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The genetic engineering of value-added traits such as the accumulation of bioproducts 21 in high biomass C4 grass stems is one promising strategy to make plant-derived biofuels more 22 economical for industrial use. A first step toward achieving this goal is to identify stem-specific 23 promoters that can drive the expression of genes of interest with good temporal and spatial 24 specificity. However, a comprehensive characterization of the spatial-temporal regulatory 25 elements of stem tissue-specific promoters for C4 grasses has not been reported. Therefore, we 26 performed an in-silico analysis on Sorghum bicolor cv BTx623 transcriptomes from multiple 27 tissues over development to identify stem-expressed genes. The analysis identified 10 genes that 28 are "Always-On-Stem-Specific," 59 genes that are "Temporally-Stem-Specific during early 29 development," and 21 genes that are "Temporally-Stem-Specific during late development." 30 Promoter analysis revealed common and/or unique cis-regulatory elements in promoters of genes within each of the three categories. Subsequent gene regulatory network (GRN) analysis revealed 32 that different transcriptional regulatory programs are responsible for the temporal activation of the 33 stem-expressed genes. The analysis of temporal stem GRNs between sweet (cv Della) and grain 34 (cv BTx623) sorghum varieties revealed genetic variation that could influence the regulatory 35 landscape. This study provides new insights about sorghum stem biology, and information for 36 future genetic engineering efforts to fine-tune the spatial-temporal expression of transgenes in C4 37 grass stems. 38 39 40 High biomass grasses, such as sorghum (Sorghum bicolor), miscanthus (Miscanthus x 41 giganteus), switchgrass (Panicum virgatum), and sugarcane (Saccharum sp.), offer an abundant 42 and renewable source of biomass for biofuels production (McLaughlin and Kszos, 2005; Rooney 43 et al.

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