3817	XM_002468250.2	18
	UGD 5	Sh01_t005970	Sh01:8657598..8660482 (- strand)	2885	XM_021464912.1	23
UDP-glucose Pyrophosphorylase	UGPase 1	Sh10_t017220	Sh10:31655313..31671316 (- strand)	16004	XM_021449324.1	1
	UGPase 2	Sh09_t013490	Sh09:26041479..26044800 (+ strand)	8212	XM_021447834.1	15
	UGPase 3	Not found in monoploid genome	n/a	n/a	XM_021447834.1	3
	UGPase 4	Not found in monoploid genome	n/a	n/a	XM_002453140.2	1
UDP-glucose-4-Epimerase	GALE 1–1	Not Annotated	Sh09:36286695..36291305 (+ strand)	4526	XM_002467816.2	19*
	GALE 1–2	Not Annotated	Sh01:49061275..49066223 (- strand)	4949	XM_002467816.2	19*
	GALE 2	Sh07_t008170	Sh07:13109984..13111148 (+ strand)	1165	XM_002445384.2	8
	GALE 3	Not Annotated	Sh02:34046443..34049056 (- strand)	2613	XM_021452554.1	0
	GALE 4	Not Annotated	Sh02:19730286..19736641 (+ strand)	6356	XM_002462165.2	1
UDP-Glucuronic Acid Epimerase	UGE 1–1	Not Annotated	Sh04:41812833..41814159 (- strand)	1327	XM_002452919.2	3
	UGE 1–2	Not Annotated	Sh10:9476275..9477597 (- strand)	1323	XM_002437940.2	0
	UGE 6 − 1	Not Annotated	Sh07:26068992..26070452 (+ strand)	1461	XM_002444670.2	0
	UGE 6 − 2	Not Annotated	Sh02:31857034..31858595 (- strand)	1562	XM_002462575.2	2
UDP-Xylose Epimerase	UXE 1	Not Annotated	Sh02:2941527..2944505 (+ strand)	2979	XM_002459288.2	3
	UXE 2	Not Annotated	Sh06:27777954..27782014 (- strand)	4061	XM_021463492.1	11
	UXE 3	Not Annotated	Sh01:53599712..53602144 (- strand)	5097	XM_002467958.2	2
UDP-xylose synthase (UDP-glucuronic acid decarboxylase)	UXS 1–1	Sh03_t025580	Sh03:43004627..43007178 (+ strand)	2552	XM_002456558.2	6
	UXS 1–2	Sh01_t033240	Sh01:55515466..55517229 (+ strand)	1764	XM_021464234.1	1
	UXS 2 − 1	Not Annotated	Sh01:49677978..49684559 (+ strand)	6682	XM_002465248.2	1
	UXS 2–2	Not found in monoploid genome	n/a	n/a	XM_002457713.2	1
	UXS 4	Sh09_t008330	Sh09:16796741..16798837 (- strand)	2097	XM_002440927.2	11
	UXS 6	Sh01_t033550	Sh01:55755363..55756710 (- strand)	7487	XM_021461997.1	7
Hexokinase	HXK 2	Sh03_t019610 & Sh03_t019620	Sh03:34702506..34705040 (+ strand)	2534	XM_021456830.1	0
	HXK 3	Not found in monoploid genome	n/a	n/a	XM_002459027.2	2
	HXK 5	Sh09_t015900	Sh09:29734562..29738896 (+ strand)	4335	XM_002440059.2	7
	HXK 6	Sh03_t020670	Sh03:36265525..36272095 (- strand)	6571	XM_002458422.2	1
	HXK 7	Sh09_t006090	Sh09:10277092..10280058 (+ strand)	2967	XM_002440690.2	2
	HXK 8	Sh03_t002520	Sh03:4682232..4685187 (+ strand)	2956	XM_021457110.1	3
	HXK 10 − 1	Sh09_t009120	Sh09:18357298..18360337 (- strand)	3040	XM_002440956.2	0
	HXK 10 − 2	Sh06_t013660	Sh06:24339191..24342711 (+ strand)	3521	XM_002440956.2	0

