The AGCVIII kinase Dw2 modulates cell proliferation, endomembrane trafficking, and MLG/xylan cell wall localization in elongating stem internodes of Sorghum bicolor

Abstract

Stems of bioenergy sorghum (Sorghum bicolor L. Moench.), a drought-tolerant C4 grass, contain up to 50 nodes and internodes of varying length that span 4-5 m and account for approximately 84% of harvested biomass. Stem internode growth impacts plant height and biomass accumulation and is regulated by brassinosteroid signaling, auxin transport, and gibberellin biosynthesis. In addition, an AGCVIII kinase (Dw2) regulates sorghum stem internode growth, but the underlying mechanism and signaling network are unknown. Here we provide evidence that mutation of Dw2 reduces cell proliferation in internode intercalary meristems, inhibits endocytosis, and alters the distribution of heteroxylan and mixed linkage glucan in cell walls. Phosphoproteomic analysis showed that Dw2 signaling influences the phosphorylation of proteins involved in lipid signaling (PLDδ), endomembrane trafficking, hormone, light, and receptor signaling, and photosynthesis. Together, our results show that Dw2 modulates endomembrane function and cell division during sorghum internode growth, providing insight into the regulation of monocot stem development.

Keywords: Sorghum bicolor; cell proliferation; cell wall heteroxylan; endocytosis; endomembrane system; mixed linkage glucan; stem growth.

Figure 1

Diagram of internode development in monocots; Dw2 phenotype. (a) Monocots grow stems in discriminate segments called internodes. Each internode is a part of a phytomer which contains the leaf blade and leaf sheath. At the base of elongating internodes is an area of active cell division, the zone of cell division (ZoD). Above the ZoD are areas of cell elongation (ZoE) and maturation (ZoM), where cell expansion and maturation/lignification occur, respectively. The apical meristem is located at the very tip of the growing stem and is where new phytomers are formed. Beneath the apical meristem are small undeveloped internodes of phytomers 1–3 that have yet to elongate. For this study, the apical meristem and Int(P1‐4) were grouped together in what we define as the ‘apical dome’. (b) Mature DYM and DDYM internodes, which are wild‐type and dw2, respectively. DYM internodes are longer. Scale bar is 5 cm. (c) Measure of maximum length of cells in a fully mature internode. There is no difference in maximum length of cells in DYM or DDYM. Error bars represent SD over three replicates. (d) Number of cells in the length of the internode, measured from nodal plane to nodal plane. DYM internodes have approximately 2.2× larger cells than DDYM internodes. Errors bars represent SD over three replicates.

Figure 2

Mutation of Dw2 decreases cell proliferation in growing internodes. (a–c) Cells in the zone of division in elongating internodes of DYM (a) and DDYM (b). Cells in DDYM are larger (c). Scale bar is 50 μm. Error bars represent SD of three replicates. (d) Cell number in internodes of phytomers 4, 5, and 6 from nodal plane to nodal plane. Cell numbers in DYM and DDYM Int(P4), Int(P5), and Int(P6) diverge during internode development. Error bars represent SD of three replicates. ns, not significant; ***P ≤ 0.001.

Figure 3

Dw2 causes uniform cell morphology and vascular bundles (VBs). (a,c) Longitudinal sections of mature internodes in DYM (a) and DDYM (dw2) (c) plants. Cells in DYM show uniform cell files and morphology, whereas mutant plants show irregular cell shapes and compromised cell files. (b,d) Horizontal sections of mature internodes in DYM (b) and DDYM (d) plants. DYM plants show typical monocot VB morphology, whereas DDYM plants show aberrations in VB morphology (orange boxes). Scale bar is the same for all images.

Figure 4

Dw2 is expressed in pith and rind, but is concentrated around vascular bundles. (a,d) Negative control of RNAscope in situ hybridization of the bacterial gene dapB. (b,e) Positive control of in situ hybridization of a cytochrome P450 in DYM plants. (c,f) RNAscope in situ hybridization of Dw2 in Int(P5) of DYM plants. Signal (red spots) is present in pith of the internode (c), but is highly concentrated at the rind, surrounding VBs, and within VBs (f). X, xylem; P, phloem.

