To identify a rapidly expanding internode, S. viridis plants were grown under highly controlled conditions and seven developmental stages were examined. The fourth developmental stage, half-head emergence, was defined as the point when half of the seed head had emerged from the leaf sheath (Fig. 1a). This was chosen as the developmental stage with the most rapid rate of stem growth, which also corresponded with the most rapid rate of sucrose accumulation (Fig. 1b). At this developmental stage, the leaf sheath was carefully stripped to expose the stem and its constituent internodes. Internode five, counted from the bottom (considering only internodes ≥5 mm in length), or, interchangeably, the second internode from the top (not including the flag internode), was selected as a rapidly elongating internode containing well-defined elongation and mature zones (Fig. 1c). To ensure this internode was rapidly elongating, the same internode was also measured in mature plants and was shown to increase from an average length of 3.8 cm at harvest (half-head emergence) to 10.2 cm at maturity (Fig. 1d).
To define the meristematic, cell expansion, transitional, and mature zones of internode five, nuclei density and cell size were examined using fluorescence microscopy. It was also essential to develop a method for visually identifying these zones of internode five so that harvest for RNAseq analysis could be achieved rapidly, without any need for microscopy, and therefore without excessive tissue damage.
Internodes were photographed (Fig. 2a), whilst intact and then embedded in agarose and sectioned longitudinally. Nuclei were stained with DAPI, illuminated with UV light and viewed using a long-pass DAPI filter that allowed red chlorophyll fluorescence (Fig. 2b) and blue DAPI fluorescence of nuclei (Fig. 2c) to be visualised simultaneously. Processing DAPI images allowed nuclei density (a proxy for the number of cells) and also cell size to be plotted along the length of the internode (Fig. 2d). A spike in nuclei density mapped to a well-defined white band at the base of the internode, and this was defined as the meristematic zone (MsZ) (Fig. 2a) allowing for rapid identification of this zone. Directly above the meristematic zone, cell size increased and nuclei density decreased, both asymptotically. It was observed that this cell expansion zone was dark green in colour, which was also indicated by the increased red chlorophyll fluorescence observed in longitudinal sections of this region (Fig. 2b). The base of the internode was flexible (Fig. 2a), likely due to the lack of lignified secondary cell walls. The point where the internode became rigid corresponded with the end of the chlorophyll-enriched zone and the approach of nuclei density and cell size towards their asymptote. Both the end of the chlorophyll-enriched zone and the point where the internode became rigid were therefore used as markers to dissect the cell expansion zone (CEZ) (Fig. 2a) of the internode without the need to stain or section the tissue. The region of the internode directly above this point, cut to ~85 % the length of the CEZ, was dissected and defined as the transition zone (TZ) (Fig. 2a). A zone immediately below the node above internode five, cut at the same length as the CEZ, was dissected as the mature zone (MZ) (Fig. 2a).
Using brightfield microscopy, it was evident that there were many different cell types within these zones. The cell expansion zone contained immature vascular bundles with protoxylem and very little cell wall thickenings in any cell types (Fig. 2e). The transition zone contained fully developed vascular bundles with fully developed xylem, but only modest cell wall thickenings (Fig. 2f). The mature zone contained fully developed vascular bundles with fully developed xylem and more cell wall thickening than the transitional zone (Fig. 2g). Based on evidence from these images, the dissected zones contained a variety of cell types, but were sufficiently enriched in expanding, transitioning, and mature tissues to reveal differences in metabolite levels and gene expression between each developmental stage.
Primary metabolites were measured in the four zones of the developing internode to establish carbon pools (Fig. 3). Starch was found only in the developmentally young meristematic and cell expansion zones. The ratio of sucrose to hexoses was high in the meristematic zone, reduced in the growing cell expansion and transitional zones, and increased in the mature zone, which was likely at the beginning of a sugar storage phase as sugar levels rise towards the maximal concentration observed in stage 6 (Fig. 1b). High levels of the soluble C4 acid malate were also measured and correlated strongly with sucrose levels. The role of malate in this developing C4 internode is unclear; however, it may be involved in a functional C4 pathway, used as a pH regulator, a storage molecule in vacuoles, or an osmolyte for turgor regulation [28].
The lignin content of cell walls was also measured in the cell expansion, transitional, and mature internode zones as an indicator of the presence of primary or secondary cell walls (Fig. 3). Very low levels of lignin were observed in the cell expansion zone; however, the cell walls rapidly became lignified in the transitional zone, and continued to accumulate lignin up to the mature zone (Fig. 3). This suggested that the cell expansion zone contained primary cell walls, whereas the transitional zone was rapidly synthesising secondary cell walls, and that this synthesis was maintained, up to higher levels in the mature zone where it has, likely, completed lignification.