Gene-specific and gene family specific expression profile throughout the sugarcane plant

Sucrose to UDP-glucose and UDP-glucose/hexose phosphate to sucrose associated gene families

In the SuSy gene family, cumulative expression was significantly higher (p ≤ 0.05) in R and TI compared to other organ samples of both Q208 and KQ228 genotypes (Fig. 2). The 4 gene isoforms of SuSy displayed differing degrees of expression throughout the sugarcane plant. SuSy 1, 2 and 7 had significantly higher expression in TI organs in comparison to leaf and mature internodal organs. No significant difference was observed between R and TI samples, however, differences between R and all other organs were not significant due to the high variance of triplicate values. SuSy 1 and 2 were the most prominent gene isoforms in terms of expression.

In the sucrose synthetic sucrose phosphate synthase (SPP) gene family, cumulative expression was higher in some leaf samples in comparison to the mature internodal samples, in both genotypes. The SPP 2 isoform had significantly higher expression values in roots in comparison to leaf and mature internodal samples. The SPP 1 isoform had heightened expression in L5 and L1 leaf samples in genotypes KQ228 and Q208, respectively. Among the two SPP isoforms, SPP 1 had the highest volume of expression.

Cumulative sucrose phosphate synthase (SPS) gene expression was not significantly different throughout the KQ228 genotype, whereas a significantly higher degree of expression was observed in the Q208 genotype. In the KQ228 genotype, SPS gene isoform-specific expression did not differ between SPS 2, 3, 4 and 5 genes, only SPS 1 displayed significantly higher expression in L5 in comparison to root and internodal organs. Distinctly different isoform expression patterns were observed in the Q208 genotype with SPS 2, 4 and 5 having significantly higher expression in TI sample in comparison to R, all leaf and BI samples. SPS 1 expression was significantly higher in L1 sample in comparison to R, BI and L5 samples. The expression of SPS 1 and 4 was the highest of all isoforms.

Cellulose synthase and cellulose synthase-like gene families

In general, comparing between two genotypes, cumulative expression of gene families related to cellulose biosynthesis including CesA, CSLA, CSLC and CSLD showed a significantly higher expression in TI and R samples compared to other samples used in this study (Fig. 3). Cumulative expression of the CesA gene family was significantly higher in TI samples compared to other samples, while roots had significantly higher CesA expression than leaves. Isoform-specific expression for CesA 1–1, 1–2, 2, 3, 5, 7 and 8 was also significantly higher in TI sample, whereas, CesA 6 expression was significantly higher in root sample compared to other samples. The volume of CesA 1–1, 3, 6 and 8 expression was the highest of all isoforms.

Cumulative expression of the CSLA gene family was significantly higher in TI samples, followed by R sample which had significantly higher expression than mature internode and leaf samples. Among the isoforms, CSLA 1 and 7 expression was significantly higher in TI sample, and CSLA 1 expression was significantly higher in R sample, in comparison to leaf and mature internodal samples. CSLC cumulative expression was significantly higher in TI sample than all other samples in both genotypes, followed by R sample which was significantly higher than mature internodal and leaf samples. Isoform-specific expression displayed enhanced expression of CSLC 2 and 7 in TI samples, followed by R sample. In the KQ228 genotype, CSLC 9 expression was significantly higher in R sample, whereas in Q208 there was no significant difference between TI and R sample, both of which had significantly higher expression values than the other samples.