Figure 5

Phosphoproteomics of internode tissue revealing endomembrane and cytoskeleton proteins. (a) Volcano plot of the phosphoproteomic experiment on growing Int(P5) tissue. Most differential phosphorylation events occur in DDYM tissue. (b) GO enrichment analysis of the phosphoproteins in the right section of (a). Significant enrichment is observed for many processes involved in cytoskeletal maintenance. (c) GO enrichment analysis of the phosphoproteins in the left section of (a). Significant enrichment is observed for cytoskeletal organization, as well as localization, exocytosis, and vesicle transport. A complete list of enriched terms for both phosphoprotein datasets can be found in the supplementary information.

Figure 6

The phospholipase D inhibitor n‐butanol causes DYM to phenocopy DDYM. (a) Internodes from control and treated DYM and DDYM plants. (b) Treated DYM internodes are indistinguishable from DDYM internodes from either condition. Scale bar is 5 cm. (c) Images of cells from the zone of division (ZoD) of treated and control DYM and treated or control DDYM. (d) Treated DYM internodes have significantly longer cells in the ZoD than control DYM cells and are indistinguishable from DDYM internodes in either condition. Error bars are the SD of three replicates in the control and four replicates in the treated conditions.

Figure 7

Dw2 mutants have altered root hair morphology and reduced endocytosis. (a, top) 12‐day‐old DYM seedling have straight root hairs. (a, bottom) 1‐day‐old DDYM seedlings have bent or wavy root hairs. (b, top) DYM root hair after 15 min of FM4‐64 treatment. Most of the signal has been taken up into the cytoplasm. (b, bottom) DDYM root hair after 15 min of FM4‐64 treatment. Most of the signal is localized to the plasma membrane (PM). (c) Time course of the endocytic tracking dye FM4‐64 of root cells. At 5 min after treatment, both genotypes show a strong signal in the PM. (d) As time progresses to 30 min after treatment, DYM roots show strong internalization of the dye, with DDYM plants showing a higher fluorescent signal in the PM, indicating slower uptake. Whiskers represent minimum and maximum values and the box indicates the interquartile range of 50 cells over five replicates. (e) DYM roots treated with Brefeldin A (BFA), an inhibitor of endosomal cycling. (f) After 1 h of treatment, DYM roots contain many ‘BFA bodies’ per cell. DDYM roots contain fewer BFA bodies per cell. Whiskers represent minimum and maximum values and the box is the interquartile range of 50 cells over three replicates. ***P ≤ 0.001. Scale bar in (c,e) is 25 μm.

Figure 8

Dw2 signaling regulates the localization of cell wall components trafficked by the endomembrane system. (a–c) Immunolocalization of mixed linkage glucan (MLG) in DYM (a) and DDYM (b) internode cells. MLG shows strong localization in the cellular vertices of elongating DDYM internodes (b,c). DYM plants show uniform MLG localization (a). Cell wall fluorescence intensity is normalized to DYM levels. The point in (c) is an outlier. (d–f) Heteroxylan (HX) immunolocalization in DYM (d) and DDYM (e) internode cells. In DDYM, HX shows strong localization at the planes where adjacent cells meet (faces) (e,f). DYM plants show uniform HX localization (d). Cell wall fluorescence is normalized to DYM levels. Both MLG and HX are polysaccharides that are trafficked through the endomembrane system. (g–i) Calcofluor White staining in DYM (g) and DDYM (h) internode cells. Calcofluor White preferentially stains cellulose and callose. Both DYM and DDYM cells show a uniform signal in their cell walls, with total fluorescence intensity reaching 90% of that in the DYM cells (i). Scale bar is 10 μm. Whiskers represent the minimum and maximum values, and the box indicates the interquartile range of 50 cells over three replicates. DDYM fluorescence intensity values are normalized to DYM values. ***P ≤ 0.001.