Bamboo, like most of vascular plants, generally has cylinder trunk since it is essential to anti-interference of exterior harsh environments such as strong wind, the load of snow, and the severe competition of light. However, the Chimonobambusa utilis presents a different geometrical shape of stem, which closer to square or four-angle shape. Then, the species was assigned an informal nomenclature as ‘square bamboo’ for its “four-angled” culm (Dyer 1885). The underlying reason of this exception is attractive to biological scientists since how to control the geometrical shape of stem or trunk is an important and interesting research target. Besides that, a hyperelliptic formula have been applied for fitting the geometrical constructions of square-like culm to superellipse data (Gielis 2017; Huang et al. 2020).It was suggested that the geometrical character of the square-like culm was meaningful to optimization of structure of thin-walled tube, which is a useful energy-absorbing component in constructing frame for preventing from collision (Chen et al. 2021). So, an understanding of the exception also has contribution to design protecting system with resisting impact damage function. Therefore, it is essential to explore the knowledge of formation of Chimonobambusa utilis stem, namely square-like stem.
As the first step toward understanding a species, anatomical characteristics represented important parameters that reflected the microstructure of natural biomass materials (Jiang, 2020). The anatomical structures of a bamboo culm can be characterized by vascular bundles and fibre morphology (Grosser and Liese, 1971; Lin et al. 2004). Arrangement of typical vascular bundles displayed as ‘open’ type (Li and Chin, 1960; Jiang et al. 2021), and morphology of fibre changed at different circumferential and radial position in square bamboo (Lin et al. 2004; Jiang et al. 2021).Meanwhile, since the biomolecular function and regulation mechanisms is the fundamental to understand the structure of plant, which started at nano-scale and ended at macro-scale, it is important to revealing the formation mechanism of square-like stem at a molecular level. However, previous work main focused on the phenotypic traits of square bamboo, for example, anatomical characteristics, geometrical structures, but its formation and development mechanism at molecular level remains to be discussed.
The exploration of the gene expression level and molecular mechanism on formation of square-like stems is meaningful to know how square-like stem form and reveal the morphogenetic process (Wang et al. 2018). The application of a multi-omics approach can be applied for investigating the molecular biological mechanism systematically and comprehensively (Hori et al. 2020). By considering the most effective method, analyses of phenotypic characters combined with transcriptome and metabolome has been extensively validated (Kasirajan et al. 2018; Hu et al. 2020a, 2020b). A transcriptome represents the intermediate state of gene expression and offered a relatively holistic approach to interpreting molecular mechanism of plant development (Shi, 2011; Wang et al. 2009). RNA-Seq experiment has been successfully applied to identify transcriptional levels of each expressed gene and drawing generalized conclusions about differential characteristics observed in plant groups (Costa et al. 2010). Metabolites, the final products of gene transcription, formed the material basis of all organismal phenotypes (Oliver et al. 1998; Fiehn et al. 2000; Fiehn 2002; Kopka et al. 2004). It can help us to characterize the activities of cells at the functional level, providing a great theoretical impact on our understanding of metabolic pathways in processes of plant development (Fiehn 2000; Oliver 2002; Cornish-Bowden and Cárdenas 2000). Combined analysis of transcriptome and metabolome can be applied to reveal internal relationship between the regulation mechanism and biomolecular function systematically (Jewett et al. 2005; Conesa et al. 2016). Such approach has been successfully employed in the exploration of stem growth and development of Bambusoideae (Yue 2017; Xu 2017; Wang 2018; Guo 2018; Hu et al. 2020a, 2020b; Yang et al. 2020; Hou et al. 2021). According to transcriptome analysis, abnormal thickening of stem wall in Phyllostachys edulis ‘Pachyloen’ and dwarfing and internode shortening traits in Phyllostachys heterocycla ‘Pubescens’ related candidate genes were identified (Yue 2017; Wang et al. 2018; Zhang 2016; Xu 2017). Nitrogen metabolism pathway and tyrosine metabolism metabolic pathways and their uniquely expressed genes were related to culm wall thickness in Phyllostachys edulis ‘Pachyloen’ (variant of moso bamboo) (Hu et al. 2020a, 2020b). These studies provide a reference for detecting core genes related to bamboo morphology and anatomical characteristics, and exploring the gene regulation mechanism in the malformation of bamboo stem. However, unfortunately, these approaches haven’t been applied to clarify the formation of square bamboo at present.
To investigate the biomolecular function and regulation mechanisms associated with the formation of square stem, Chimonobambusa utilis was selected as study material. The stem was divided into eight micro areas in circumferential direction at first. Comprehensive analysis of anatomy, spectroscopy, transcriptome, and metabolome was performed simultaneously at each individual micro area later then. Morphology of fibre and vascular bundles was observed by using the anatomical method, the relative lignin content was detected using Fourier transform infrared (FTIR) spectroscopy, and transcriptomic and metabolomic responses in different circumferential position of the bamboo shoots were analyzed. Finally, the formation mechanism of square-like stems was revealed comprehensively and systematically, through analyzing the link between macro and micro structures, chemical components, expression level of genes, and metabolites. This work identified square-like stem related candidate genes and revealed its formation mechanism from macro-scale to nano-scale, furthermore, it is meaningful to reference to other stem mutant species in vascular plants.