BIOMASS YIELD 1 regulates sorghum biomass and grain yield via the shikimate pathway

Abstract

Biomass and grain yield are key agronomic traits in sorghum (Sorghum bicolor); however, the molecular mechanisms that regulate these traits are not well understood. Here, we characterized the biomass yield 1 (by1) mutant, which displays a dramatically altered phenotype that includes reduced plant height, narrow stems, erect and narrow leaves, and abnormal floral organs. Histological analysis suggested that these phenotypic defects are mainly caused by inhibited cell elongation and abnormal floral organ development. Map-based cloning revealed that BY1 encodes a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) that catalyses the first step of the shikimate pathway. BY1 was localized in chloroplasts and was ubiquitously distributed in the organs examined, particularly in the roots, stems, leaves, and panicles, which was consistent with its role in biomass production and grain yield. Transcriptome analysis and metabolic profiling revealed that BY1 was involved in primary metabolism and that it affected the biosynthesis of various secondary metabolites, especially flavonoids. Taken together, these findings demonstrate that BY1 affects biomass and grain yield in sorghum by regulating primary and secondary metabolism via the shikimate pathway. Moreover, our results provide important insights into the relationship between plant development and metabolism.

Keywords: Biomass yield; flavonoids; metabolic profiling; metabolism; shikimate pathway; sorghum; transcriptome.

Fig. 1.

Biomass production and grain yield in wild-type sorghum and the by1 mutant. (A) Phenotypes of the wild-type (WT) and by1 plants at the flowering stage. Scale bar is 20 cm. (B) Phenotypes of panicles at maturity. Scale bar is 10 cm. (C) Biomass yields in terms of fresh and dry weight. (D) Gain yield (DW). Data are means (±SD), n=20. Significant differences were determined using two-tailed Student’s t-test: *P<0.05, **P<0.01.

Fig. 2.

Histological analysis of wild-type sorghum and the by1 mutant. (A) Stems of the wild-type (WT) and by1. White arrowheads indicate nodes. Scale bar is 20 cm. (B, C) Longitudinal sections of the panicle neck internodes of the WT and by1. Scale bars are 50 µm. (D) Comparison of internode lengths between the WT and by1. (E) Leaves of the WT and by1 (scale bar is 10 cm), and (F, G) corresponding SEM images, with cell morphologies highlighted (scale bars are 30 µm). (H) Leaf cell lengths and (I) cell widths in the WT and by1. (J, K) Images of fertile florets in the WT and by1. Scale bars are 1 mm. (L, M) Images of pollen grains of the WT and by1. Scale bars are 50 µm. (N, O) Staining for pollen viability in the WT and by1. (P) Pollen integrity, as determined from images in (L, M). (Q) Pollen viability, as determined from images in (N, O). Data are means (±SD), n=10, except n=20 in (D). Significant differences were determined using two-tailed Student’s t-test: *P<0.05, **P<0.01.

Fig. 3.

Map-based cloning of sorghum BY1. (A) Rough and (B) fine-mapping of BY1, where n is the total number of plants used for recombinant screening and mapping, and R is the number of recombinant individuals identified by screening recombinants between flanking markers. (C) Genes in the fine-mapping region of BY1. (D) The structure of BY1 and the by1 mutation site. The unfilled boxes at the left and right ends represent the 5´-UTR and 3´-UTR, respectively. The black boxes represent exons and the lines between them represent introns. The red line and text at the second exon indicate the position and form of base mutation in the by1 mutant. (E) The amino acid structure of BY1 and the mutation site in by1. The blue box represents the functional domain region, and indicates the position and form of the amino acids in the by1 mutant. (F) Conservation analysis of the amino acid substitution region and frequency in homologous plant genes. The red box indicates the position of the amino acid substitution in by1. (G–I) The japonica rice cultivar ZH11 was gene-edited using CRISPR/Cas9. Osby1-1 is a line containing target 1 and Osby1-2 is a line containing target 2. (G) Phenotypes of the lines. The scale bar is 5 cm. (H) Biomass yield and (I) grain yield of the lines (both DW). Data are means (±SD), n=10. Significant differences compared with the ZH11 wild-type were determined using two-tailed Student’s t-test: **P<0.01.