Cumulative expression in the CSLD gene family was significantly higher in R samples. Isoform-specific expression of CSLD 1 and 2 was significantly higher in root sample. CSLD 1 expression was significantly higher than CSLD 2 expression. The sum of CSLE expression was significantly higher in leaf samples. Isoform-specific expression of CSLE 6 − 1 was significantly higher in leaf samples, whilst CSLE 6 − 2 was significantly higher in leaf sample only in the Q208 genotype. CSLE 6 − 2 had the most pronounced expression of the CSLE isoforms. Cumulative expression of the CSLF gene family was significantly higher in R and TI samples. Expression of the CSLF 3 isoform was significantly higher in R samples, whilst expression for both CSLF 6 and 8 was significantly higher in R and TI samples. CSLF 6 had the most pronounced expression of the CSLF isoforms. Expression in the single CSLG gene was significantly higher in L1 sample in the Q208 genotype.

CSLG expression values tended to be higher in leaf samples, however due to the large variance in expression between replicates the differences were not significant, except L1 sample of Q208 genotype. Cumulative expression in the CSLH gene family did not differ between samples of the KQ228 genotype, however, in the Q208 genotype expression was significantly higher in leaf samples. Isoform-specific expression of CSLH 1–1 and 1–2 was significantly higher in leaf sample within the Q208 genotype. CSLH 1–2 expression was significantly higher in root sample in comparison to internodal samples. CSLH 1–1 had the most pronounced expression of the CSLH isoforms.

Hemicellulose synthesis associated gene families

The UDP-G consuming UGD gene family displayed significantly higher cumulative expression rates in R and TI samples (Fig. 4). Within the two UGD isoforms, UGD 5 displayed significantly higher rates of expression in TI sample in comparison to all other samples. Root organ also had significant rates of UGD 5 expression, which were significantly higher than leaf and mature internodal organs. UGD 4 expression rates were significantly higher in R samples, closely followed by TI samples. Cumulative expression rates of the UGE gene family were significantly higher in R and TI samples across both genotypes. Isoform-specific expression of UGE 1–1 and UGE 6 − 2 was significantly higher in both R and TI samples. UGE 1–1 had the most pronounced expression of the UGE isoforms. Within the UXE gene family, cumulative expression was significantly higher in R and TI samples. Expression of the UXE 1 and 3 isoforms was significantly higher in TI samples, whereas UXE 2 had significantly higher expression in R samples, followed by TI sample. UXE 2 had the most pronounced expression of the UXE isoforms.

Cumulative expression of the RHM gene family was significantly higher in root samples, in comparison to all other samples. Isoform-specific expression of RHM 1–1 was significantly higher in root samples. Expression of RHM 1–2 was not significantly different between all samples in the KQ228 genotype, whereas in the Q208 genotype expression was significantly higher in R samples. The UXS gene family had significantly higher expression in TI samples, closely followed by R samples which had significantly higher expression than mature internode and leaf samples. Isoform-specific expression of the UXS gene family was as follows: UXS 4 had significantly higher expression in the R samples, whereas UXS 6 expression was significantly higher in TI samples. Expression rates of the other UXS isoforms tended to be higher in TI samples, although in some cases the differences were not significant between all samples. UXS 6 had the most pronounced expression of the UXS isoforms. Cumulative expression of the GALE gene family did not show a clear expression pattern between samples, with all leaf, R and TI samples having pronounced expression, although expressional differences were not significant between all samples. Isoform-specific expression of GALE 2 was significantly higher in TI sample, whereas GALE 1 expression tended to be higher in R and both leaf samples, although the differences between these samples and the internodal samples were not consistent across both genotypes. The single gene with UAXS functionality had significantly higher expression in TI samples, followed by R sample which had significantly higher expression than leaf and mature internodal samples.

myo-Inositol pathway associated genes

Figure 5 displays the cumulative and gene isoform-specific expression values of genes associated with the myo-Inositol pathway. Only single genes were identified at each point in the myo-Inositol pathway. MIPS gene expression displayed distinctly different expression patterns between the two genotypes with larger rates of expression being observed in the KQ228 genotype. Whilst no significant differences were observed between samples in the Q208 genotype, within the KQ228 genotype, TI sample had significantly higher expression than R and mature internodal samples. GluK and MIP gene expression were significantly higher in TI samples in both genotypes. MIOX gene expression was significantly higher in root samples.