Once the developmental status of the four internode zones was established, RNA was extracted, sequenced, mapped (see Additional file 1 for mapping details), and expression values calculated. Principal component analysis (PCA) of global gene expression confirmed that each internodal zone had a unique gene expression profile with the meristematic and cell expansion zones clustering separately from the transitional and mature zones (Fig. 4a). Very little variation between biological replicates was also observed confirming the reproducibility of the harvesting technique.
Comparing log2 fold-change expression of each gene to that obtained from a ‘whole plant’ background transcriptome [3], lists of genes that were up-regulated (>1 log2 fold) in each zone were generated and displayed as a Venn diagram (Fig. 4b). This also revealed a dichotomy between the two younger meristematic and cell expansion zones, and the two older transitional and mature zones. Thousand eight hundred and sixty genes were up-regulated in common in MsZ/CEZ and 983 in TZ/MZ. In comparison, only 52 MsZ/TZ, 157 MsZ/MZ, 213 CEZ/TZ, and 12 CEZ/MZ genes were up-regulated in common (Fig. 4b). The number of up-regulated genes and the number expressed above a threshold of one fragments per kilobase per million reads (FPKM) also showed dichotomy with more being found in younger than older tissues (Fig. 4c). This suggests that greater biological complexity is required for meristematic activity and cell expansion than for the established biological processes of mature tissues including secondary cell wall synthesis and carbon storage.
Using criteria of gene expression above a level of 80 FPKM (chosen by visually examining read coverage of genes with ~80 FPKM), and a log2 fold change >2.5 against the whole plant (WP) background transcriptome, 418 ‘stem-enriched’, 13 ‘MsZ-enriched’, 8 ‘CEZ-enriched’, 84 ‘TZ-enriched’, and 45 ‘MZ-enriched’ genes were identified which can be used to identify promoter sequences for the construction of tissue-specific, and likely cell-specific, expression vectors (‘enriched’ genes are marked in Additional file 2).
To further characterise the segregation of biological processes within each zone of the developing internode, genes were categorised based on the most recent Mapman ontology, which was updated for this analysis (Additional file 3). The meristematic zone was enriched in processes related to cell division and cell cycle including DNA, RNA, and protein synthesis/regulation, confirming that this zone is actively undergoing cell division. The cell expansion zone was enriched in metabolism, energy production, and lipid biosynthesis, which are required to drive cell expansion. The transitional zone was enriched in secondary cell wall biosynthesis-related gene categories including lignin biosynthesis, laccases, cell vesicle transport, MYB, and NAC transcription factors (which are maintained in the mature zone), and amino acid metabolism. The mature zone was enriched in sugar transporters, photosynthesis (which begins to operate, however, not to the levels observed in the whole plant), and flavonoid biosynthesis (Fig. 5). These transcript profiles confirm that the transcriptional machinery required for stem-specific intercalary meristem cell division and differentiation is active in the meristematic zone; energy-driven cell expansion is active in the cell expansion zone; secondary cell wall synthesis, including transcriptional switches, is active in the transitional zone; and sugar unloading, storage, and possibly synthesis (photosynthesis) is active in the mature zone.
To encapsulate the effectiveness of this experimental system for gene discovery and manipulation in a variety of fundamental biological processes, Mapman was used to visualise the expression of gene families relating to three major carbon demands of a developing grass culm (Fig. 6). In the meristematic zone, gene expression suggested predominantly symplasmic import of photoassimilate and utilisation of starch for cell metabolism and energy production (demand 1), since expression of sugar transporters, including SWEETs (sugar effluxers [29], SUTs (sucrose-H+ symporters [30]), HTs (hexose transporters [31]), PLTs (polyol transporters [32]), and CWI (cell wall invertase [33]) was low (Fig. 6). Gene expression in the cell expansion zone also suggested that photoassimilate is imported predominantly symplasmically [i.e. low expression of SWEETs, SUTs, HTs, PLTs, and CWIs and higher expression of plasmodesmatal located proteins (PDLPs)] not only in this zone and is directed towards demand 1 (energy/metabolism), but also primary cell wall synthesis, including hemicellulose, pectin, and cell wall protein synthesis (demand 2) (Fig. 6). The transitional zone is a pivotal developmental stage switching from predominantly metabolism (demand 1) and symplasmic photoassimilate import to a highly significant up-regulation of genes associated with apoplasmic sugar transport, coupled with a major switch and a re-direction of carbon flow to secondary cell wall synthesis (demand 2) (Fig. 6). The mature zone showed down-regulation of genes linked to metabolism (demand 1) and cell wall synthesis (demand 2), whilst apoplasmic transport genes (SWEETs, SUTs, HTs, PLTs, and CWIs) and vacuolar sugar storage (demand 3) genes (tonoplast monosaccharide transporters—TMTs [34]) showed further up-regulation. This suggested that the mature zone had ceased most of its transcriptional activity related to secondary cell wall synthesis and is actively transcribing genes that are associated with apoplasmic unloading of photoassimilate for storage.