Fig. 4.

Transcriptional and functional characterization of BY1. (A) Relative expression of BY1 in different tissues of the sorghum wild-type (WT) and the by1 mutant. (B) Subcellular localization of the BY1 and by1 proteins in maize protoplasts. 35S::GFP is the control (top row), 35S::BY1-GFP fused with the full-length sequence of BY1 is the WT gene fusion (middle row), and 35S::by1-GFP fused with the full-length sequence of by1 is the mutant gene fusion (bottom row). Scale bars are 10 μm. (C) Comparison of the tertiary structure of the BY1 protein in the WT with by1 in the mutant. The structures were predicted using SWISS-MODEL (https://swissmodel.expasy.org/). Black arrows indicate the obvious structural alterations between the WT and by1 due to the conversion of the 192nd amino acid from Pro to Leu. (D) Reaction scheme for 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS, BY1). PEP, phosphoenolpyruvate; E4-P, erythrose-4-phospate. (E) Enzyme activity of DAHPS (BY1) in the WT and the by1 mutant. Data are means (±SD), n=3. Significant differences were determined using two-tailed Student’s t-test: *P<0.05, **P<0.01.

Fig. 5.

Transcriptome analysis of sorghum BY1. (A) Gene ontology (GO) analysis of differentially expressed genes (DEGs). The numbers of up- and down-regulated DEGs in each functional pathway are indicated on the right axis, and their percentages are indicated on the left axis. (B) q-values of individual groups of enriched KEGG pathways. Each point represents the degree of enrichment of the KEGG category according to the color scale, whilst the number of DEGs is indicated by the size of the point. (C–G) Percentages of up- and down-regulated DEGs related to (C) photosynthesis, (D) phenylpropane metabolism, (E) the cell cycle, (F) nucleic acids, and (G) mitosis and meiosis.

Fig. 6.

Metabolic profiling analysis of sorghum wild-type BY1 and the by1 mutant. (A) Volcano plot of the different metabolites. For each metabolite, the variable importance in projection (VIP) value is plotted against the logarithm of the quantitative difference multiple of the metabolite in two samples: the larger the absolute value, the greater the difference multiple of the content of the metabolite. The larger the VIP value, the more significant the difference in the metabolite levels. Metabolites down-regulated in by1 relative to the wild-type (WT) are shown in green, metabolites up-regulated are shown in red, and those with no significant difference are shown in gray. (B) Clustering heatmap of metabolites with significantly different contents (MSDCs) in by1 compared with the WT. The metabolite classes are grouped according to the color scale and their contents are indicated by the red–green scale. WT lines are to the left and by1 lines are to the right. (C) KEGG classification of MSDCs. The number of the MSDCs annotated to each pathway is shown together with its percentage of the total number of MSDCs. (D–G) Fold-changes of (D) phosphoenolpyruvate, (E) shikimate, (F) phenylalanine, and (G) chalcone levels between the WT and by1. Data are means (±SD), n=3. Significant differences were determined using two-tailed Student’s t-test: **P<0.01.

Fig. 7.

A working model of the role of BY1 in regulating sorghum growth and development. (A) In the wild-type (WT), a portion of carbon dioxide fixed during photosynthesis is transformed into phosphoenolpyruvate (PEP) and erythritol-4-phosphate (E4-P) through glycolysis and the pentose phosphate pathway, respectively. PEP and E4-P enter the shikimate pathway under the control of BY1, pass through aromatic amino acid (AAA) biosynthesis and the flavonoid metabolism pathway, and form flavonoids, which regulate plant growth and development. (B) In the BY1 loss-of-function mutant (by1), the shikimate pathway is weakened, and feedback regulation leads to a decrease in the flow of carbon source the shikimate pathway, which leads to abnormal contents of secondary metabolites, thereby adversely affecting plant growth and development. The widths of the black arrows represent the amount of the carbon flow in the pathway. Scale bars in the images are 10 cm.