Invertase gene families

Figure 6 displays the cumulative and gene isoform-specific expression values of invertase associated gene families. Within the ANINV gene family, cumulative and isoform-specific expression did not display clear expression patterns between the sample types. Cumulative expression patterns of the CINV gene family were higher in R samples in both genotypes, although differences were only significant in the KQ228 genotype. Isoform-specific expression of CINV 1 and 2 was significantly higher in root sample in the KQ228 genotype, however, in the Q208 genotype these differences were not significant. Expression of the CINV 1 isoform was higher than that of CINV 2. Cumulative expression of the CWI gene family was significantly higher in R samples in the KQ228 genotype, whereas R and both leaf samples had tended to have higher expression in the Q208 genotype, although only L5 sample had significantly higher expression than internodal samples. CWI isoforms 1, 2, 5 and 7 had significantly higher expression in R across both genotypes. CWI 4 expression tended to be higher in leaf sample, with significantly higher expression being observed in the Q208 genotype. In the KQ228 genotype, only L5 sample had significantly higher expression than that of internodal samples. CWI isoforms 1 and 4 had the most pronounced expression of all CWI isoforms. VINV expression was significantly higher in roots in both genotypes.

Phosphorylated sugar synthesis /degradation associated gene families

Figure 7 displays the cumulative and gene isoform-specific expression values of genes associated with the synthesis and degradation of phosphorylated sugars. Cumulative UGPase expression was significantly higher in TI samples in comparison to leaf and mature internodal samples in the KQ228 genotype. In the Q208 genotype whilst expression was observed to be higher in TI samples, this result was not significant. Isoform-specific expression of the UGPase gene family did not display any conclusive evidence of significant differences between samples in both genotypes. The UGPase 2 isoform had pronounced expression in comparison to the 3 other UGPase isoforms. Cumulative AGP expression tended to be higher in leaf samples, closely followed by TI sample, however, this trend was only significant in the KQ228 genotype. Isoform-specific expression of AGP 4 and 6 was significantly higher in leaf samples. Cumulative expression of the PGM gene family did not display any consistent expressional trends between samples in both genotypes. Isoforms AGP 1, 2 and 6 had far more expression than other AGO isoforms. The chloroplastic PGM 1 isoform had significantly higher expression in leaf sample, whilst bi-functional PGM 1 and cytoplasmic PGM 2 had significantly higher expression in TI sample. A significant amount of bi-functional PGM 2 expression was observed in both L1 and L5 samples of the Q208 genotype, however, this trend was not observed in the KQ228 genotype.

G6PI cumulative and isoform-specific expression did not display any consistently significant expressional trends between samples across both genotypes. Cumulative expression of the FK gene family was significantly higher in TI and R samples across both genotypes. Expression of the FK 1 isoform was significantly higher in R sample, whereas expression for FK 2 and 6 was significantly higher in TI sample. FK 1 and 2 had pronounced expressional values in comparison to the FK 3 isoform. Cumulative expression in the HXK gene family was significantly higher in TI and R samples. Expression of HXK 6 and 8 isoforms was significantly higher in TI samples across both genotypes. There was a significantly higher expression of HXK 7 isoform in R samples. HXK 3 expression was significantly higher in both R and TI samples.

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 [13–19]. 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 (source organ, young and mature), internodes (sink organ, young, maturing and mature) and root (a meristematic sink organ).

Expression of genes associated with the UDP-G metabolism in the major storage sink organ internodes

Sucrose synthesis and degradation in internodal samples by the SPP, SPS and SuSy enzymes, 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 [44]. As indicated in this study and previous studies of SuSy genes, expression and enzymatic activity are significantly higher in immature internodes [44, 45], 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. 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), GALE, UAXS, UGE, UXE and UGD. Some of the aforementioned genes code for enzymes that directly consume UDP-G, including UGD, CesA and some CSL gene families [46–48], 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, and CesA subunit genes 1–1, 1–2, 3, 5, 7 and 8.

In conjunction with the heightened sucrose cleavage to UDP-G, cumulative invertase expression in CINV, NAINV and CWI gene families was significantly higher in immature internodes in comparison to mature internode samples. 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 [49]. The heightened levels of reducing sugars in the immature internodes [31], displays the heightened activity of invertases in the immature internodes in this sample. The heightened expression of the CWI 1 and 7 genes in the immature internodes, suggests prominent activity of C fixation into parenchyma tissue from conducting tissues, and the apoplastic/symplastic transfer of sucrose [50, 51]. Lower expression of CWI in mature internodes may suggest that C importation facilitated by the activity of invertases is less pronounced in this sample. The heightened expression of genes encoding phosphorylating enzymes, FK and HXK in immature internodes, 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 [52]. The indication of enhanced C flux into these pathways that may be mediated by gene expression is important, as results from [53] 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, which is in contradiction to several studies that have found an opposite trend in enzymatic activity of SPS [44, 54]. It must be noted that enzymatic activity and gene expression are not necessarily correlated [55]. 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 [44, 56]. 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 [44], 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 non-storage sink organ root

Despite root and immature internodal samples both being major sinks and moving a large proportion of fixed C into the cell wall, protein and respiratory pools, there were large expressional differences likely indicating spatial regulation of specific gene family isoforms. The transcriptional differences suggest that meristematic/immature sink samples in different organs likely have a differing mode of C partitioning. Compositional analyses of these two samples [57] indicated differences in simple sugar and hemicellulosic monomer content. Concerning the simple sugar content, i.e. fructose, glucose and sucrose, it was postulated that the reduced levels in root sample indicate the efficient breakdown and movement of fixed C into the cell wall, protein and respiratory pools in roots. It must be noted that in immature internodes, there is an underlying trend for the accumulation of sucrose that does not exist in roots. This is made clear by the high expression of VINV in roots, which has been implicated as a key enzyme negatively affecting the accumulation of sucrose in samples in several plant systems, as reviewed by [58]; and the heightened expression of SPS 1 and 4 genes (only significant in Q208 genotype). In this study, low VINV expression in all internodes and leaf samples, indicating low VINV activity, likely allows the accumulation of sucrose for storage or transportation in source samples, the high expression in roots suggests a low requirement for sucrose bioaccumulation in this sample. 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. 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 source sample [59]. Interestingly, in sugarcane, the activity of CWI in internodes is correlated with higher sucrose levels [60, 61]. Enhanced invertase activity is counterintuitive to enhanced sucrose levels, however, the sucrose cleavage and resynthesis model as proposed by Glasziou and Gayler [62], may explain this. Higher expression of CWI genes in roots suggests the intercellular sucrose cleavage and intracellular resynthesis model to not be relevant in root sample, as there is no evidence for large degrees of sucrose resynthesis to be occurring [31]. This notion is further supported by the lower expression of SPS observed in roots (only significantly different in Q208 genotype) in comparison to immature internodes and leaf samples.

Equivalent expression of SuSy genes between immature internodes and roots samples likely indicates a high degree of UDP-G formulation from sucrose, 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. Additionally, downstream steps of hemicellulose synthesis displayed a significant difference between the two samples, which could be responsible for the heightened amount of arabinose and galactose mixed linkages in roots, particularly GALE 1 and UXE 2. Although as shown in related analyses, there was also a higher expression in other hemicellulose related transcripts in roots, that do not result in differences in composition [31, 53]. Interestingly, CesA expression was significantly higher in immature internodes. The significant differences in gene expression related to hemicellulose and cellulose synthesis are likely related to the presence of specialised cells in both roots and internodes [63–65], having different requirements for C, or differing metabolic conditions, i.e. access to substrates or the presence of feedback inhibitory molecules [21]. Notably, compositional analysis of roots and internodes, as presented in [31], 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 the source organ leaf

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 had significant expression in leaves including CSLE, CSLG and CSLH, specifically CSL6-2, CSLG2 and CSLH1-1 genes. 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 in these samples. 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, as reviewed by [66], although it is likely still associated with hemicellulose synthesis, whereas the CSLH group encodes mixed linkage glucan synthases [67]. 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, 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. SPP and SPS form a complex which transfers UDP-G to sucrose and is the key enzyme complex of sucrose biosynthesis in source and sink samples [44, 68, 69]. 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, with a smaller amount derived from UGPase activity [70], as reviewed by [7]. In support of this notion, SuSy activity was significantly higher in meristematic sinks. However, the pronounced role of UGPase in sucrose biosynthesis in leaves did not coincide with significantly higher expression. This likely 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, 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. In a previous experiment of photosynthetic regulation by sugars in sugarcane leaves, fed radiolabeled sucrose was rapidly converted into hexoses, which was stipulated to be due to the activity of SuSy and invertase enzymes [71]. 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 [72, 73]. In the case of the CWI gene family, most expression in leaves was contributed by the CWI 4 gene, 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 [31], 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.

This study represents the first effort to quantify the expression of gene families associated with UDP-G metabolism in a sugarcane plants. The data presented provides a quality reference for future efforts in altering UDP-G metabolism and in turn carbon partitioning in sugarcane. Transcriptional analysis displayed the likelihood that carbon partitioning in sugarcane is closely related to the transcription of genes associated with the UDP-G metabolism in the four major modes of carbon partitioning as defined in leaves (source), roots (non-storage immature/meristematic sink), mature internodes (storage sink) and immature internodes (immature sink that will become a storage sink). The data presented may provide an accurate genetic reference for future efforts in altering UDP-G metabolism and in turn C partitioning in sugarcane.

C: Carbon; NGS: Next Generation Sequencing; mRNA: messenger RNA; cDNA: complementary DNA; BI: Bottom Internode; MI: Middle Internode; TI: Top Internode; L1: 1^st visible dewlap leaf; L5: 5^th visible dewlap leaf; R: Root; UDP-G: Uridine Diphosphate Glucose; CWI: Cell Wall Invertase; VINV: Vacuolar Invertase; CINV: Cytosolic Invertase; ANINV: Alkaline/Neutral Invertase; FK: Fructokinase; MIPS: myo-Inositol phosphate synthase; MIP: myo-Inositol phosphatase; MIOX: myo-Inositol oxygenase; GluK: Glucuronokinase; PGM: Phosphoglucomutase; UGPase: Uridine Diphosphate Glucose pyrophosphorylase; UGD: UDP-Glucose Dehydrogenase; AGP: Adenosine Diphosphate Glucose pyrophosphorylase; CesA: Cellulose Synthase; CSL: Cellulose Synthase-Like; G6PI: Glucose-6-Phosphate Isomerase; SPP: Sucrose Phosphate Phosphatase; SPS: Sucrose Phosphate Synthase; SuSy: Sucrose Synthase; RHM: Uridine Diphosphate Glucose 4,6-dehydratase; UAXS: Uridine Diphosphate Glucose Apiose/Xylose Synthase; GALE: Uridine Diphosphate Glucose 4-Epimerase; UGE: Uridine Diphosphate Glucuronic Acid Epimerase; UXS: Uridine Diphosphate Xylose Synthase; UXE: Uridine Diphosphate Xylose Epimerase; HXK: Hexokinase

ACKNOWLEDGEMENTS

We thank the staff at the SRA Burdekin branch for their assistance in preparing and sampling the plant material. Research was supported by Sugar Research Australia (SRA), providing access to plant material. Also, the high-performance computing resources required for in silico analysis was provided by the Research Computing Centre (RCC), University of Queensland.

AUTHORS' CONTRIBUTIONS

Patrick J Mason: Conceptualisation, Methodology, Investigation, Data Curation, Writing-Original Draft, Writing-Review and Editing, Visualisation Nam V Hoang: Conceptualisation, Methodology, Investigation, Data Curation, Writing-Review and Editing, Visualisation Frederick C Botha: Conceptualisation, Methodology, Writing-Review and Editing, Supervision, Resources Agnelo Furtado: Conceptualisation, Methodology, Investigation, Writing-Review and Editing, Supervision Annelie Marquardt: Methodology, Investigation, Writing-Review and Editing Robert J Henry: Conceptualisation, Methodology, Writing-Review and Editing, Supervision, Resources

FUNDING

This research was supported by a Sugar Industry Postgraduate Research Scholarship (SPRS) from Sugar Research Australia to P.J.M.

PERMISSION TO COLLECT PLANT MATERIAL

All plant material utilised are commercial sugarcane varieties protected by Plant Breeders Rights. Identification of plant material was undertaken by Sugar Research Australia. Full descriptions are available (https://www.ipaustralia.gov.au/plant-breeders-rights accessed 14/04/2022) and voucher specimens are held by Sugar Research Australia as required for plant breeders rights.

AVAILABILITY OF DATA AND MATERIALS

The version of the SUGIT database used in this study can be accessed in Figshare under the DOI https://doi.org/10.6084/m9.figshare.4981655. The RNA-seq data used for expression profiling has been deposited in the NCBI Sequence Read Archive (SRA) under the BioProject ID PRJNA479814, Study Accession Number SRP152893.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

None declared

CONSENT FOR PUBLICATION

None declared

COMPETING INTERESTS

The authors declare that they have no competing interests.

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No competing interests reported.

Supplementarydatafile1.docx
Figure S1. Schematic of sugarcane transcript gene family homologue assignment and quantification.Table S1. Numerical Identifier for Tukey t-test results and one-way ANOVA significance values.Figure S2. One-way ANOVA and Tukey t-test results from SuSy, SPS, SPP and CSLA gene family expression comparisons.Figure S3. One-way ANOVA and Tukey t-test results from CesA, CSLC, CSLD and CSLE gene family expression comparisons.Figure S4. One-way ANOVA and Tukey t-test results from CSLF, CSLG, CSLH and UGD gene family expression comparisons.Figure S5. One-way ANOVA and Tukey t-test results from UGE, UXE, RHM and GALE gene family expression comparisons.Figure S6. One-way ANOVA and Tukey t-test results from UXS and UAXS gene family expression comparisons.Figure S7. One-way ANOVA and Tukey t-test results from MIPS, GluK, MIP and MIOX gene family expression comparisons.Figure S8. One-way ANOVA and Tukey t-test results from Invertase gene families ANINV, CINV, CWI and VINV.Figure S9. One-way ANOVA and Tukey t-test results from UGPase, AGP, PGM and G6PI gene family expression comparisons.Figure S10. One-way ANOVA and Tukey t-test results from HXK and FK gene family expression comparisons.

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Organ-specific expression of genes associated with the UDP-glucose metabolism in sugarcane (Saccharum spp. hybrids)

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Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

Plant material collection, data generation and transcript profiling

Extraction of transcripts of interest and subsequent matching to gene family isoforms

Genomic location and gene length determination using the sugarcane STP monoploid genome

Collapsing transcripts into single expression value

Data processing of gene family expression values

Results

Identification of gene families associated with UDP-glucose metabolism in sugarcane

Gene-specific and gene family specific expression profile throughout the sugarcane plant

Sucrose to UDP-glucose and UDP-glucose/hexose phosphate to sucrose associated gene families

Cellulose synthase and cellulose synthase-like gene families

Hemicellulose synthesis associated gene families

myo-Inositol pathway associated genes

Invertase gene families

Phosphorylated sugar synthesis /degradation associated gene families

Discussion

Expression of genes associated with the UDP-G metabolism in the major storage sink organ internodes

Expression of genes associated with the UDP-G metabolism in non-storage sink organ root

Expression of genes associated with the UDP-G metabolism in the source organ leaf

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1